MassXpert User Manual

version 10.5.10

PDF document

Filippo Rusconi, 2026

Version 10.5.10
Published: 27/10/2026

Revision history

1 Preface

1.1 Software feature offerings and intended audience

This manual is about the MsXpertSuite mass spectrometric software suite, a software environment that contains two modules:

• MassXpert: a module that allows users to define brand new polymer chemistries. The polymer chemistry definitions are then used to model linear polymer sequences. Once modelled, a polymer sequence can undergo chemical reactions (enzymatic or chemical cleavages, gas-phase fragmentations...). The obtained results are a model of what a mass spectrum would look like if the modelled experiment had actually been carried over up to the mass spectrometry analysis.

• MineXpert: a module that allows users to load mass spectrometry data from mass spectrometry data files. The loaded data are then visualized and explored throughout all their MSⁿ depth. The mass data visualization starts either at the TIC chromatogram level or the MS data table view and deepens to the MSⁿ mass spectra, the drift spectra, the XIC chromatograms, various color maps relating m/z values with either retention times or drift times. A wide array of mass spectral integrations are available to ease the MSⁿ data finest scrutiny.

This manual is therefore intended for people willing to learn how to use the comprehensive MsXpertSuite software package.

Mass spectrometry has gained popularity across the past twenty years or so. Indeed, developments in polymer mass spectrometry have made this technique appropriate to accurately measure masses of polymers as heavy as many hundreds of kDa, and of any chemical type.

There are a number of utilities—sold by mass spectrometer constructors with their machines, usually as a marketing “plus”—that allow predicting/analyzing mass spectrometric data obtained on polymers. These programs are usually different from a constructor to another. Also, there are as many mass spectrometric data prediction/analysis computer programs as there are different polymer types. You will get a program for oligonucleotides, another one for proteins, maybe there is one program for saccharides, and so on. Thus, the biochemist/massist, for example, who happens to work on different biopolymer types will have to learn to use several different software packages. Also, if the software user does not own a mass spectrometer, chances are he will need to buy all these software packages.

The MsXpertSuite mass spectrometric software is designed to provide free solutions to all these problems by providing the following features:

• MassXpert:

  • Model ex nihilo polymer chemistry definitions (in the module that is part of the MassXpert program);

  • Peform simple yet powerful mass computations to be made in a mass desktop calculator that is both polymer chemistry definition-aware and fully programmable (that’s the module also part of the MassXpert program);

  • Edit polymer sequences on a polymer chemistry definition-specific basis, along with chemical reaction simulations, finely configured mass spectrometric computations... (all taking place in the module that is the main module of the MassXpert program);

  • Customize the way each monomer will show up graphically during the program operation (in the module);

  • Edit polymer sequences with immediate visualization of the mass changes elicited by the editing activity (in the module);

  • Open an unlimited number of polymer sequences at any given time and of any given polymer chemistry definition type (in the module);

• MineXpert:

  • Load mass spectometry data files in the mzML format, thanks to the excellent library of ProteoWizard¹ fame; mass data file loading can be performed in full-memory mode (all the data read from file are stored in memory) for faster operations or in streamed mode when loading files largers than the available memory.

  • Display the data in powerful ways in a unified graphical user interface. The interface was designed to integrate all the most useful characteristics of the various proprietary environments known by the author, thanks to the excellent library;²

  • Configure the way mass spectrometry data integrations are performed from a combination standpoint and optionally apply a Savitzky-Golay smo

  • Configure the level of MS data (MSⁿ) for which the integrations are performed. Optionally filter data by precursor ion m/z values or precursor spectrum indices;

  • Perform data mining by performing data integrations in various ways;

  • Ion mobility mass spectrometry data are supported with a int=f(m/z,dt) color map plot calculation;

  • A specific data integration mode allows easy mass spectral intensity determination for any feature (peak in a TIC chromatogram, a mass spectrum, a drift spectrum, any color map region);

1.2 Feedback to the developers

We are always grateful to any constructive feedback from the users.

The MsXpertSuite software team might be contacted via the following addresses:

1.3 Brief history of MsXpertSuite.

• 1998–2000

  The name MassXpert comes from a project I started while I was a post-doctoral fellow at the École Polytechnique (Institut Européen de Chimie et Biologie, Université Bordeaux 1, Pessac, France);

  The MassXpert program was published in Bioinformatics (Rusconi, F. and Belghazi, M. Desktop prediction/analysis of mass spectrometric data in proteomic projects by using massXpert. Bioinformatics, 2002, 644–655.

  At that time, was at the version and the next big release was going to be you will see what you'll see: .

  When I tried MassXpert on that new version (one colleague had gotten it with a new machine), I discovered that my software would not run normally (the editor was broken). The Microsoft technical staff would advise to buy a new version of the compiler environment and rebuild. This was a no-go: I did not want to continue paying for using something I had already produced with legitimate software.

• 2001–2006

  During fall 1999, I decided that I would stop using products for my development. At the beginning of 2000 I started as a CNRS research staff in a new laboratory and decided to start fresh: I switched to (I never looked back). After some months of learning, I felt mature to start a new development project that would eventually become an official GNU package: .

  The software, much more powerful than what the initial MassXpert software used to be, was published in BMC Bioinformatics in 2006 (Rusconi, F. GNU polyxmass: a software framework for mass spectrometric simulations of linear (bio-) polymeric analytes). BMC Bioinformatics, 225–.

  Following that publication I got a lot of feedback (very positive, in a way) along the lines: —Hey, your software looks very interesting; only it’s a pity we cannot use it because it runs on GNU/Linux, and we only use .

• 2007–2016

  In december 2006, I decided to make a full rewrite of . The software of which you are reading the user manual is the result of that rewrite. I decided to “recycle” the MassXpert name because this software is written in C++, as was the first MassXpert software. Also, because the first -based MassXpert project is not developped anymore; taking that name was kind of a “revival” which I enjoyed. However, the toolkit I used this time is not the Microsoft Foundation Classes (first MassXpert version) but the Trolltech framework (see menu Help$⇨$About Qt in MassXpert ).

  Coding with the libraries has one big advantage: it allows the developer to code once and to compile on the three main platforms available today: , , . Another advantage is that the libraries are wonderful software, technically and philosophically (Free Software).

  The rewritten software was published in 2009 (Rusconi, F. massXpert 2: a cross-platform software environment for polymer chemistry modelling and simulation/analysis of mass spectrometric data.Bioinformatics, 2009, 2741–2742.

• 2016–2019

  In 2016, I started a new project about visualization of mass spectrometric data. The project developed pretty quickly, as we needed at the mass spectrometry facility a software that would allow to cope efficiently with ion mobility mass spectrometric experimental data. MineXpert was thus started.

  To bundle both MassXpert and MineXpert in a single software suite, I bought the MsXpertSuite domain name http://msxpertsuite.org and created that new name.

• 2019–2021

  The MineXpert software was published in 2019. The reviewers acknowledged that it was a useful piece of software but complained about its not handling the MS/MS data. Reference to cite: Filippo Rusconi. mineXpert: Biological Mass Spectrometry Data Visualization and Mining with Full JavaScript Ability. J. Proteome Res. 2019, 18, 5, 2254-2259.

In response to the reviewers and for my personal project that I had started a little earlier, I decided to fully rewrite MineXpert into MineXpert. The new version of the software had to support MSⁿ data.

• 2021–2022

  The MineXpert software was published in 2019. The main new capability is now the full handling of MSⁿ data. This new feature required the full rewriting of the software internal data model. See Langella O., Rusconi F. (2021) mineXpert2: Full-Depth Visualization and Exploration of MSⁿ Mass Spectrometry Data. J. Am. Soc. Mass Spectrom., 4 (32) 1138-1141.

• 2022–2025

  A major work is now undertaken: the native handling of the timsTOF data from the Bruker instrument vendor. This kind of data contains ion mobility data.

• 2025–2026

  Rewrite of major portions of the software to ease evolution of the code base, in particular with a strong enhancement of code reuse by putting large portions of code in the and libraries, for use by MineXpert and MassXpert .

• 2026–

  Following the almost full rewrite, development of new features has resumed, all along regular feature requests by the users of the software. In particular, data processing features have been added to &MineXp;, such as a new automatic deconvolution of low mass analytes with analyte identification (with fully labeled analytes handling), peak labeling management in the plot widgets, new plot management inside a plot widget, network-based communication between MineXpert and the sister application MassXpert .

1.4 Program and Documentation Availability and License

The programs and all the documentation that are shipped along with the MsXpertSuite software suite are available at http://www.msxpertsuite.org. Most of the time, a new version is published as source, and as binary install packages for (64-bit systems only). No binary packages are created outside of the autobuilder of the various distributions. As a Debian Developer, the author creates ³ packages that are uploaded on the distribution servers. These packages are available using the system’s software management infrastructure (like using the ‘s apt command, for example, or the graphical application).

The software and all the documentation are all provided under the Free Software license GNU General Public License, Version 3, or later, at your option. For an in-depth study of the Free Software philosphy, I kindly urge the reader to visit http://www.gnu.org/philosophy.

2 Generalities

In this chapter, I wish to introduce some general concepts around the MassXpert program and the way data elements are named in this manual and in the program.

The MassXpert mass spectrometry software suite has been designed to be able to “work” with every linear polymer. Well, in a certain way this is true... A more faithful account of the MassXpert’s capabilities would be: “The MassXpert software suite works with whatever polymer chemistry the user cares to define; the more accurate the polymer chemistry definition, the more accurate MassXpert will be”.

For the program to be able to cope with a variety of possibly very different polymers, it had to be written using some abstraction layer in between the mass calculation engine and the mere description of the polymer sequence. This abstraction layer is implemented with the help of “polymer chemistry definitions”, which are files describing precisely how a given polymer type should behave in the program and what its constitutive entities are. The way polymer chemistry definitions are detailed by the user is the subject of a chapter of this book (see menu XpertDef of the program). However, in order to give a quick overview, here is a simple situation: a user is working on two polymer sequences, one of chemistry type “protein” and another one of chemistry type “DNA”.

The protein sequence reads “ATGC”, and the DNA sequence reads “CGTA”.

Now imagine that the user wants to compute the mass of these sequences. How will MassXpert know what is the formula (hence mass) of each monomer code in each polymer sequence? There must be a way to inform MassXpert that one of the sequences is a protein while the other is a DNA oligonucleotide: this is done upon creation of a polymer sequence; the programs asks of what chemistry type the sequence to be created is. Once this “chemical parentage” has been defined for each sequence, MassXpert will know how to handle both the graphical display of each sequence and the calculations for each sequence.

2.1 On Chemical Formulæ and Chemical Reactions

Any user of MassXpert will inevitably have to perform two kinds of chemical simulations:

• Define the formula of some chemical entity;
• Define a given chemical reaction, like a protein monomer modification, for example.

While the definition of a formula poses no special difficulty, the definition of a chemical reaction is less trivial, as detailed in the following example. The lysyl residue has the following formula: C₆H₁₂N₂O. If that lysyl residue gets acetylated, the acetylation reaction will read this way:

This can also read:

.

The representation of that reaction is:

When the user wants to define that chemical reaction, they can use that representation: “−H₂O+CH₃COOH”, or even the more brief but chemically equivalent one: “−H+CH₃CO”.

There is another interesting feature of formulæ in MassXpert: the title of a formula. Anywhere a formula might be entered, it can be entered with a title in the following form: “Acetylation”−H₂O+CH₃COOH. This is of particular interest in cases where the user wants to store formulæ for later use when that is offered by the MassXpert software.

2.2 The MassXpert Framework Data Format

All the data dealt with in MassXpert are stored on disk as XML-formatted files. XML is the eXtensible Markup Language.

This “language” allows to describe the structure of a document. The structure of the data is first described in a section of the document that is called the Document Type Definition, DTD the data then follow in the same file. One of the big advantages of using an XML format in MassXpert is that it is a text format, and not a binary one. This means that any data in the MassXpert package is human-readable (even if the XML syntax makes it a bit difficult to read data, it is actually possible). Try to read one of the polymer chemistry definitionXML-formatted files that are shipped with this software package, and you’ll see that these files are pure text files (the same applies for the *.mxp XML-formatted polymer sequence files). The advantages of using text file formats, with respect to binary file formats are:

• The data in the files are readable even without the program that created them. Data extraction is possible, even if it costs work;
• Whenever a text document gets corrupted, it remains possible to extract some valid data bits from its uncorrupted parts. With a binary format, data are chained from bit to bit; loosing one bit leads to automatic corruption of all the remaining bits in the file;
• Text data files are searchable with standard console tools (, ...), which make it possible to search easily text patterns in any text file or thousands of these files in one single command line. This is not possible with binary format, simply because reading them requires the program that knows how to decode the data and the powerful console-based tools would prove useless.

2.3 Chemical Entity Naming Policy

Unless otherwise specified, the user is strongly advised not to insert any non-alphanumeric-non-ASCII characters (space, %, #, $...) in the strings that identify polymer chemistry definition entities. This means that, for example, users must refrain from using non-alphanumeric-non-ASCII characters for the atom names and symbols, the names, the codes or the formulæ of the monomers or of the modifications, or of the cleavage agent definitions, or of the fragmentation pathway definitionss… Usually, the accepted delimiting characters are - and _. It is important not to cripple these polymer data for two main reasons:

• So that the program performs smoothly (some file-parsing processes rely on specific characters (like # or %, for example) to isolate sub-strings from larger strings);
• So that the results can be easily and clearly displayed when time comes to print all the data.

3 Configuring the application preferences

A large application like MassXpert has lots of configurable options. The Application preferences window described in this chapter is the central place for these customizations. To enter the application preferences window, select Preferences$⇨$Edit preferences.

3.1 Setting the application preferences - general options

As shown in Figure 2 – Application preferences - General page the general options encompass the following notions:

• Maximum number of threads: Set the maximum number of threads when performing resource intensive tasks, like mass spectrum integration.

• Decimal places for number representation: Set the numer of decimal digits when representing numbers. Note that not all numbers need the same decimal precision.

3.2 Setting the application preferences - shortcuts

As shown in Figure 3 – Application preferences - Shortcuts page it is possible to configure the shortcuts for a large number of tasks throughout the application.

The shortcuts can be configured by double-clicking the item of interest in the cell of the Shortcut column and then entering the desired shortcut. Then simply click somewhere else.

When all the shortcuts have been configured, click the button labeled Apply and restart the program.

3.3 Setting the application preferences - filesystem hierarchy structure

The MassXpert program comes with a number of predefined polymer chemistry definitions and polymer sequence files. The locations of these data elements are set during the installation of the software by the system’s package manager. It is however possible to configure any directory of the filesystem hierarchy so that the program continues to run even with changed (but documented) locations. In particular it is possible to configure the location of the user’s data directories.

4 Basics in Polymer Chemistry

This chapter will introduce the basics of polymer chemistry. The way this topic is going to be covered is admittedly biased towards mass spectrometry and biological polymers. Moreover, the aim of this chapter is to provide the reader with the specialized words that will later be used to describe and explain the (inner) workings of the MassXpert program. This manual is not a “crash course” in biochemistry.

4.1 Polymers? Where? Everywhere!

Indeed, polymers are everywhere. If you ask somebody to show you something polymeric, he/she will point you at the first plastic object in the vicinity. Right, plastic materials are made of hydrocarbon polymers. We also have many different polymers in our body. Proteins are polymers, complex sugars are polymers, DNA (the so-called “molecule of heredity” is a huge polymer. There are polymers in wine, in wood... Where? Everywhere!

My Oxford Advanced Learner’s Dictionary of Current English gives for polymer the following definition: —“natural or artificial compound made up of large molecules which are themselves made from combinations of small simple molecules”.

A polymer is indeed made by covalently linking small simple molecules together. These small simple molecules are called monomers, and it is immediate that a polymer is made of a number of monomers. A general term to describe the process that leads to the formation of a polymer is polymerization. It should be noted that there are many ways to polymerize monomers together. For example, a polymer might be either linear or branched. A polymer is linear if the monomers that are polymerized can be joined at most two times. The first junction links an “entering” monomer to an “elongating” polymer (thus making it the new end of the elongating polymer which, by the way, is longer than before by one unit) and the second junction links the new elongating polymer’s end to a new monomer. This process goes on until the reaction is stopped, the point at which the polymer reaches its finished state. A branched polymer is a polymer in which at least one monomer is able to contract more than two bonds. It is thus clear that a single monomer linked three times to other monomers will yield a “T-structure”, which is nothing but a branched structure.

In the following sections we’ll describe a number of different kinds of polymers. Each time, they will be described by initially detailing the structure of their constitutive monomers; next the formation of the polymer is described. At each step we shall try to set forth each polymer characteristics in such a manner as to introduce the way MassXpert “thinks polymers” and to introduce specialized terminologies. Once the basic chemistries (of the different polymers) have all been described, we will enter a more complex subject that is of enormous importance to the mass spectrometry specialist: polymer chain disrupting chemistry. We shall see that this terminology actually involves two kinds of chemistries: cleavage, on the one hand, and fragmentation, on the other hand.

While MassXpert is basically oriented to linear single-stranded polymer chemistries, it can also be used to simulate highly complex polymer chemistries. Biological polymers are the main focus of this manual, however all the concepts described here may be applied with no modification to synthetic polymer chemistries.

4.2 Various Biopolymer Structures

Biopolymers are amongst the most sophisticated and complex polymers on earth and it certainly is not a mistake to take them as examples of how monomers (be these complex or not) can assemble covalently into life-enabling polymers. In this section we will visit three different polymers encountered in the living world: proteins, nucleic acids and polysaccharides. We shall be concerned with 1) the structure of the monomers, 2) the polymerization reaction and 3) the final end-capping reaction responsible for putting the polymer in its finished state.

4.2.1 Proteins

These biopolymers are made of amino acids. There are twenty major amino acids in nature, and each protein is made of a number of these amino acids. The combinations are infinite, providing enormous diversity of proteins to the living world.

A protein is a polar polymer: it has a left end and a right end, and polymerization actually occurs from left to right (from N-terminus to C-terminus, see below).

The bond formed by condensation of two amino acids is an amide bond, also called—in protein chemistry— a peptidic bond. The elongation of the protein is a simple repetition of the condensation reaction shown in

Now we should point at a protein chemistry-specific terminology issue: we have seen that a protein is a polymer made of a number of monomers, called amino acids. In protein chemistry, there is a subtlety: once a monomer is polymerized into a protein it is no more called a monomer, it is called a residue. We may say that a residue is an amino acid less a water molecule.

From what we have seen until now, we may define a protein this way:

This definition is still partly inexact, however. Indeed, from what is shown in

Now comes the question of unambiguously defining the structure of a protein. It is commonly accepted that the simple ordered sequence of each residue code in the protein, from left to right, constitutes an unambiguous description of the protein’s primary structure (that is, of its sequence). Of course, proteins have three-dimensional structures, but this is of no interest to a program like MassXpert, which is aimed at calculating masses of polymers. To enunciate unambiguously the sequence of a protein, one would use a symbology like this:

• Using the 3-letter code of the amino acids: Ala Gly Trp Tyr Glu Gly Lys

• Using the 1-letter code of the amino acids: A G W Y E G K

  Alanine is thus the residue 1 and Lysine is the last residue (n = 7)

4.2.2 Nucleic Acids

These biopolymers are more complex than proteins, mainly because they are composed of monomers (nucleotides) that have three different chemical parts, and because those parts differ in DNA and RNA. A nucleotide is the nucleic acid’s brick: a nucleotide consists of a nitrogenous base combined with a ribose/deoxyribose sugar and with a phosphate group. There are two different kinds of nucleic acids: deoxyribonucleic acid (DNA, the sugar is a deoxyribose) and ribonucleic acid (RNA, the sugar is a ribose). DNA is most often found in its double stranded form, while RNA is most often found in single strand form. There are four nitrogenous bases for each: Adenine, Thymine, Guanine, Cytosine for DNA; in RNA only one of these bases changes: Thymine is replaced by Uracile. As for proteins, nucleic acids are polar polymers: the polymerization process is polar, from left to right (sometimes left is up and right is down in certain vertical representations found mainly in textbooks).

This manual is not to teach biochemistry, which is why the structure of the monomers is not described in atomic detail. However, since it is important to understand how the polymerization occurs,

The conventional representation of a nucleic acid involves showing the 5′ end on the left, and the 3′ end on the right, horizontally. Sometimes, to clearly indicate that the left end is phosphorylated, while the right end is not, the ends are indicated as “5′P” and “3′OH”.

Now comes the question of unambiguously defining the structure of a nucleic acid. It is commonly accepted that the listing of the named nitrogenous bases in the nucleic acid—from left (5′ end) to right (3′ end)—constitutes an unambiguous description of the nucleic acid sequence. To enunciate the sequence of a gene, one would use a symbology like this:

for a DNA, using the 1-letter code of the nitrogenous bases:

A T G C A G T C

for an RNA, using the 1-letter code of the nitrogenous bases:

A U G C A G U C

Adenine is thus the base 1 and Cytosine is the last base (n = 8)

4.2.3 Saccharides

These biopolymers are certainly amongst the most complex ones in the living world. This is mainly due to the fact that saccharides are usually heavily modified in living cells with a huge variety of chemical modifications. Furthermore, the ramifications in the polymer structure are more often the normal situation than not. Interestingly, these molecules are first thought of as the “fuel” for the cell, which is certainly far from being total nonsense, but it is also undoubtful that their structural role is extremely important (often in combination with proteinaceous material). Another interesting aspect of their ability to form complex structures is their use as “key” systems for identification processes: a number of complex sugars are located on the cell walls and provide “recognition patterns” for the other cells to deal with…

Nonetheless, the general picture is not that complex, if the way monomers are polymerized together is the only concern (which is the case in this manual). As far as we are concerned, in fact, the polymerization mechanism is a simple condensation (much like what has been described for proteins), yielding a sugar bond. Indeed, some people use the same terminology: a monomeric sugar becomes a residue once polymerized in the saccharidic chain. There are two main different kinds of sugars: pentoses (in C₅) and hexoses (in C₆); it should be noted, however, that there is a variety of other common molecules, like sialic acids, heptoses…

Like already seen for proteins and nucleic acids, a saccharidic polymer is polar: it has a left end and a right end. The terminology regarding the ends of a saccharidic polymer is rather unexpected at first sight: the left end is said to be the non-reducing end while the right end is said to be the reducing end. Historically this was observed with monosaccharides (also called monoses), which reduced cupric (Cu²⁺) ions, thus getting oxydized themselves on the carbonyl (when in the open ring aldehydic form).

Now comes the question of unambiguously defining the structure of a saccharidic polymer. It is commonly accepted that the simple ordered sequence of the named monoses in the saccharidic polymer, from left (non-reducing end) to right (reducing end), constitutes an unambiguous description of the glycan sequence. To enunciate the sequence of a glycan, one would use a symbology like this:

Using a 3-letter code:

Ara Gal Xyl Glc Hep Man Fru

Arabinose is thus the monose 1 and Fructose is the last monose (n = 7)

Incidentally, this is where the ability of MassXpert to handle monomer codes of non-limited length comes in handy!

4.3 To Sum Up

We made a rapid overview of the three major polymers in the living world. A great many other polymers exist around us.

4.4 Polymer Chain Disrupting Chemistry

The “polymer chain disrupting chemistry” was mentioned earlier as a complex subject that was of enormous importance to the mass spectrometry scientist. This is why that subject will be treated in a pretty thorough manner. First of all it should be noted that a chemical modification of a polymer does not necessarily involve the perturbation of the chain structure of the polymer. Here, however, we are concerned specifically with a number of chemical modifications that yield a polymer chain perturbation; cleavage and fragmentation:

CleavagesThese are chemical processes by which a cleaving agent will act directly on the polymer chain making it fall into at least two separated pieces (the oligomers). As a result of the cleavage reaction, groups originating in the cleavage agent molecule may remain attached to the polymer at the precise cleavage location;

FragmentationsThese are chemical processes by which the polymer structure is disrupted into separated pieces (the fragments) mainly because of energy-dependent electron doublet rearrangements leading to bond breakage.

4.4.1 Polymer Cleavage

We said above that, upon cleavage of a polymer, the cleaving molecule reacts with it, and by doing so directly or indirectly “dissolves” an inter-monomer bond. A polymer cleavage always occurs in such a way as to generate a set of true polymers (smaller in size than the parent polymer, evidently, which is why they are called oligomers). Indeed, let us take the example shown in

• In the first case the molecule that is responsible for the cleavage is water, while in the second case it is cyanogen bromide;
• In both cases the bond that is cleaved is the inter-monomer bond (in protein chemistry this is a peptidic bond);
• In both cases the Oligomer 2 has the same structure;
• The structures of the Oligomer 1 species differ when produced using water or cyanogen bromide as the cleaving molecule.

The difference between hydrolysis and cyanogen bromide cleavage is in the generation of the Oligomer 1 species: the cyanogen bromide cleavage has a side effect of generating a homoserine as the right end monomer of Oligomer 1, while hydrolysis generates a genuine methionine monomer. This is because water reverses in a very symmetrical manner what polymerization did (hydrolysis is the converse of condensation), while cyanogen bromide did some chemical modification onto the generated Oligomer 1 species.

Nonetheless, the reader might have noted that—interestingly—all the four oligomers do effectively have their left cap (a proton) and their right cap (the hydroxyl). This means that in both water- and cyanogen bromide-mediated cleavages, all the generated oligomers are indeed true polymers in the sense that: 1) they are a chain of monomers (modified or not) and 2) they are correctly capped (i.e. they are polymers in their finished state). This is important because it is the basis on which we shall make the difference between a cleavage process and a fragmentation process. Thus, the MassXpert definition of an oligomer can be formulate like so:

When the polymer cleavage reaction precisely reverses the reaction that was performed for the same polymer’s synthesis, there is no special difficulty. But when the cleavage reaction modifies the substrate, then this should be carefully modelled. How? To answer this question we might start by comparing the two different Oligomer 1 species that were yielded upon the water-mediated and the cyanogen bromide-mediated cleavage reactions. The difference might be worded like so:

Turns out that this is a big difference! The observations we did so far might be worded this way:

In MassXpert’s jargon, this logical condition is called a cleavage rule (described later; see Section 5.2.4 – The cleavage agent definitions.

Well, all this sounds reasonable. But what about the “normal” case, when the cleavage is done using water? Nothing special: the mass of the oligomer is calculated by summing the mass of each monomer in the oligomer (since the monomers are not modified, this is easily done) and the masses corresponding to the left and right caps (these are defined in the polymer chemistry definition; in our present case it would be a proton on the left end, and a hydroxyl on the right end). In this way, the oligomer complies with its definition, which states that it is a faithful polymer made of monomers and that it is in its finished state.

Yes, but then how will MassXpert manage to calculate the mass of the modified oligomer, like our Oligomer 1 in the case of the cyanogen bromide-mediated cleavage? Simple enough: in a first step it does exactly the same way as for the unmodified oligomer. Next, each oligomer is checked for presence or absence of a methionine residue on its right end. If a methionine is found, the mass corresponding to the “−C1H2S1+O1” chemical reaction is applied. And that’s it.

In the previous cyanogen bromide example, the logical condition was involving the identity of the oligomers’ right end monomer, but other examples can involve not the right end monomer, but the left end monomer, if some chemical modification was to occur to the monomer sitting right of the cleavage location. In this case the user would have to analyse the situation and provide MassXpert with the proper chemical reaction by stating something analog to: “If and only if they have a Xyz monomer at their left end”. This introduction to polymer cleavage abstraction should be enough to later delve into the cleavage specification definition as MassXpert conceives it and that is thoroughly detailed at Section 5.2.4 – The cleavage agent definitions.

4.4.2 Polymer Fragmentation

In a fragmentation process, the bond that is broken is not necessarily the inter-monomer bond. Indeed, fragmentations are oft-times high energy chemical processes that can affect bonds that belong to the monomers’ internal structure. This is one of the reasons why fragmentations do differ from cleavages: they are specific of the polymer type in which they occur. Hydrolyzing a protein and an oligosaccharide is just the same process, from a chemical point of view. But fragmenting a protein or an oligosaccharide are truly different processes because the way that the fragmentation happens in the polymer sequence is so much dependent on the nature of each monomer that makes it.

Another peculiarity of the fragmentations, compared with the cleavages that were described above, is the fact that there is no cleaving molecule starting the process. Instead, a fragmentation process is often initiated by an intra molecular electron doublet rearragement that propagates more or less in the polymer structure to eventually break it. Fragmentations are mainly a gas phase process, not some reaction that happens in solution as a result of putting in contact the polymer and some reagent. It is precisely because no cleaving molecule is involved in the fragmentation process that the fragments are not necessarily capped like a normal polymer should be; and this is another really important difference between cleavage and fragmentation. The following examples should illustrate these concepts using protein and nucleic acid fragmentation pathways.

4.4.2.1 Protein Fragmentation

There is a pretty important number of different kinds of fragments that can be generated upon fragmentation of peptides. We are going to detail the most common ones; the user is invited to use the MassXpert fragmentation-specification grammar to add less frequent (or newly discovered) fragmentation types. Note that the fragmentation schemes below apply to positively-charged precursor ions. To compute the product ions’ masses obtained in negative mode fragmentation experiments, then, simply remove as many protons as required. For example, to switch from a fragment positively charged once (+H), then remove a first proton to go back to the uncharged state and then remove another proton to yield the deprotonated (thus singly negatively charged) ion product. The requirement to be able to computed masses for the positively- and negatively-charged ion products imposes a specific way to define fragmentation specifications in the module (to be detailed later in this manual, see Section 5.2.5 – The fragmentation pathway definitions.

As can be seen from

The two observations above should make clear to the reader that calculating masses for fragments is a more difficult process than what was described earlier for the cleavate-generated oligomers. Indeed, while it was simple to calculate the mass of an oligomer (by simply adding the masses of its constitutive monomer units, plus the left and right caps, plus ionization), here there is no chemical formalism generally applicable to all the fragment types. This is why the specification of the fragmentation is left to the user’s responsibility.

By looking at

The mass calculations for the different fragment product ion types is shown in

If we take the a fragment series,

For the x series of fragments we do not add the left cap anymore, but replace it with the right cap, since the fragments hold the right end of the precursor polymer. Note also that the numbering of the monomers using the variable i in the following mathematical expressions goes from right to left (contrary to what happened for the a, b, c fragment series. All the fragments that hold the precursor polymer right end are numbered this way, so this applies to fragments x, y, z.

In low energy CID, the z fragments are expressed in a distinct way, when compared to high energy CID.

In the immonium fragment series, the fragments are internal fragments in the sense that they do not hold neither of the two precursor polymer’s ends. MassXpert understands that the user is speaking of this kind of fragment when the “from which end” piece of data in the fragmentation specification states “NE” instead of “LE” or “RE” (see Section 4.4.2 – Polymer Fragmentation. The mass calculation for these fragments does not take into account the monomers surrounding the one for which the calculation is done. The mass for an immonium ion—at position i in the precursor polymer—will be the mass of the monomer at position i, less the mass of a CO, plus the mass of a proton.

4.4.2.2 Nucleic Acids Fragmentation

The fragmentations that can be obtained with nucleic acids are numerous and it is more complicated than with proteins to describe them fully. The main reason for this is that there are a big number of fragmentation combinations because of the loss of nitrogenous bases from the skeleton. The mechanisms by which this loss happens are fairly complex, and I am not going to detail any of them.

Another remark pertaining to the ionization mode of the ion products: the fragmentation schemes below apply to negatively-charged precursor ions (by loss of a proton, typically). To compute the product ions’ masses obtained in positive mode fragmentation experiments, then, simply add as many protons (or any other cationic ionization agent) as required. For example, to switch from a fragment negatively charged once (-H), then add a first proton to go back to the uncharged state and then add another proton to yield the monoprotonated (thus singly positively charged) ion product. The requirement to be able to computed masses for the positively- and negatively-charged ion products imposes a specific way to defined fragmentation specifications in the module (to be detailed later in this manual; see Section 5.2.5 – The fragmentation pathway definitions).

The reader might have noticed at the bottom of

Exactly as done earlier for the protein fragments, the mathematical expressions used to calculate the mass of different series of nucleic acid fragments are provided in

In these calculations it is assumed that the left end of the precursor polymer is phosphorylated (5′P) and the reader should bear in mind that this precise phosphate might itself be expelled by the fragmentation. The fragment naming schemed detailed earlier for proteins applies to nucleic acids in the very same manner.

In the a fragment series, the fragments most often appear with base loss. There are also a variety of fragments for which a base is lost.

4.4.2.3 More Complex Patterns Of Fragmentation

Before finishing with fragmentations, it is necessary to describe a powerful feature of the fragmentation specification grammar available in MassXpert. This feature was required for the fragmentation of oligosaccharides and also sometimes for proteins. When the fragmentation (the bond breakage reaction itself) occurs at the level of certain monomers, it might be necessary to be able to specify some particular chemistry that would arise on the monomer in question.

We have seen in the cleavage documentation that, upon cleavage of a protein sequence with cyanogen bromide, for example, a particular chemical reaction had to be applied to the oligomers that were generated with a methionine monomer as their right end monomer. Well, in a fragmentation specification it is possible to apply comparable chemical reactions but in a more thorough manner. Indeed, while in the cleavage it was possible to say something like: —“Apply a given chemical reaction to the oligomer if the right end monomer is Xyz”, in the fragmentation the logical condition can be bound not only to the identity of the currently fragmented monomer, but also (optionally) to the identity of the previous and/or next monomer in the precursor polymer sequence. For example:

These logical conditions are called fragmentation rules. A fragmentation specification can hold as many rules as necessary. All of this is described in great detail at Section 5.2.5.2 – More complex fragmentation pathway patterns.

4.4.2.4 To Sum Up

To sum up all what we have seen so far with polymer chain disrupting chemistries:

• Fragments are never ionized automatically; ionization (gain/loss of a charged group) is necessarily integrated post-fragmentation as a configuration bit to mass calculations.

• A polymer sequence gets fragmented into fragments when a bond breakage occurs, without the help of any exterior molecule, at any level of the polymer structure, with no limitation to the inter-monomer bond; monomer-specific chemical reactions can be modelled into the fragmentation specification using any number of fragrules;

• Oligomers are automatically capped—on both ends—using the rules described in the precursor polymer’s definition;

• Fragments are capped automatically only—on the end they hold, if any—using the rules described in the precursor polymer’s definition;

• Oligomers are automatically ionized (if required by the user) using the rules described in the precursor polymer’s definition;

• Fragments are never ionized automatically; ionization (gain/loss of a charged group) is necessarily integrated in the fragmentation specification.

5 XpertDef: Definition of Polymer Chemistries

After having completed this chapter the reader will be able to accomplish the very first steps needed to use MassXpert’s features at best: the normal workflow, indeed, is to first make a polymer chemistry definition, in order to be able to edit polymer sequences of that specific definition. The module is made available in MassXpert by pulling down the XpertDef menu item from the program’s menu. It is possible to start a new polymer chemistry definition from scratch, but it is certainly usually easier to first duplicate a polymer chemistry definition shipped with MassXpert and then open that copy and edit it. Please, refer to Section 10 – Data Customizations for an explanation of how this is safely done.

To open a polymer chemistry definition, the user may either select one that is already registered with the system, and that appears listed in the drop-down list widget shown in

• Define the name of the polymer chemistry definition;
• Define “singular” data like the left cap and the right cap of the polymer, the ionization rule governing the default ionization of the polymer sequence;
• Define the isotopes needed to operate the different polymer chemistry entities (these are “plural” data);
• Define all the polymer chemistry entities needed to work on polymer sequences (all these are also “plural data”).

The definition of the isotopes and of all the chemical entities belonging to a given polymer chemistry are collectively called a polymer chemistry definition. The polymer chemistry definition window that shows up is shown in figure 

5.1 The isotopes

The definition of the isotopes is performed through the user interface shown in figure 

The table view is editable. Each cell, when double-clicked, switches to an edit mode in which the user might enter a value. A series of buttons below the table view trigger various actions. To add a new isotope, either above or below the currently selected one, click one of the two Insert isotope... buttons. To remove isotopes, select them and click the Remove selected button. The three buttons on the right hand side of the window, allow loading, saving and saving with a new file name the isotopes currently displayed in the table view.

When the user selects one or more rows of the table, the program updates some data specific to the selected isotopes. As long as the selected isotopes all have the same chemical element, they are taken into account for the calculation of the average mass (Average mass). The Probabilities line edit widget displays the sum of the abundances of the selected isotopes (only if these are of the same chemical element).

5.2 The polymer chemical entities

Once the isotopes have been properly defined (note that such isotopes are already available in the distributed package), it is possible to start entering data for the other polymer chemical entities (figure 

The following are the data that need to be entered so as to obtain a usable polymer chemistry definition:

• The polymer chemistry definition’s name: protein-1-letter

• The chemical capping reactions that should happen on the left end and on the right end of the polymer sequence:

  • +H: left capping of the polymer sequence. Proteins are capped at the N-terminal end with a proton;
  • +OH: right capping of the polymer sequence. Proteins are capped at their C-terminal end with a hydroxyl group;

• The Ionization rule describes the manner in which the polymer sequence should be ionized by default, when the mass is calculated. This rule actually holds three elements:

  • Formula: +H: chemical reaction that ionizes the polymer sequence.In the example, all the polymer sequences of polymer chemistry definition protein-1-letter are protonated by default;
  • Charge: 1: charge that is brought by the chemical agent ionizing the polymer (the formula above). In the example, a protonation reaction brings a single positive charge.
  • Level: 1: ionization level, that is, the number of times that the ionization reaction above must be performed by default on any polymer sequence of this chemistry definition. In this example, monoprotonation is set as the default ionization rule.

At this point, time has come to deal with “plural” data. The first chemical entities to deal with are monomers.

5.2.1 The monomers

The monomers are the constitutive blocks of the polymer sequence. In the MassXpert’s jargon, “monomer” stands not for the molecule that may be used to perform a polymer synthesis; it stands for this molecule less the chemical group(s) that were eliminated upon polymerization. In the case of the biological polymers, the creation of chemical link between two monomers invariably leads to the loss of a water molecule (a condensation reaction, in organic chemistry).

Click onto the Monomers button, which triggers the opening of the dialog window shown in

The way this dialog is operated is similar to what was described for the atom definition, unless it is simpler, because monomers are non-deep objects: there are no contained objects. One data element is critical: the number of characters that might be used to define the code of the monomer cannot be greater than the value entered in the Code length spinbox widget.⁴

Each time a formula is either displayed by selecting a new monomer in the list or modified by editing it in its line edit widget, the monoisotopic and average masses are recalculated.

As of version 2.3.5, it is possible to calculate the mass difference between any two monomers in the definition. This is useful, for example, to grasp the resolution and mass accuracy requirements for a given polymer definition. The user sets a threshold to filter the results (in the example, that Mono mass threshold was set to 0.1. The results of such a calculation are displayed in figure 

Once all the monomers have been added to the polymer chemistry definition, it is always a good idea to validate them by clicking Validate.

5.2.2 The modifications

Polymers are often either chemically or biochemically modified. In nature, biopolymers are modified more often than not. Some of the more common modifications in the protein realm are phosphorylation, acetylation and methylation, for example. Nucleic acids are modified with a sheer number of chemical modifications, saccharides also. The MassXpert software provides entire freedom to define any number of intelligent modifications, that is, modifications with any chemical formula but also that are knowledgeable of what monomers they can modify. Indeed, it would make no sense to phosphorylate a glycyl residue in a protein, for example.

Click the Modifications button to open the dialog window shown in figure 

• Name: the name of the modification;
• Targets: a ‘;’-separated list of codes of the monomers that might be modified by this modification:;
• Max. count: the maximum number of times a given monomer might be modified with this modification. This feature is essential when working on methylation of proteins, for example, with arginyl and lysyl residues being multi-methylated;
• Formula: the formula that defines the modification chemical reaction, as explained in Section 2.1 – On Chemical Formulæ and Chemical Reactions.

Note that in the example of the figure, for LysMethylation, the C1H2 formula is a net formula. That formula could be more explicit by entering “−H+CH3”. The net formula is thus the one visible on the figure. The LysMethylation reaction can thus be read like this:

The LysMethylation is being defined as having K targets only, that means that when the user will try to modify non-lysyl monomers, the program will complain that these monomers are not targets of LysMethylation. There is, however, and for maximum flexibility, the possibility to override these target-limiting data when modifying monomers ( See overriding the maximum target count limitation).

When a monomer is modified with this modification, its masses will change according to the net mass of this LysMethylation modification.

5.2.3 The cross-linkers

Polymers are often either chemically or biochemically modified by interconnecting monomers from the same polymer sequence. In the protein reign, one classical example of intra-sequence cross-linking is the formation of disulfide bonds. Another wonderful example is the formation of the fluorophore in the fluorescent proteins: there is a chemical reaction involving the side chains of three consecutive residues going on, resulting in the formation of a complex intra-sequence cross-link. Each side chain of the three monomers involved undergoes a chemical modification.

Cross-linkers are defined in the dialog window shown in

The formation of a cross-link between one or more monomers often involves chemical reactions to occur at the level of the engaged monomers. Cross-linkers defined in MassXpert should refer to these modifications as modification objects already available in the polymer chemistry definition. Note that, in some cases, it is not necessary to define modifications to occur at the level of the cross-linked monomers.

The example described in figure 

5.2.4 The cleavage agent definitions

It is common practice—in biopolymer chemistry, at least—to cut a polymer into pieces using molecular scissors like the following:

• Proteases, for proteins;
• Nucleases, for nucleic acids;
• Glycosidases, for saccharides...

For each different polymer type, the molecular scissors are specific. Indeed, a protease will not cleave a polysaccharide. This is why cleavage specifications belong to polymer chemistry definitions. In the example of

• Name: CyanogenBromide: the name of the cleavage agent;
• Pattern: M/: the sequence pattern recognized by the cleaving agent. In this case, the cleavage agent cleaves the protein right after M residues (mind that the ‘/’ character is right of ‘M’);
• The Cleavage rules group box allows the user to define the cleavage rules that might be added to the cleavage agent. When a new cleavage rule is added, the corresponding data must be entered in the Cleavage rule group box, specifically with a Name and specifications for the chemical reactions that might occur at the left or right end of the oligomers resulting from the polymer cleavage (Left end and Right end). The chemical reactions are described by inserting the Code at which the reaction should occur and the Formula.

The notion of “Left end” or “Right end” are to be construed in the sense that when the oligomer is generated upon cleavage, the “Left end” rule applies to the left end of the generated peptides and the “Right end” rule applies to the right end of the generated peptides.

The case of the cyanogen bromide reagent is interesting in this regard. The cleavage occurs right of methionyl residues. The generated peptides thus systematically end with a M methionyl residue. The cleavage reaction by cyanogen bromide involves a chemical modification of the methionyl residue at which the cleavage has occurred. This means that each generated peptide will have a modified methionyl residue on its right end. This is exactly what is described in

• the Left end chemical reaction data comprise no specific reaction data because once the reagent has cleaved right of a methionyl residue, the generated peptide has no modification on its left end.

• the Right end chemical reaction data comprise value M for Code and -CH2S+O3 for Formula, respectively. Same explanation as above. This cleavage rule stipulates that upon cleavage of a protein using cyanogen bromide, the methionyl residue that gets effectively cleaved must be converted to a homoseryl residue. See below for a detailed explanation.

Here are some examples of more complex cleavage patterns:

• Pattern: K/;R/;-K/P, which reads like

• Pattern: /D, which reads like

• Pattern: Hypothetical = T/YS; PGT/HYT; /MNOP; -K/MNOP:

  .

Now comes the time to explain in more detail what the Left end and Right end specifications mean, along with their Code and Formula fields, are for. For this, we shall consider that we have the following polymer sequence (1-character monomer codes): THISMWILLMBECUTMANDTHATMALSOM. If that sequence had been cleaved using cyanogen bromide and if the cleavage had been total,⁵ that would have generated the following oligomers:

THISM WILLM BECUTM ANDTHATM ALSOM.

But if there had been partial cleavages, one or more of the following oligomers would have been generated:

THISMWILLM BECUTMANDTHATM ALSOM WILLMBECUTM ANDTHATMALSOM and so on...

Now, the biochemist knows that when a protein is cleaved with cyanogen bromide, the cleavage occurs effectively right of monomer “M” (this we knew already) and the “M” monomer that underwent the cleavage is changed from a methionyl residue to an homoseryl residue (this chemical change involves this formula: “−CH2S+O”). Amongst all the oligomers generated above, there are two oligomers that should not undergo the “−CH2S+O” cleavage rule: ALSOM and ANDTHATMALSOM. Indeed, these two oligomers were generated by the CyanogenBromide cleavage, but were not actually cleaved at the right side of a methionyl residue, because they correspond the the right end terminal part of the protein sequence.

This example should clarify why the definition clearly stipulates—in the cleavage specification for CyanogenBromide—that the oligomers resulting from this cleavage should undergo the “−CH2S+O” reaction only if they have a M as their right end monomer code. These cleavage rules need to be defined in a very careful way: imagine that—in some experiments involving cyanogen bromide —that reagent would cleave right of “C” (cysteine) residues, but with no chemical modification of the “C” monomer.⁶ In this case, it would be suitable to put the flexibility of MassXpert at work by specifying that the generated oligomers should “undergo the “−CH2S+O” formula” only if they have a “M” as their right end monomer, so that “C”-terminated oligomers are not chemically modified. Thus the cleavage pattern might be safely defined: “M/;C/”...

5.2.5 The fragmentation pathway definitions

The specification of fragmentation pathways in a polymer chemistry definition is not a trivial task. In this section three different cases will be described, from simplest to most complex. One major rule is that the fragmentation pathway should be defined in such a manner that the resulting fragment is neutral. The ionization of the fragments will be then automatically performed by MassXpert upon calculation of the ion products according to the current ionization rule. This is a major improvement over previous versions, that forced the user to define fragmentation specifications by assuming a product ion of a given ionization status ([M+H]⁺ for proteins or [M−H]⁻ for nucleic acids, for example).

Will use the “dna” and “saccharide” polymer chemistry definitions to describe the notions involved in the definition of fragmentation pathways.

In the DNA chemical world, the fragmentation pathways involve the cleavage of bonds at the level of the phosphodiester link between nucleosides. There are also fragmentation pathways that also involve the decomposition of the nucleic base. This is shown in

5.2.5.1 Simple fragmentation pathway patterns

One simple example of a polymer chain fragmentation pathway is the formation of a fragments within a nucleic acid molecule (DNA, in this example). Bond cleavage occurs right before the sugar-carbon-linked oxygen of the phosphodiester bond linking one deoxyribonucleoside to the next. Thus, the molecular weight of the fragment corresponds to the sum of the monomer masses from the left end of the polymer up to and including the monomer being decomposed less one oxygen. Note that this specification yields a nucleic acid ion product that is protonated. We thus need to remove a proton to change its charge to 0, thus the formula of the a fragmentation pattern is “−OH”. This is illustrated in figure 

5.2.5.2 More complex fragmentation pathway patterns

In nucleic acids gas-phase chemistry, it often happens that not only the fragmentation occurs at the level of the phospho-ribose skeleton, but also at the level of the nucleic base.

These fragmentation pathways are said to be “abasic”. The decomposition of the base occurs at the monomer position where the fragmentation occurs. For example, if a “ATGC” oligonucleotide is fragmented according to pattern a but with nucleic base decomposition, and that fragmentation occurs at position 1, then the computation of the mass should occur like represented in

Using a monomer-generic specification In this manner of defining a base decomposition for a fragmentation pathway in the “dna” polymer chemistry definition, the base decomposition is performed in two steps (see

• Apply an adapted specification for a fragments: removal of the H due to the insaturation on the sugar and also removal of the OH related to the formation of an a fragment: the −HOH component of the formula;

• Remove one full monomer with Monomer set to −1 (this equals to the removal of both the skeleton and the side chain—the adenine, here);

• Add back the skeleton: the +C5H8O5P component of the formula;

• Add the left end cap, since a fragments start at the left end of the fragmented polymer sequence (End is set to LE). That LE bit of information will be transformed into a +OH formula, since this is the formula of the left end cap for nucleic acids.

The advantage of working this way is that we need not specify a fragmentation rule for each different monomer in the sequence (see below, for how this might be done). Indeed, by specifying Monomer to be -1, we tell the mass calculation engine that once the fragmentation has occurred in the polymer chain, the mass of the monomer that got fragmented—without knowing its identity—should be subtracted from the fragment mass. But since removing the whole monomer is more than what is actually decomposed, we add back the skeleton, so that we actually only decompose the nucleic base (adenine, in our example). This is why we ask in the fragmentation specification formula that the skeleton be added back (the +C5H8O5P component of the Formula). Because the skeleton does not change along the polymer chain, even if the base itself changes, this computation method is generalizable, and because of this the polymer chemistry definition works.

It might happen that the base decomposition only occurs at specific nucleotide sequence patterns. In this case it might be necessary to define a specific base decomposition rule that accounts for a specific nucleic base. This is described below.

Using a monomer-specific specification Another way of achieving what was described above is by using fragmentation rules, whereby the fragment’s mass computation is made conditional to one or more conditions that should be verified as shown in

In this example, the a-b@C fragmentation pathway definition involves one specific rule (decompose-C) that instructs the mass calculation engine to remove specifically the formula of the “C” nucleic base whenever the fragmentation occurs exactly at this nucleotide (Curr. code in the Local logic group box has value C). Here is the detail of this fragmentation pathway definition:

• For the fragmentation specification part, everything is like for fragments of type a, that is, the formula is merely −HOH and the End is set to LE (see above, for explanations);

• The Monomer field has value 0 because we are not going to remove the monomer’s mass;

• One rule named a-b-decomp-at-c adds some Local logic for the fragmentation specification: the formula −C4H4N3O should be applied upon calculation of the fragment’s masses if the monomer at which the fragmentation actually occurs is a cytosine (Curr code is C). The Formula of the fragmentation rule is set to −C4H4N3O, which is the formula of cytosine (the nucleic base, not the monomer).

• There should be other fragmentation rules specified for the remaining three bases A, T, G, but adapting the formula of the fragmentation rule to the actual formula of each base. In this way, the fragmentation rule-based definition of fragmentation pattern a-b described here is equivalent to the more generalizable method described earlier in
  

  .

5.2.5.3 Even more complex fragmentation patterns

In saccharide chemistry the fragmentation patterns are extremely complex, and often totally depend on the nature of the monomers local to the fragmentation site. For example, the fragmentation behaviour at position E in a sequence DEAR might be different than in a sequence DERA.

MassXpert had to be able to model these complex situations, and this is done using fragmentation rules where the local logic involves defining the Prev code and/or the Next code for a given Curr code at which the fragmentation occurs. For example, one specific fragmentation pattern for fragmentation at E in sequence DEAR might be defined this way:

• Prev code: D
• Curr code: E
• Next code: A.

Instead of that fragmentation rule, one would have for fragmentation at E in sequence DERA the following rule:

• Prev code: D
• Curr code: E
• Next code: R.

Note the change for Next code, from A to R.

Also, be aware that the “prev”, “curr” and “next” notions are polar, that is, they depend on the value of End (that is, LE or RE). For example, if we wanted to model the fragmentation pattern at E for a fragment of End type RE, similar to what was done above with sequences DEAR and DERA, we would have set the local logic like this:

For sequence “DEAR”:

• Prev code: A
• Curr code: E
• Next code: D.

For sequence “DERA”:

• Prev code: R
• Curr code: E
• Next code: D.

This highly flexible fragmentation specification allows for highly complex fragmentation pathway defintions matching the complex gas phase fragmentation mechanisms of biopolymers.

5.3 Saving the definition

Once the polymer chemistry definition is completed, the user can save it to an XML file. Prior to actually writing to the file, the program checks the validity of all the chemical entities in the definition. This check can be triggered manually by clicking the Validate button. If an error is found, it is reported so that the user may identify the problem and fix it.

The location where the file should be saved, and the manner that it may be made available to MassXpert is to be described in a later chapter. It is, in fact, very important that MassXpert knows where to find newly defined polymer chemistries so as to be able to use them when sequences of that polymer chemistry are created or used.

6 XpertEdit: a Powerful Editor and Simulation Center

After having completed this chapter you will be able to perform sophisticated polymer chemistry simulations on polymer sequences—that can be edited in place—along with automatic mass recalculations.

6.1 Invocation

The module is easily called by pulling down the XpertEdit menu item from the MassXpert program’s menu. The user may start the module by:

• Opening a sample polymer sequence;
• Creating a new polymer sequence;
• Loading a polymer sequence from disk.

6.2 Operation: In Medias Res

The first manner to start an session is by opening a sample sequence out of the list of sequences that were shipped along with MassXpert. The XpertEdit$⇨$Open Sample Sequence menu opens the dialog box shown in Figure 28 – Selection of a sample polymer sequence

The drop-down widget in this dialog window lists all the polymer sequence files that were shipped along with MassXpert. Simply select one item and click OK. To select another polymer sequence file, click Cancel, which will trigger the system’s file selection dialog for you to browse to the location where the polymer sequence file is stored. The process is identical to the normal polymer sequence file opening (see below).

The second way to start an session is by creating a new polymer sequence using the XpertEdit$⇨$New sequence menu. The program immediately asks to select a polymer chemistry definition, as shown in Figure 29 – Selection of the polymer chemistry definition The drop-down widget lists all the polymer chemistry definitions currently registered on the system. If the polymer chemistry definition is not listed, clicking onto Cancel will let the user browse the disk in search for a polymer chemistry definition file.⁷ Once the polymer chemistry definition has been selected and successfully parsed by the program, the user is presented with an empty sequence editor.

The third way to start an session is by opening an existing polymer sequence file. Once the sequence file has been opened, the user is presented with a sequence editor as represented in Figure 30 – The main window At this point, when the user starts editing a sequence, the characters entered at the keyboard, or pasted from the clipboard, will be interpreted using the polymer chemistry definition that was selected in the initialization window described above.

Now, of course, editing a polymer sequence is not enough for a mass spectrometric-oriented software suite; what we want is to compute masses! The mass calculation process is immediately visible on the right hand side of the sequence editor shown in Figure 30 – The main window Two group box widgets are located at the top of the right hand side of the window:

• Whole sequence masses: A group box widget displaying the Mono and Avg masses of the whole polymer sequence, irrespective of the current selection;

• Selected sequence masses: A group box widget displaying the Mono and Avg masses of the currently selected region of the polymer sequence.

The user may change the mass calculation engine configuration at any point in time using the widgets in the Calculation Engine tool box that contains the following configurable parameters:

• Polymer:

  • If Left Cap is checked, the left cap of the polymer sequence will be taken into account;

  • If Right Cap is checked, the right cap of the polymer sequence will be taken into account;

  • If Left Modif is checked, the modification of the polymer sequence’s left end will be taken into account. Note that if Force is checked also, then the modification is taken into account even when selecting a region of the sequence that does not encompass the monomer located at that end;

  • The behaviour of Right Modif is the same as above, but for the right end modification;

• Selections and regions:

  See Section 6.7 – Multi-region Selections for a detailed explanation of multi-region selection and multi-selection regions.

  • If Multi-region is checked, the sequence editor allows more than one region to be selected at any given time (there is no limitation whatsoever on the number of selected regions);

  • If Multi-selection is checked, the sequence editor allows not only the selection of multiple regions at any given time, but also the selection of totally or partially overlapping regions.

  • When multiple regions are selected, each selected region behaves like an oligomer if Oligomers is selected; that is, it gets its left and right end caps added (if the corresponding calculation engine configuration item is activated);

  • When multiple regions are selected, the different regions behave like residual chains if Residual chains is selected; that is, the left and end caps are added only once (if the corresponding calculation engine configuration item is activated).

• Monomers:

• If Modifications is checked, the monomer modifications will be taken into account;

• If Cross-links is checked, the cross-links in the polymer sequence will be taken into account.

• Ionization:

• The +H formula represents the ionization agent formula (that is, a protonation) ;

• The Unitary charge value is set to 1 because, by definition, a protonation brings a single positive charge;

• The Ionization level is set to 1, that is, in the example, the polymer must undergo a single protonation.

When any parameter listed above is changed, the recalculation of the masses—for both the Whole sequence and the Selected sequence—is triggered and the new masses are updated in their respective line edit widgets described earlier. The fact that the user can specify ionization rules should make it clear that the values that are displayed are actually m/z ratios (as long as one ionization is set).

6.3 The Editor Window Menu

The menu bar in the polymer sequence editor displays a number of menu items, reviewed below:

• File:

  • File$⇨$Close: close the sequence;

  • File$⇨$Save: save the sequence under a new file name. If the sequence has no filename yet, the user is invited to select a filename;

  • File$⇨$Save As: save the sequence in a new file;

  • File$⇨$Import$⇨$Import Raw: try to import the sequence. If invalid monomer code characters are found, the user is given a chance to revise the imported sequence;

  • File$⇨$Export to Clipboard: copy the sequence and all the data (masses and calculation options) to the clipboard, in the form of simple text;

  • File$⇨$Export to File: write the sequence to file and all the data (masses and calculation options), in the form of simple text (if a file name was

  already selected, otherwise the user is invited to select a file into which the data are to be written);

  • File$⇨$Select export file: select a file into which the data are to be written.

• Edit:

  • Edit$⇨$Copy: Copies the current selected region(s) (if any) to the clipboard. If there are more than one region currently selection, then the user is informed that the copied sequence will correspond to these two sequences joined together.

  • Edit$⇨$Cut: copy the current selection (if any) to the clipboard and removes it from the sequence. Note that it is not yet possible to cut more than one selected region in one single operation;

  • Edit$⇨$Paste: paste the sequence from the clipboard into the sequence at the current cursor location. If the pasted sequence is found to contain characters not valid for the current polymer chemistry definition, the user is given a chance to revise the pasted sequence. If one sequence region was selected, it is replaced with the pasted sequence. If more than one sequence region was selected, the operation cannot be performed and the user is informed;

  • Edit$⇨$Find Sequence: find a sequence motif in the polymer sequence.

• Chemistry:

  • Chemistry$⇨$Modify Monomer(s): modify (or unmodify) one or more monomers in the polymer sequence;

  • Chemistry$⇨$Modify Polymer: set (or unset) the left (or right, or both) modification(s) of the polymer sequence;

  • Chemistry$⇨$Cross-link Monomers: cross-links monomers;

  • Chemistry$⇨$Cleave: perform a chemical/enzymatical cleavage of the polymer sequence;

  • Chemistry$⇨$Fragment: perform the gas phase fragmentation of the currently selected oligomer;

  • Chemistry$⇨$Mass Search: search in an arbitrary manner for any sequence having a mass matching the searched mass;

  • Chemistry$⇨$Compute m/z Ratios: calculate a range of m/z ratios with a given ionization agent starting from a given m/z ratio and a given ionization status;

  • Chemistry$⇨$Determine Compositions: calculate the monomeric/elemental composition of the whole polymer sequence or of the current selection;

  • Chemistry$⇨$pKa pH pI: perform acidity, pH and isoelectric point calculations on the whole sequence or on the current selection.

• Options:

  • Options$⇨$Decimal places: set the number of decimal places to be used to display the numerical values.

6.4 Editing Polymer Sequences

As described earlier, in the chapter about the module, a polymer chemistry definition may allow more than one character to qualify the codes of the monomers (see Section 5 – XpertDef: Definition of Polymer Chemistries). It was noted also that it is not because the number of allowed characters is 3, for example, that all the monomer codes of the polymer chemistry definition must be defined using three characters: 3 is the maximum number of characters that may be used.

6.4.1 Multi-Character Monomer Codes

This section deals with the editing of a polymer sequence for which monomer codes can be made of more than one character. Figure 31 – Multi-character code sequence editing in shows the case of a polymer sequence for which the polymer chemistry definition allows three characters to define monomer codes. The example is based on the following real-world situation: the user wants to edit the sequence by insertion—at the end of the sequence (right of the Leu residue). The new monomer to be appended to the sequence is “Asn”.

After keying-in (panel 1), no sequence modification is visible in the sequence editor. Instead, an “A” character is now displayed in the left line edit widget under the sequence. The reason of this apparently odd behaviour is that the polymer chemistry definition allows up to 3 characters to describe a monomer code. If no monomer vignette is displayed in the polymer sequence, that means that more than one monomer code start with an “A” character: cannot figure out which monomer code was actually meant by the user when keying-in .

There is a way, called code completion, to know which monomer code(s)—in the current polymer chemistry definition—do start with the keyed-in character(s) (currently, “A”). The user can at any moment always enter the “code completion mode” by hitting the key. This is what is shown in the panel 1st, right hand side Monomer List listview widget (click on that Monomer
List label to show that list if it is not already visible). We see that, in the current polymer chemistry definition, four monomer codes start with an “A” character, and these are “Ala”, “Arg”, “Asp” and “Asn” (as highlighted in the code completion monomer list).

Because we now know that the code we are to key-in is “Asp”, we key-in a . The result is shown in the small panel 2. What we see here is that, this time also, nothing changed in the polymer sequence. What changed is that the character string in the left line edit widget below the sequence is now “As”. Let’s key-in once more the key. This time, only two items are highlighted: “Asp” and “Asn” in the code completion monomer list (panel 2nd). This is easily understood: there are only two monomer codes that start with the two letters “A” and “s” (“As”) that we have keyed-in so far. At this time, we key-in a last character: . At this point, the monomer is effectively inserted in the polymer sequence, as the “Asn” monomer left of the cursor, as shown in panel 3. Note how the bottom edit widget is now cleared: there are no more letters in the buffer awaiting to be completed to form a full monomer code. Also, by entering , all the monomer codes are selected in the list of monomer codes available for editing the sequence: since a new monomer code might be entered all possibilities are open.

6.4.2 Unambiguous Single-/Multi-Character Monomer Codes

Let’s imagine that we have a polymer chemistry definition that allows up to 3 characters for the definition of monomer codes, but that we have one of these monomer codes (let’s say the one for the “Glutamate” monomer) that is one-letter-long: “E”. This monomer code “E” is the only one in the polymer chemistry definition to start with an “E” character. In this case, when we key-in , we’ll observe that the monomer code is immediately validated and that its corresponding monomer vignette is also immediately inserted in the polymer sequence. This is because, if there is no ambiguity, will immediately validate the code being edited.

The mechanism described above means that the user is absolutely free to define only single-character monomer codes in a polymer chemistry definition that allows multi-character monomer codes; the behaviour of the program is thus to behave exactly as if the multi-character code feature were inexistent in the program: each time a new uppercase letter is keyed-in, it is automatically validated and the corresponding monomer is created in the sequence.

6.4.3 Erroneous Monomer Codes

The typing error detection system triggers immediate alerts whenever the code being keyed-in is incorrect. This is described in

If you start keying-in valid monomer character codes, like for example we did earlier with “As”, and you want to erase these characters because you changed your mind, hit the key to remove any character entered previously. These characters will disappear sequentially, at each key press, from the line edit widget below the sequence. For example, let’s say you have already keyed-in and . In this case the left line edit widget displays these two characters: “As”. Now, if you change your mind, not willing to enter “Asp” monomer code anymore, but “Gly” instead, all you have to do is to key-in once for the “s” character (which disappears) and once more to remove the remaining “A” character. At this point it is possible to start fresh with the “Gly” monomer code by keying-in sequentially , and finally .

6.4.4 Simplified Editing

When the monomer codes of a given polymer chemistry definition are too numerous or too long to remember, one simplified editing strategy is by using the list of available monomers located on the right side of the sequence editor (widget labelled Monomer list ; Figure 33 – Simplified editing with the monomer list). The items in the list are active: if double-clicked, an item will see its corresponding monomer code inserted in the sequence at the current cursor location. This list thus makes it easy to “visually” edit the polymer sequence without having to remember all the codes in the polymer chemistry definition.

6.5 Finding sequence motifs

Finding sequence motifs in the polymer sequence is performed by selecting the Edit$⇨$Find Sequence menu item. The dialog window is shown in Figure 34 – Finding a sequence motif in the polymer sequence. When performing the first search in a polymer sequence, the Find button should be used. This will trigger a search starting at the beginning of the polymer sequence. For each successive search, the Next button should be used.

Each searched sequence motif will be stored in a history list that is made available by dropping down the combo box widget where the sequence motif is entered. The Clear history button will erase all the searched sequence motifs from the history, thus resetting it.

6.6 Importing Sequences

Very often, the user will make a sequence search on the web and be provided with a polymer sequence that is crippled with non-code characters. That web output might either be saved in a text file for future reference or copied to the clipboard for immediate use in MassXpert. The two cases are reviewed below.

6.6.1 Importing From The Clipboard

provides a convenient way to spot non-valid characters in a text and to let the user “purify” the imported sequence. A clipboard-imported sequence is systematically parsed. When invalid characters are found, the window depicted in Figure 35 – Clipboard-imported sequence error-checking is presented to the user for them to make appropriate adjustments (in this example we tried to copy from clipboard the following sequence into a polymer sequence of the “dna” polymer chemistry definition: “!100 ATGCATGC ATGCATGC ATGCATGC ATGCAUGC anotherSilly-Text;”).

As soon as a character does not correspond to any valid monomer code, it is tagged, and the sequence is presented to the user in a text edit widget (Initial Sequence) with the all the invalid characters tagged by underlining. At that point, if the user clicks the Remove Tagged From Initial button, all the tagged characters will be automatically removed and the purified sequence will show up in the Purified Sequence text edit widget.

Also, the user is provided with automatic “purification” procedures whereby it is possible to remove one or more classes of characters from the imported sequence (Purification Options frame widget). Checking one or more of the Numerals or Spaces or Punctuation or LowerCase or Uppercase checkbuttons, or even entering other user-specified regular expressions in the Other (RegExp) line edit widget, will elicit their removal from the imported sequence after the user clicks the Purify Initial
(Options) button.

When you are confident that almost all the erroneous characters have been removed (Figure 36 – Clipboard-imported sequence purification), you can click the Test Purified button, which will trigger a “re-reading” of the sequence in the Purified Sequence text edit widget. If erroneous characters are still found, they are tagged.

Once the sequence is finally depured from all the invalid characters, you can select it in the text edit widget and paste it in the sequence editor. This time, the paste operation will be error-free.

6.6.2 Importing From Raw Text Files

It might be of interest to be able to import a sequence from a raw file. To this end, the user is provided the menu File$⇨$Import$⇨$Import Raw that opens up a file selection window from which to choose the file to import. The program then iterates in the lines of that file and checks their contents for validity. If errors are found, then the same process as described earlier for clipboard-imported sequences is started (Section 6.6.1 – Importing From The Clipboard). The user can then purify the sequence imported from the file and finally integrate that sequence in the polymer sequence currently edited. Note that if any sequence portion is currently selected, it will be replaced by the one that is being imported.

6.7 Multi-region Selections

MassXpert implements a sophisticated multi-region selection model. Two selection modes are available:

• Multi-region selection mode:: In this mode, it is possible to select more than one region in the polymer sequence. In all cases below, make sure that the Multi-region checkbutton is checked in Selections and regions group box (see Selections and regions). This is how these selections are performed:

  • With the mouse: Left-click and drag to make the first selection. Go with the mouse cursor at the beginning of desired new selection, hold the key down while left-clicking and dragging to perform the second region selection. Continue as may times as necessary;

  • With the keyboard: Position the cursor at the beginning of the first region to be selected, hold the two and keys pressed while moving the cursor with the direction keys (the arrow keys of the keyboard). Hold the key down and use the direction keys to go to the beginning of the new region selection, press the key and hold it down while moving the cursor with the direction keys to actually perform the region selection.

• Multi-selection region mode: In this mode (which requires the multi-region selection mode to be enabled), it is possible to perform selections that overlap. For example, one could select the sequence “MAMISGM” and then select the sequence “SGMSGRKAS”. The overlapping sequence is thus “SGM”.

Being able to select multiple regions and/or to select multiple times the same region involves some configurations, as far as calculating relevant masses is concerned. Indeed, whatever the selection mode that is enabled, each time one selection (overlapping with another or not) is added or removed, masses are recalculated for the current selection.⁸ The way the multi-region selections and the multi-selection regions are handled, from the mass calculation standpoint, is configured as follows:

• Regions are oligomers: In this configuration, each selection behaves as an oligomer, and is thus capped on both its left and right ends. This is typically the situation when the user wants to simulate the formation of a cross-linked species arising from the cross-linking of two oligomers: each oligomer is capped on both its ends;

• Regions are residual chains: In this configuration, each selection behaves as a residual chain, and thus the oligomer resulting from the multi-region selections is capped on its left and right ends only once. This situation is typically encountered when simulating partial cleavages by first selecting an oligomer, checking its mass and then continuing selection to simulate a longer oligomer resulting from a partial cleavage. Also, the situation might be encountered when there are multiple repeated sequence motifs in a polymer sequence and mass data are difficult to analyze.

6.8 Polymer Sequence Modification

It very much often happens that (bio) chemists use chemical reactions to modify the polymer sequence they are working on. Mass spectrometry is then often used to check if the reaction proceeded properly or not. Further, in nature, chemical modifications of biopolymer sequences are very often encountered. For example, protein sequences get often modified as a means to regulate their function (phosphorylations, for example, or acetylations, methylations...). Nucleic acid sequences are very often and extensively modified with modifications such as methylations...

It is thus crucial that MassXpert be able to model with high precision and flexibility the various chemical reactions that can be either made in the chemistry lab or found in nature. The MassXpert program provides two different chemical modification processes:

• A process by which monomers belonging to the polymer sequence can be individually modified;

• A process by which the whole polymer sequence can be modified, either on its left end or on its right end or even on both ends.

6.8.1 Selected Monomer(s) Modification

There are a number of manners in which monomers can be modified in a polymer sequence. Figure 39 – Selected monomer modification shows the simplest manner: the user first selects the monomer vignette to be modified and calls the Chemistry$⇨$Modify Monomer(s) menu. A window shows up where all the modifications currently available in the polymer chemistry definition are listed. Because a monomer vignette was initially selected in the editor window, the Current selection target radiobutton is on by default.

It is then simply a matter of choosing the right modification from the Available modifications list and clicking onto the Modify button. The target(s) of a given modification (as selected in the Target frame widget) can be identified according to:

• The Current selection group box will show if a single monomer vignette was selected at the time the monomer modification action was invoked (exactly as in Figure 39 – Selected monomer modification).

• If the Monomers Of Same Code radiobutton widget is

selected, all the monomers in the sequence that have their code identical to the one shown in the Current selection line edit widget are modified, as shown below;

• If the Monomers from the list radiobutton widget is selected, all the monomers in the polymer sequence having a code corresponding to any code selected in the Available Monomers list are modified;

• If the All Monomers radiobutton widget is selected, all the monomers of the polymer sequence are modified;

Note that there is one checkbox widget (Override limitations) that requires an explanation. In the chapter about the definition of polymer chemistries (Section 5 – XpertDef: Definition of Polymer Chemistries) the definition of modifications was detailed, and the “target” notion was explicited.

If, during a monomer modification, MassXpert detects that the user is trying to modify a monomer that is not a target of the modification at hand, it will complain, as shown in the Messages text edit widget of Figure 42 – Modification of all the monomers.

Another situation where target limitations might show up, is when trying to modify a monomer more times than authorized by the Max. count number of times that the monomer might be modified at once with that modification. For example, when working on methylation of proteins, it might happen that lysyl residues get methylated more than once at a time (tri-methylation occurs often in histones). If the chemical modification was defined in with a maximum modification count of 2 and a third chemical modification is asked on a given target monomer, then the program refuses to perform the modification. To override this limitation, check the Override target limitations checkbox widget.

The general concept about this is: the Override limitations checkbox widget is unchecked by default so that the user does not do mistakes without knowing. However, flexibility is desirable, and that checkbox widget can be checked if required.

As a result of the monomer modification, the monomer vignette gets modified. Figure 44 – Modification of a monomer in a polymer sequence shows one phosphorylated Seryl residue at position 13: a transparent graphics object (a red “P”) was overlaid onto the corresponding seryl monomer vignette. If the user modifies a monomer with a modification that has no corresponding SVG-formatted file defined for its graphical rendering in file modification_dictionary, then a default modification rendering is used.

The user is responsible for correctly reading the messages that might be published in the Messages text edit widget. It is important to understand that, when a monomer is modified, its previous modification (if any) is overwritten with the new one. The user is invited to experiment a bit with the monomer modification process, so as to be confident of the results that she is going to obtain when real polymer chemistry work is to be modelled in MassXpert.

It is perfectly feasible to modify a single monomer more than once (with the same modification or not; for example a tri-methylation with a methylation modification). This is why when the window depicted in Figure 46 – Seryl residue modified twice with Phosphorylation shows up, the two lists at the right hand side show the monomers currently modified and the modification(s) that are currently set to these modified monomers. Selecting one item from the Modified monomers list will show only the modifications set to that monomer in the Modifications list. If all the modifications in the polymer sequence are to be displayed then, checking the All modifications check box widget will trigger the display of all the modifications set to any monomer in the whole polymer sequence.

Unmodification of monomers is easily performed by selecting any number of items from the Modifications list and clicking the Unmodify button.

6.8.2 Polymer Modification

As described above, it is possible to modify any monomer in the polymer sequence; when any modified monomer is removed, the modification associated to it goes away with it. The modifications that we describe here are not of this kind. They can be applied either to the left end of the polymer sequence or to its right end (or both). But these modifications do belong to the polymer sequence per se and are not removed from it—even if the polymer sequence is edited by removing the left end monomer or the right end monomer. This is why these modifications are polymer modifications and not monomer modifications.

The way in which a polymer sequence is modified using polymer modifications is much easier than the previous monomer modifications case. The modification window is opened by choosing the Chemistry$⇨$Modify Polymer menu as shown in Figure 47 – Modification of the left end of a polymer sequence.

If a formally defined modification is not available, you can define manually the modification that is needed (check the Define modification checkbox to that effect) and then apply to the polymer end of interst. This is illustrated in Figure 47 – Modification of the left end of a polymer sequence for the C-terminal end (the right end) of the protein. The modification object used is created on-the-fly by the program and gets saved in the file as if the user had selected a modification out of the list of available modifications. In the example, the polymer sequence was modified on its left end using the Acetylation available in the polymer chemistry definition. The polymer sequence editor window displays the left end and right end modifications as labels of buttons located in the Polymer
modifications groupbox widget right above the Calculation engine configuration widget.

6.9 Monomer Cross-linking

A cross-link is a covalent bond that links a monomer with one other monomer. A monomer might be cross-linked more than once. The dialog window in which the user might define cross-links is shown in Figure 48 – Cross-linking of monomers.

Cross-linkers were defined in the chapter ( Section 5.2.3 – The cross-linkers). A cross-linker might either define no modification to be applied to the cross-linked monomers or the same number of modifications as there are monomers cross-linked. For example, fluorescent proteins have a chromophore that is made by reaction of three residues (Threonyl [or Seryl]–Tryptophanyl [or Tyrosinyl or Phenylalanyl]–Glycyl), as shown in Figure 48 – Cross-linking of monomers. When cross-linking with the fluorescent protein cross-linker, there must be three monomers involved as these are three modifications defined in the cross-linker.

When any monomer involved in a cross-link is edited off a polymer sequence, the cross-link(s) it was involved in are automatically dissolved and destroyed. Destruction of a cross-link might be performed by selecting the cross-link in the Cross-links list widget at the right hand side of the dialog window depicted in Figure 48 – Cross-linking of monomers and by clicking the Uncross-link button.

6.10 Sequence Cleavage

It happens very often that polymer sequences get cleaved in a sequence-specific manner. These specific cleavages do occur very often in nature, and are made by enzymes that do cleave biopolymer sequences, like the glycosidases (cleaving saccharides), the proteases (cleaving proteins) or the nucleases (cleaving nucleic acids). But the scientist also uses purified enzymes or chemicals to perform such cleavages in the test tube. MassXpert must be able to perform those cleavages in silico.

Starting a polymer sequence cleavage is a matter of having a polymer sequence opened in an editor window and selecting the Chemistry$⇨$Cleave menu. The user is provided with a window where a number of cleavage agent names are listed (Figure 49 – Polymer sequence cleavage window), along with options that permit the customization of the production of oligomers. The parameters that can be set are the following:

• Available cleavage agents: list of the cleavage agents available in the polymer chemistry definition (see Section 5.2.4 – The cleavage agent definitions).;

• Oligomer coordinates: when the window is opened, this group box widget lists the coordinates of the currently selected region of the polymer sequence. Either leave the values as they are shown or check Whole sequence. If Whole sequence is checked, the cleavage will be performed over all the sequence length. Otherwise it will be performed over the sequence in the Start–End range. This feature, which was introduced in version 2.3.0, is useful so as to simulate a first cleavage of a polymer sequence and then a second cleavage of a selected oligomer using a different cleavage agent. In protein chemistry, that would be useful to explore possibilities of double sequential cleavages of a protein, first with EndoAspN, for example, and then with Trypsin.

• Setting the Partial cleavages number defines if the cleavage must be total (value of 0) or if missed cleavages are allowed.

• Setting the Ionization level range defines what charge state the generated oligomer will have.

A number of other features might be configured, either on the way oligomers should be exposed or on the way they should be exported to a number of destinations:

• Checking With sequence tells the cleavage engine to store their sequence in the generated oligomers; see Cleavage oligomer details.

• In normal operation, when multiple cleavages are performed by clicking Cleave, the new oligomer set replaces the one obtained previously. However, it might be useful to generate in the same unique list all the oligomers generated for different cleavages. To stack oligomers from different cleavages, check Stack oligomers.

• It is possible to export the oligomers selected in the tableview widget in a number of ways. Select the destination in the drop down menu widget. A specific export format might be defined and the mass type to be exported might be selected. When exporting for the module, check For XpertMiner.

• Mass spectrum synthesis: This group box is about the configuration of the way a mass spectrum might be calculated and displayed in another program. The Trace color button allows one to select a color with which to plot the mass spectrum’s trace once exported to a mass data visualizing program (MineXpert). The line edit widget above the button allows one to define a title for the mass spectral trace plotted. Finally, the drop down menu holds three menus, as described below:

  • Configure the mass peak shaper: Click this menu item to display the Mass peak shaper configuration dialog window as shown on Figure 49 – Polymer sequence cleavage window. The configuration of the mass peak shaper works in this dialog window exactly as described in Figure 72 – Setting-up of the centroid mass peak shaping process.

  • Synthesize the mass spectra: This button actually triggers the computation of the mass spectra (the isotopic clusters) of all the selected fragmentation oligomers currently selected in the table view. The calculation is only performed if the previous configuration step has yielded a valid isotopic cluster shaper configuration.

  • Mass spectrum to clipboard: This menu item copies the mass spectrum to the clipboard. For this menu to be available, a mass spectrum synthesis must have been performed successfully, otherwise the menu reads Mass spectrum not yet available.

The Details frame widget at the bottom of the window displays a number of informative data. In particular, the Sequence tab widget displays the sequence of the oligomer currently selected in the Oligomers table view along with the name of the cleavage agent which it arose from. The Cleavage Details tab widget displays the mass calculation engine configuration at the time the last cleavage was performed (checked items mean that the corresponding feature was on, unchecked items mean that the related feature was off). In the example (Figure 49 – Polymer sequence cleavage window, the mass calculation for the oligomers did not account for the monomer modifications nor for the left/right end modifications of the polymer, nor for the cross-links.

When the user triggers a cleavage, the mass calculation engine configuration currently set in the sequence editor is used for the calculation of the mass of the oligomers obtained per the cleavage. This process allows an easy change in the mass calculation engine configuration between one cleavage and another so as to allow comparison of masses obtained for the same cleavage but with different mass calculation engine configurations.

For oligomer data filtering, please refer to Section 6.13 – Oligomer Data Filtering.

6.11 Oligomer Fragmentation

It happens very often that polymer sequences need to be fragmented in the gas phase (in the mass spectrometer) so that structure characterizations may be performed. For protein chemistry, this happens very often in order to get sequence information for a given peptide ion selected in the gas phase. MassXpert must be able to perform those fragmentations in silico. Let’s see how an oligomer can be fragmented using MassXpert.

Starting an oligomer fragmentation is a matter of having a polymer sequence opened in an editor window, selecting the sequence region to be fragmented and finally selecting the Chemistry$⇨$Fragment menu. The user is provided with a window where a number of fragmentation pathway names are listed (Figure 50 – Oligomer fragmentation window). As detailed for the cleavage of polymers, these fragmentation pathways are listed by looking into the polymer chemistry definition corresponding to the polymer sequence of which an oligomer is to be fragmented.

Select the fragmentation pathways(s) of interest, set the ionization range required for the generated fragment oligomers (the same as for polymer sequence cleavage) and click Fragment. Upon successful termination of the fragmentation reaction, the generated fragments are displayed in the Oligomers tableview widget.

As detailed for the cleavage of polymer sequences, the Details frame widget displays data about the fragments generated and the way masses were calculated for them.

It is possible to take into account cross-links that are beared by monomers contained in the oligomer.

The cross-links are accounted for during the fragmentation of an oligomer if Cross-links is checked in the calculation engine configuration panel of the sequence editor window (Figure Figure 51 – Starting a fragmentation that accounts the cross-links). This is a partial view of the cyan fluorescent protein, with the “TWG” chromophore tripeptide.

If we select the oligomer region [60–99] and that we ask for a fragmentation, the fragmentation results will take into account the cross-link only in the case the generated fragments encompass fully the cross-link.

The following calculation rationale applies:

• Fragments b (left end) from b₁ (D) to b₂₉ (D), sequence [⁶⁰DATY…QCFS⁸⁸], do not take into account the cross-link as all the monomers involved in it are outside of these be fragments;

• Fragments b₂₉ to b₃₁ do not take into account the cross-link because not all the monomer involved in it are contained in these fragments;

• Fragments b₃₂ (up to C) to b₄₀ do take into account the cross-link because all the monomers involved in it are contained in these fragments;

• Likewise, the only y fragments (right end) that do take into account the cross-link are the fragments y₉ and all the remaining, as for these fragments, the cross-link is fully contained since all the monomers involved in it are contained in these fragments.

A more complex cross-linked structure example is shown in Figure Figure 52 – Complicated cross-linking situation, where the 17-mer oligomer has 4 fully-encompassed cross-links and one partial. This oligomer is used as an example of how the fragmentation computation is handled by MassXpert.

The calculation of the fragments for this oligomer involves the following steps:

• Calculate regions of the oligomer that involve cross-links either overlapping or not. The regions are thus the following: [3–5], [8–11] and [13–15]. Note that the cross-link involving monomer 12 is never taken into account as it involves also a monomer outside of the oligomer;

• For fragments that have the left end of the oligomer ($⟶$ “Left-end nomenclature”), the following rationale is used:

  • Fragments $⟶$1 and $⟶$2 do not have any cross-link;

  • Fragments $⟶$3 to $⟶$4 do not account for cross-link a because that cross-link is not fully encompassed by the fragments;

  • Fragments $⟶$5 to $⟶$10 account only for the cross-link a as this is the only cross-linked region to be fully encompassed by these fragments;

  • Fragments $⟶$11 to $⟶$14 account for cross-links a, b and c as they are all fully encompassed in the fragments;

  • Fragments $⟶$15 to $⟶$16 account for all cross-links, a, b, c, d as they are all fully encompassed in the fragments;

• For fragments that have the right end of the oligomer ($⟵$ Right-end nomenclature), the following rationale is used:

  • Fragments 1$⟵$ and 2$⟵$ do not have any cross-link;

  • Fragments 3$⟵$ and 4$⟵$ do not account for cross-link d because that cross-link is not fully encompassed by the fragments;

  • Fragments 5$⟵$ and 6$⟵$ account for cross-link d because it is fully encompassed in these fragments;

  • Fragments 7$⟵$ to 9$⟵$ only account for cross-link d because cross-links b and c (which make one cross-linked region) are not fully encompassed by these fragments;

  • Fragments 10$⟵$ to 14$⟵$ account for cross-links d, c and b, but not for cross-link a as this last cross-link is not fully encompassed in these fragments;

  • Fragments 15$⟵$ and 16$⟵$ account for all the cross-links of the oligomer.

The various widgets in the Actions groupbox widget are very similar to the ones found in the polymer sequence cleavage window (Figure 49 – Polymer sequence cleavage window).

For oligomer data filtering, please refer to Section 6.13 – Oligomer Data Filtering.

Exactly as described for the cleavage oligomer mass spectrum synthesis, it is possible to synthesize a mass spectrum corresponding to all the fragmentation oligomers currently selected in the table view widget. This process has been described at Mass spectrum synthesis.

6.12 Mass Searching

It may happen that the scientist needs to know if some arbitrary sequence region would have a given mass. MassXpert allows for mass searching operations in the polymer sequence. This is done by using the menu Chemistry$⇨$Mass Search. The window in Figure 53 – Searching masses in a polymer sequence shows up and the user enters masses to search for. A number of parameters are to be detailed:

• Targets: should the masses be searched for in the whole sequence or in the currently selected region only?

• Ionization: should different levels of ionization be calculated when calculating masses for the potential oligomers matching the searched mass? For example, one finds in an electrospray ionization experiment mass spectrum a peak at m/z 1245. It is not possible to know the ionization level for that ion. On could imagine that this value is for a monoprotonated or for a multiprotonated species. If we wanted to assess this, we might ask that the mass be searched for by computing a range of possible ionization levels between Start level 1 and End level 4 (admitting that for that experiment this is what one would expect).

Once the masses have been searched for, if results are found they are displayed in the same window in the Oligomers table view widgets (the left one for the mono masses and the right one for the avg masses).

The various widgets in the Actions groupbox widget are very similar to the ones found in the polymer sequence cleavage window (Figure 49 – Polymer sequence cleavage window).

For oligomer data filtering, please refer to (Section 6.13 – Oligomer Data Filtering).

6.13 Oligomer Data Filtering

Oligomer-generating simulations, like polymer sequence cleavages or fragmentations or mass searches, produce a very large amount of data. It is often desirable to be able to filter quickly some specific data out of these bunch of data... In all these three simulation contexts, the results that are displayed in the corresponding dialog windows are easily filtered using the mechanism illustrated in Figure 54 – Oligomer data filtering. To enter filtering mode, check Filtering options the line edit widgets will appear for you to start entering filters.

Filtering on the data is easily performed by entering the options in the Filtering options group box (Figure 54 – Oligomer data filtering). For any filtering operation, only one criterium can be used, that is, for example, filtering can occur only on the basis of the monoisotopic mass or of the average mass, but not on both masses.

For example, if one wanted to filter a huge set of data against a specific monoisotopic m/z of 850 +/− 0.33 u, it would simply be a matter of setting the monoisotopic m/z to be 850 with a tolerance of 3 AMU in the corresponding line edit widgets contained in the Filtering options group box. To perform that filtering action, first set the tolerance value (0.33) in its line edit widget below the drop down list that reads AMU and next set the monoisotopic mass value to be 850 in the corresponding Mono line edit widget. While the cursor is still in the Mono line edit where 850 was entered, press the keyboard key combination  +  .

The filtering will be immediate and the table view will show the data that passed the filter. Note that the combo box widget holding the unit of the tolerance (in the example, that unit is AMU, that is, “atomic mass unit”) and the line edit widget where the tolerance value proper is set (0.33 in the example) do not trigger any filtering by themselves; these widgets are only useful in conjunction with other oligomer data: Mono, Avg, Error line edit widgets (depending on the dialog window where the filtering occurs: cleavage, fragmentation or mass search). In our example, thus, the filtering would be spelled like this:

.

To exit the data filtering mode, uncheck Filtering options and all the initial data will be displayed, irrespective of any data in the line edit widgets described above.

6.14 Monomeric And Elemental Composition

The Chemistry$⇨$Determine Compositions menu triggers the window shown in Figure 55 – Determination of the compositions. The elemental composition is determined using the calculations engine configuration currently set in the polymer sequence editor window.

6.15 pKa, pH, pI and Charges

When preparing biochemical experiments, very often users need to know how many charges a given polymer sequence will bear at any given pH. Equally important is the ability to know at which pH value the polymer sequence will have a net charge near to zero. The pH value for which a given polymer sequence has a net charge near to zero (typically this means that the number of positive charges equals the number of negative charges) is called the isoelectric point—the pI.

Such computations are pretty computer-intensive and require a very precise knowledge of the chemical structure of the different monomers that take part in the definition of the polymer chemistry. A file, called pka_ph_pi.xml is located in the polymer chemistry definition directory. This file lists all the chemical groups that are possibly charged; each monomer of the polymer definition is represented by a “<monomer>” element in which data are defined for any chemical group of that monomer that might bear a charge at any given pH. You can find the listing of the pka_ph_pi.xml file in the appendices. We’ll discuss any aspect of this file’s contents in the next sections with enough detail that the user will be able to write one such file for her specific polymer chemistry.

At the moment, two entities in the polymer chemistry definition might have chemical groups bearing charges: monomers and modifications. We will first review monomers, and modifications next.

6.15.1 Ionized Group(s) In Monomers

Monomers are the building blocks of polymer sequences. These blocks must have at least two reactive groups so that they can be polymerized into a polymer sequence thread. Reactive groups are often chargeable groups; for example, the amino group of amino-acids is such that it gets protonated (positively charged) at a pH inferior to its pKa. Similarly, the carboxylic acid group of amino-acids is deprotonated (negatively charged) at physiological pH.

6.15.1.1 Some Theory First

For the non-biochemist reader, amino-acids involved in the formation of proteins have always at least two chemical groups that are of inverted electrical charge, at physiological pH values (Figure 56 – Different pKa values of chemical groups).

• The amino group (called $𝛼$NH₂) has a typical pKa value of 9.6. This means that, at physiological pH values (between 6.5 and 7.5), the amino group will find the environment rather acidic, and will thus be protonated, leading to a positively-charged species ($𝛼$NH₃⁺);

• The carboxylic group (called $𝛼$COOH) has a typical pKa value of 2.35. This means that, at physiological pH values, the carboxylic group will find itself in a rather basic environment, and will thus be deprotonated, leading to a negatively-charged species ($𝛼$COO⁻).

It should be clear that, at physiological pH values the two $𝛼$-chemical groups have a naught net charge ($$z=0). But proteins are charged, and this is because some of the twenty common amino-acids have other chemical groups beyond the two others already described. Indeed, some amino-acids have lateral chains that bear groups that might be charged depending on the pH: seryl residues have an alcohol group that has a pKa of 13, for example; that means that it is almost always uncharged (form ROH at physiological pH values). The lateral chain of lysine has a pKa of 10.53, which means that at pH values below this pKa value, the εNH₂ gets protonated, introducing a positive charge in the protein. Similarly, amino-acids glutamate and aspartate do have a lateral chain ending with a γCOOH and a βCCOOH, respectively. Their pKa values are below 4.5, and thus the groups are negatively charged a physiological pH values.

When the net charge of a polymer sequence has to be computed for a given pH condition, the program iterates in the sequence, and for each monomer will check which one of its chemical group(s) is possibly charged. For this to happen, it is required that a number of data be known for each monomer’s chemical group that might play a role in the determination of the polymer sequence’s electrical charge. Thus, for each chemical group a number of data should be listed in the pka_ph_pi.xml file. In the XML file format code below, the examples of the protein’s glutamate (left column) and lysine (right column) amino-acids are detailed in the next page.

• The chemical group’s <name> element is required. For example, “αNH₂” or “εNH₂” or “αCOOH”

• The chemical group’s <pka> element is optional, but is the basis for the charge calculation. For example, 9.6 for the “αNH₂” or 2.35 for “αCOOH”

• The <acidcharged> element is required if the <pka> element is given. This element is responsible for telling if the chemical group is charged (positively) when the pH is lower than pKa (that is when the medium is acidic with respect to the pKa). For example, an amine is positively charged when it is in its acidic form (protonated); a carboxylic acid is not charged when it is in its acidic form;

• There can be none, one, or more <polrule> element(s) for each chemgroup. The <polrule> element gives informations about the way the chemical group at hand might be “trapped” (or not) in the formation of inter-monomer bonds (while the monomer is polymerized into the polymer sequence). The value left_trapped means that the chemical group ceases to be involved in charge calculations as soon as it has a monomer at its left end. The value right_trapped means the same as above, but when a monomer is polymerized at its right end. For a chemical group that is left_trapped, we understand that it is only effectively evaluated if it is at the left end of the polymer sequence, since in this case it does not have a monomer at its left side. Conversely, a chemical group that has a <polrule> element with value right_trapped, will be evaluated only if the monomer is actually the right end monomer in the polymer sequence. Finally, the typical lateral chains of amino-acids have a <polrule> element with a value never_trapped, as these chemical groups do not take part in the formation of the inter-monomer bond;

• There can be none, one or more <chemgrouprule> element(s) for each chemical group. Such an element should contain the following:

  • There must be an <entity> element that indicates what is the chemical entity being dealt with in the current guilabel“” element. Valid values for this element are LE_PLM_MODIF, RE_PLM_MODIF or MNM_MODIF;

  • There must be a <name> element naming the chemical entity properly;

  • There must be an <outcome> element telling what action should be taken when encountering the <entity> on the <chemgroup>. Valid values are either LOST or PRESERVED.

6.15.2 Ionized Group(s) In Modifications

In the excerpt from the pka_ph_pi.xml file below, we see that chemical modifications can also bring charges. The example of the chemical modification Phosphorylation shows that when a monomer is phosphorylated, two chemical groups are brought in: the first has a pKa value of 1.2 (that is it will always be deprotonated at physiological pH values), the second has a pKa value of 7 (that is it will be divided by half in a protonated (not charged) form and in an un-protonated (negatively charged) form, leading to a net electrical charge of −0.5.


<modif>
<name>Phosphorylation</name>
<mdfchemgroup>
<name>none_set</name>
<pka>1.2</pka>
<acidcharged>FALSE</acidcharged>
</mdfchemgroup>
<mdfchemgroup>
<name>none_set</name>
<pka>6.5</pka>
<acidcharged>FALSE</acidcharged>
</mdfchemgroup>
</modif>


At this point we should be able to study the way computations are actually performed in the module.

6.15.3 pH, pI and Charge Calculations

The user willing to compute charges (positive, negative, net) or the isoelectric point for the current polymer sequence uses the menu Chemistry$⇨$pKa pH pI which triggers the appearance of the window shown in Figure 57 – Acido-basic computations: net charges.

This figure shows that the user can calculate the charges (positive, negative and net) beared by the polymer sequence (either the whole sequence or the current selection) by setting the pH value at which the computation should take place. It is also possible to calculate the isoelectric point by clicking onto the Isoelectric Point button.

Note that the computations might involve the permanent left/right modifications of the polymer sequence, as well as the monomer chemical modifications. To configure the way net charge—or pI—calculations are performed, use the calculation engine configuration of the sequence editor window.

6.16 General Options

One of the options that are valued most by users is to be able to set the number of decimal places used to diplay numbers. The settings should apply in a distinct manner depending on the different entities for which numerical values are to be displayed (see Section 3.1 – Setting the application preferences - general options).

7 : a Powerful Mass Calculator

After having completed this chapter you will be able to perform sophisticated polymer chemistry-aware mass calculations.

7.1 Invocation

The module is easily called by pulling down the $⇨$Open calculator menu item from the MassXpert program’s menu. The user is presented with a window to select the polymer chemistry definition that should be used for the calculations (Figure 58 – Selecting a polymer chemistry definition for use with ).

7.2 An Easy Operation

Once the polymer chemistry definition has been correctly selected, it is parsed by the module and its entities are automatically made available in the calculator window. The way is operated is very easy. This is partly due to the very self-explanatory graphical user interface of the module, which is illustrated in Figure 59 – Interface of the module.

can handle a number of features that are reviewed below:

• The user may (is not obliged to) seed the calculation by setting masses manually in the Seed masses line edit widgets (the left line edit is for mono and the right one for avg);

  For example, imagine that a mass spectrum analysis session ends up like this:

  As shown in Figure 59 – Interface of the module, the mass spectrometry scientist would seed the mass calculator with mass 1000.55 and ask that one Phosphorylation modification be added to it by setting 1 in front of the corresponding drop-down widget. Clicking the Apply button triggers the calculation, with the resulting masses being displayed in the Result masses line edit widgets. We can see that the phosphorylation of our analyte shifts its m/z value from 1000.55 to 1080.5163, which allows the operator to evaluate the phosphorylation hypothesis by comparing the calculated value with the observed one.

• The Formula group box widget contains two widgets: a line edit widget where the formula is typed and a count spin box widget where the user sets the number of times that the formula should be applied. Setting the formula to H2O and the count to 2 would hydrate the analyte twice.

• The Polymer Chemistry Definition Entities group box widget contains two drop-down widgets and a line edit widget. The drop-down widget on the left lists all the monomers defined in the protein-1-letter polymer chemistry definition that was selected when starting the calculator; the drop-down widget on the right lists all the modifications defined in the protein-1-letter polymer chemistry definition. Each drop-down widget has its corresponding count spin box widget. In the example, the user asked that one (1) Phosphorylation modification be applied during the calculation. The line edit widget below the first row of widgets is the polymer sequence widget where the user might enter a sequence of monomers. It is possible to apply many times the sequence by setting the count spin box widget value to something greater than 1 (either positive or negative);

It is possible to perform a set of calculations in one go, that is, the user may ask for a formula, a monomer, a modification, a sequence to be accounted in one single calculation operation. Once all the chemical entities to be taken into account have been set, the user clicks the Apply button: all the entities are parsed in sequence and their mass equivalents are added to the result masses. Other prominent features of are described in the following sections.

7.3 The Programmable Calculator

For the scientists who work on molecules that are often modified in the same usual ways, features a built-in mechanism which enables them to easily program their calculator. This programming involves the definition of how a chemical pad (or chempad) may be arranged, exactly the same way as a desktop calculator would arrange its numerical keypad.

The chemical pad can be shown/hidden by using the Show Chemical Pad check box. An example of such a chemical pad is shown in Figure 60 – Interface of the chemical pad, where a “protein-1-letter” polymer chemistry definition-associated chempad is featured. As shown, the user has programmed a number of chemical reactions that may be applied to the masses in the calculator window by simply clicking on their respective button. The configuration of the chemical pad is very easy.

One example of such a configuration file is shown below along with explanations:

The first three lines of the code above are color definitions. One line specifies the arrangement of the keys on the chemical pad. The remaining lines are definitions for groups of keys and the keys themselves. Below are the feature descriptions:

• It is possible to define as many colors as necessary according to the standard red, blue, green format, on a scale of 0–255).

• The calculator chemical pad shall have its buttons organized in three columns.

• This separator will create a group box widget labelled “Generic” that will be populated with all the items found below (until another separator is encountered).

• A button definition is introduced by the chempadkey= string. Separated by % characters, follow the name of the chemical reaction that will label the button (protonate), the chemical formula of the reaction (+H2O1) and finally the tooltip text that displays when the cursor stays on the button.

• chempagroup is a separator that starts a new button group box, labelled Hexoses in the example. This syntax allows for the coloring of the group box widget (seagreen in the example).

• The last two lines describe buttons by also specifying their color. lawngreen is the background color and black is the color of the text.

These buttons might be used in two distinct ways:

• Upon clicking the button, its formula is evaluated and the corresponding masses are added to (or subtracted from) the Result masses

• If the key is maintained pressed upon clicking on a chemical pad button, its formula is inserted into the Formula line edit widget of the calculator, so that the formula is not directly evaluated and the Result masses are not modified.

7.4 The Log Book Recorder

Each time an action that is chemically relevant—from a molecular mass point of view—is performed, the program dumps the calculations to the recorder window (Figure 61 – The recorder window). The recorder can be shown/hidden by using the Show Recorder check box widget. The text in the recorder window is editable for the user to edit the output, and selectable also, so that pasting to text editors or word processors is easy via the clipboard.

7.5 The m/z Ratio Calculator

It very often happens that the mass spectrometrist doing electrospray analyzes is faced with a challenging task: to compute by mind all the m/z ratios for a given family of charge peaks. To ease that daunting task, contains a m/z ratio calculator that is called by clicking onto the m/z
calculation button.

7.6 m/z Ratio Calculations

In electrospray ionization, a given polymer sequence might be charged a large number of times. The tool shown in Figure 62 – Calculation of ranges of m/z ratios details how to compute a range of m/z ratios starting from one m/z value for a given charge and a given ionization agent. It is also possible to switch ionization agent on-the-fly.

The sequence with which data should be entered in the upper part of the window is the following:

• Ensure that the Ionization rule is correct;

• Enter the Mono m/z and or the Avg m/z values

• Set the Target ionization status

• Click the Calculate button.

8 Isotopic cluster calculations

When the resolving power of the mass spectrometer is good, zooming-in on a mass peak may reveal that a given ion has given rise not to one peak but to a set of peaks that together form an “isotopic cluster”. The ions responsible for the different cluster peaks are called “isotopologues” because they are homologues of the analyte that have different numbers (and therefore positions) of the different isotopes.

While the richness of mass spectra acquired with high resolving power machines has been neglected for many years, it is now well established that using the resolved isotopic cluster peaks for the detailed analysis of mass spectra is of great use.

When performing mass spectrometric data analyses, it is very often necessary to calculate the isotopic cluster of a given analyte. Indeed, the isotopic cluster can be a criterion helping to establish if a given detected signal (the acquired isotopic cluster) does indeed correspond to a given chemical entity, and matching the experimental isotopic cluster with one that was simulated for the chemical entity under scrutiny might prove useful. This way of ascertaining the assignment of mass peaks to specific analytes is becoming more and more desirable as mass spectrometers have ever-greater resolving power capabilities, thus providing the user with neatly resolved isotopic clusters even for oligomers of intermediate masses. In this section, the process of calculating the isotopic cluster of a given analyte defined by its chemical composition (also called elemental composition or formula) and optionally its charge is described in detail.

It is possible to predict how a given ion (of known elemental composition and charge) is supposed to be revealed as an isotopic cluster in a high resolution mass spectrum. One such cluster is shown in Figure 63 – Simulation of the isotopic cluster for a protein, for a protein in its deca-protonated form [M+10H⁺]¹⁰⁺, of ionic elemental composition C769H1222N210O218S2.

MassXpert provides a sophisticated interface to the libIsoSpec++ library.

This library performs high-resolution isotopic cluster calculations. In order to run the calculations, it is necessary to have the following items ready:

• The elemental composition of the analyte (for example, C769H1222N210O218S2). This formula needs to account for the ionization agent that is involved in the ionization of the analyte prior to its detection in the mass spectrometer. If an analyte is deca-protonated, 10 protons need to be added to its neutral-state formula.

• A detailed isotopic configuration of all the chemical elements that are found in the elemental composition. MassXpert provides three interfaces to define the isotopic characteristics of the chemical elements. These will be described in the following sections.

8.1 Generating an isotopic cluster simulation in MassXpert

To perform an isotopic cluster simulation in MassXpert, select the Isotopic clusters$⇨$Isotopic cluster generator menu (Figure 64 – Isotopic clusters main menu).

The window shown in Figure 65 – Isotopic cluster generator window contains all the interface elements required to configure the simulation starting from a formula.

An isotopic cluster calculation is most probably performed with the aim of simulating an expected isotopic cluster for an analyte that is being analyzed by mass spectrometry. It is thus logical that the analyte be in an ionized form. The way that the analyte has been ionized needs to be taken into account in the chemical formula that describes the ion for which the isotopic cluster is being calculated. For example, when determining the elemental composition of a protein in the positive ion mode, the number of protons used to ionize the protein needs to be taken into account in the formula.

The Gaussian apex intensity value (a.u.) check box widget allows the user to set the intensity (y axis) that the isotopic cluster will have for its highest peak. This is useful when the isotopic cluster is to be simulated for direct comparison with an isotopic cluster in an experimental mass spectrum.

The Max. cumulative probability spin box widget serves to configure the extent to which simulates the theoretically expected isotopic cluster. A value of 0.99 tells the software to simulate enough combinations of the analyte isotopes to represent 99 % of the theoretically expected combinations.

The Cluster’s centroid sorting group box widget contains a few settings to configure the way the generated cluster’s (centroid, intensity) pairs are sorted in the output.

The Mass spectrum synthesis group box widget contains a few settings to configure the title and the color of the generated spectrum (the actual generation of the spectrum on the basis of the output is described in detail below). The title and color of the mass spectrum are used when the spectrum is displayed in the companion MineXpert software program. The process by which the mass spectrum is transferred from MassXpert to MineXpert is described at Section 9.1 – Configuration of the MassXpert and MineXpert intercommunications.

To perform isotopic cluster calculations, the simulation software needs to be aware of the whole set of isotopes of all of the chemical elements that enter in the composition of the ionized analyte. An isotope is defined by its mass and by the probability that it is found in nature. Carbon has two major isotopes that can be found in nature: the ¹²C most abundant isotope and the ¹³C least abundant isotope (the ¹⁴C isotope is irrelevant for conventional mass spectrometry because its abundance in nature is way too low).

There are three ways that the user might define the isotopic data to be used for the simulations. Two of them involve a configuration preparation on the part of the user. The third one, that we’ll describe first, does not involve any chemical element configuration. The other ways are reviewed in the following sections.

8.1.1 Static Standard Element Tables Shipped within the Library

In order to document all the chemical elements’ isotopes’ characteristics, the library has, in its own software code headers, a number of arrays that MassXpert automatically loads up when opening the Figure 65 – Isotopic cluster generator window dialog window.⁹ These data are displayed in the IsoSpec standard static data tab. The table view widget data are loaded from source code each time the window is opened, hence the static qualifier in the tab name, meaning that the user won’t be able to change these data and keep the changes between sessions.

In this tab, apart the configuration on the left hand side pane of the dialog window, all that is left to do is enter in the Formula combo box widget (which looks like a line edit widget with an arrow at its right side) the elemental composition for which the isotopic cluster calculation is to be performed.

Once the formula has been set to its combo box widget, the charge of the ion needs to be set (Ion charge spin box widget) along with the Max. cumulative probability. To run the calculation, click Run.

8.1.2 User-Modified Standard Element Table Shipped within the Library

While the previous section showed how to use the static element table from the library, this section shows a way to customize these elemental data for a specific experiment.

The user-modified version of the standard static isotopic data is available in the IsoSpec standard user data tab of the dialog window, which, upon opening has an empty table view widget, as shown in Figure 66 – User-modified standard element table.

The simplest way to fill in the user-modified standard isotopic table is to export the isotopic data from the IsoSpec standard static data tab to a text file (Save table data to file for further editing button). The data are output to the file in the CSV (comma-separated-values) format, in a layout that closely mimicks the data visible in the table view. An example of the exported text is shown below after modifications applied to the carbon and nitrogen isotopes for heavy labeling. One would typically modify the isotope definition file with a simple text editor.

Isotope definition for full labelling with heavy C and N isotopes

Now that the modifications have been performed, the file needs to be saved under the same format. It is then available for loading in the IsoSpec standard user data tab of the dialog window by clicking the Load isotopic data from file push button. The table view now shows the new isotope mass-abundance pairs. From now on, this tab may be used exactly like described above for the standard isospec static data-based work.

Once the formula has been set to its combo box widget, the charge of the ion needs to be set (Ion charge spin box widget) along with the Max. cumulative probability. To run the calculation, click the Run button.

8.1.3 User hand-made isotopic configuration of an elemental composition

The last way the user might configure the chemical elements to be used for an isotopic cluster calculation is based on the fully manual description of the isotopes present in the elemental composition of interest. That configuration is performed in the Manual configuration tab of the dialog window. This method is slightly more involved than the previous one but provides also for a much greater flexibility: it allows one to create “new chemical elements” that might be required in specific labelling experiments. The manual configuration is carried over in the Manual configuration tab of the dialog. Upon starting, this tab is empty, as shown in Figure 67 – Hand-made user isotopic configuration of an elemental composition (the working sheet is blank).

Upon creation of the window, the Manual configuration tab is empty, with only two rows of buttons at the bottom of the tab. To start configuring chemical elements, click Add element to create an “element group box” that will contains a number of widgets organized in two rows, as described in Figure 68 – Hand-made user isotopic configuration of an elemental composition.

• Top row, a line edit widget to receive the chemical element symbol, like C, for example;

• On the same row, a spin box widget in which to set the number of such atoms in the formula for which the isotopic cluster is being calculated;

• The Remove element removes all the “element group box” in one go;

• The bottom row contains a set of widgets to configure the first isotope of the current element. The left spin box widget is for the mass and the right one is for the abundance;

• To add another isotope for the current chemical element, click the Add isotope button. To remove that isotope row, click the Remove isotope button. There is no limitation on the number of isotopes that may be created to fully qualify a chemical element.

Once all the isotopes for a given chemical element have been defined, a new element might be needed. For this, click Add element and start the configuration of the new element as described above.

The manual isotopic configuration of the chemical elements required to perform an isotopic cluster calculation for a given formula is tedious. The user may want to save a given configuration to a file (click the Save configuration button) so that it is easier to recreate automatically all the widgets upon loading of that saved configuration (click Load configuration).

The final configuration is shown in Figure 69 – Typical manual configuration of the isotopic composition of the chemical elements. The experiment that was configured above is the labelling of a glucose molecule with Cz, an imaginary chemical element that is like carbon but that has a ¹⁴C. The glucose molecule (normal formula: C6H12O6) is labelled on one single carbon atom with an efficieny of 95 %. This means that, when the labelling fails (in 5 % of the cases) the carbon atom has its isotopes with usual probabilities (compounded by the fact that the unlabeled atom is found at that position only in 5 % of the cases). The isotopic abundances for the Cz element are thus:

• For ¹²C: 0.05 * natural ¹²C abundance;
• For ¹³C: 0.05 * natural ¹³C abundance;
• For ¹⁴C: 0.95;

The example of has been exported and is available as a text file depicted in Figure 70 – The user manual configuration might be exported to a text file and loaded back.

As described above, once the analyte’s elemental composition has been defined, as just described, click Run.

8.1.4 The results of the computation are centroids

Once the configurations have been terminated, the isotopic cluster calculations can finally be performed. In the manual configuration setting, the formula is automatically handled, since each chemical element that is defined goes along with the count of the corresponding atoms. In the case of the standard configuration (either modified or not), the user has to enter the chemical formula of the analyte in the Formula line edit widget.

Click the Run button. If the configuration was correct and the calculation could run properly, then the window switches to the IsoSpec results tab (Figure 71 – Results from the isotopic cluster calculation). That tab contains a text edit widget in which the results are displayed.

The results output by represent the centroids of the peaks that make the isotopic cluster. The results are thus a set of (m/z,i) pairs that have not the characteristic shape (the profile) that is found in mass spectra. MassXpert features the ability to give a shape to the centroid peaks. For that, click the To isotopic cluster shaper button to open the Isotopic cluster shaper window preloaded with the list of peak centroids.

The workings of this peak shaping feature is described in Section 8.2 – Shaping the centroids of the isotopic cluster peaks into well-profiled peaks.

8.2 Shaping the centroids of the isotopic cluster peaks into well-profiled peaks

As mentioned earlier, the isotopic cluster calculation produces the isotopic cluster in the form of a set of (m/z,i) pairs that represent the centroids of the isotopic cluster’s peaks and their relative intensity (probability). There are other data simulations or analysis processes that lead to having mass peaks defined by a single centroid m/z value and a corresponding intensity. Plotted to a graph, a centroid mass peak yields a bar, which is inadequate if the user is willing to simulate isotopic clusters to later compare these with experimental profiled mass peaks. The features in this dialog window aim at shaping each mass peak by creating a shape around the centroid (in specialized terms, this is the profile of the peak).

The shape of mass peaks is typically Gaussian or Lorentzian (or a mix thereof). In order to convert mass peak centroids into something that resembles a real “profile” mass peak, a mathematical formula can be applied, with some parameters to configure the shapes generated. MassXpert makes the shaping of mass peaks accessible via the Isotopic clusters$⇨$Isotopic cluster shaper menu item (Figure 64 – Isotopic clusters main menu). The window that opens up is shown in Figure 72 – Setting-up of the centroid mass peak shaping process.

The mass centroid peaks are listed in the Data centroid points (m/z,i) text edit widget. These values may come from different origins. They can be set automatically from the isotopic cluster calculation dialog window (see Section 8.1.4 – The results of the computation are centroids); they can come from the user editing them in place; finally they can come from the user pasting text from the clipboard.

The width of the “profile” mass peak is determined either by setting the resolving power of the instrument or by setting the width of the peak at half maximum of its height (FWHM).

The profile mass peaks generated can be either of a Gaussian or of a Lorentzian shape. That parameter is configured by selecting the corresponding radio button widget. The number of points used to actually craft the shape of the peak is configurable in the Shape the peak using points spin box widget.

There are two ways to configure the width of the Gaussian/Lorentzian shape around the centroid peak. Either one knows the resolving power of the instrument, or one can measure the full width at half maximum (FWHM) of the experimental peak. The Resolving power group box widget contains all the widgets required to define that FWHM. Because the FWHM actually depends on the resolving power and the resolving power is computed using the m/z value, there needs to be a Reference mass provided for the calculation. If the data in the text edit widget on the left hand side pane of the window is filled with data from an isotopic cluster calculation, the reference mass is the m/z value of the most intense peak. If the data come from the user either pasting or editing text in the text edit widget, then it is their responsibility to fill in the reference mass.

To define the FWHM value, one can just enter it in the corresponding spin box widget. When the editing is finished and the focus leaves the spin box widget, the Bin size logic group box widget will have the FWHM radio button widget selected. The Resolving power spin box widget now contains the proper resolving power value, as computed using the FWHM value set to the Reference mass spin box widget.

To define the resolving power value, one can just enter it in the Resolving power spin box widget. When the editing is finished and the focus leaves the spin box widget, the Bin size logic group box widget will have the Resolving power radio button widget selected. The FWHM spin box widget now contains the proper FWHM value, as computed using the resolving power value and the value set to the Reference mass spin box widget.

The end point of the configuration procedures described above is to have a proper m/z bin size for the mass spectrum that is generated by the combination of the various peak shapes. The greater the resolving power of the instrument, the smaller the FWHM value and thus the smaller the bin size. The bin size that is computed is displayed in the Bin size (Th) spin box widget inside of the Manually set the bin size group box widget. If checked, this group box widget becomes active to let the user set the bin size manually. Its computation does not occur anymore upon modification of the values of FWHM or of the resolving power or of the reference mass.

The successful configuration of the bin size, via the definition of the FWHM or of the resolving power takes a bit of patience. The Check parameters push button triggers a verification of the configuration and provides feedback about it in the line edit widget below the push button.

The Charge spin box widget allows setting the charge of the ions of which the centroid (m/z,i) pairs are listed in the text edit widget on the left hand side of the dialog window.

Once the peak shaping parameters have been checked, click the Run button. The dialog window shifts tab to Results, as shown in Figure 73 – Shaped peaks mass spectral data.

Once the mass spectrum has been generated by the peak shaper, it might be copied to the clipboard, for visualization in the companion MineXpert software or any other software capable of reading space-separated-values files. It is also possible to actually trigger the automatic display of the spectrum in MineXpert by clicking Display mass spectrum. In this case, MassXpert will check if the MineXpert program is available on the system, and if so, starts it automatically and transmits via the network the mass spectral data to be plotted.

The mass spectrum corresponding to the combination of all the shaped centroid peaks is shown in Figure 74 – Isotopic cluster automatically displayed in MineXpert.

8.3 Combining multiple shaped isotopic clusters’ peaks into a single mass spectrum

It often occurs that one wants to run multiples isotopic cluster calculations in order to combine the multiple obtained shaped mass spectra into a single one. This requirement has some tricky implications. Because the formula for which the various isotopic clusters are computed is going to be different each time, the very first m/z value of the resulting mass spectrum is going to be different each time. This means that when the m/z bins are calculated, the very first bin will be rooted at a different m/z value for each one of the mass spectra obtained for the different isotopic cluster calculations. Upon combination of the various mass spectra, even if the resolving power was the same, there will be artifacts visible in Figure 75 – Combination of mass spectra obtained for different isotopic cluster calculations. This is called the “unphased bins” artifact.

One way to avoid the “unphased bins” artifact is to manage the bin calculation prior to starting the isotopic cluster centroids shaping process. This is shown in Figure 76 – Reusing the isotopic cluster shaper dialog window.

In the isotopic cluster shaper dialog window, there are some configuration steps that will ensure that all the isotopic cluster mass spectra share the same m/z bin width and the same set of bins (they are thus “phased”). The configuration steps are illustrated in Figure 77 – Ensuring that the bins are “phased” in the various isotopic cluster mass spectra.

In the figure, the user has pushed the pin on the right hand size of the Reference mass value, so that upon loading a new set of isotopic cluster centroids from the isotopic cluster generator window that value is not modified.

Another setting that is crucial is the m/z range set in the Phase the bins to the m/z range group box widget. The start and stop values descring the m/z range need to encompass the whole set of m/z values in all the mass spectra generated during the isotopic cluster generation session.

Upon editing of the second m/z range value (the range end value), the Reference mass m/z value is updated to correspond to the middle value of that range. If that value is not correct, the user may modify it right in the double spin box and that value is not going to change.

By using the settings described above, one can ensure that the combination of all the isotopic cluster mass spectra works without the artifact, as shown in Figure 78 – Avoiding the combination artifact due to having “unphased bins”.

9 Utilities

The utilities made available in the Utilities menu of the main program window are shown in Figure 79 – Utilities. These are described in detail in the following sections.

9.1 Configuration of the MassXpert and MineXpert intercommunications

The two programs of the MsXpertSuite software suite, MassXpert and MineXpert can exchange data via the network, provided that both are set up for TCP/IP-based communications. In MassXpert, the configuration of these communications is performed by using the Utilities$⇨$Client-Server configuration menu item. The configuration is performed in the dialog window shown in Figure 80 – Setting-up of the centroid mass peak shaping process.

To start the server, click the Start server button and the IP address and port number will be displayed. To start the client, insert the IP address and port number in the Client configuration group box and click Start client.

9.1.1 Dynamic configuration of the MassXpert and MineXpert intercommunications

Each time a task occurring in MassXpert requires to display data in MineXpert the former program checks if the latter program is already running and if an active connection between the two is up.

If an active connection between MassXpert and MineXpert is alive, the data are immediately dislayed. Instead, if the MineXpert program is effectively running, but there is no active connection between MassXpert and MineXpert, the user is prompted with a dialog box to suggest configuring a connection (see Figure 81 – Configuration of the dynamic communication between MassXpert and MineXpert).

Whenever MassXpert cannot find a MineXpert process, it will automatically try to start if it finds the MineXpert executable installed on the computer. In that case, if the MineXpert program can be started, the data are transferred as soon as the process and a communications connection can be set up.

10 Data Customizations

In this chapter, the user will be walked trough an example of how new polymer chemistry definition data can be generated and included in the automatic “data detection system” of MassXpert (that is how new polymer chemistry definitions should be registered with the system).

Customization is typically performed by the normal user (not the Administrator nor the Root of the machine) and as such new data are typically stored in the user’s “home” directory. On machines, the “home” directory is usually the /home/username directory, where username is the login username. On , that directory is typically the C:/Windows/users/username, once again with username being the login username.

In the next sections we will refer to that “home directory” (be it on or machines) as the $HOME directory, as this is the standard environment variable describing that directory in .

When MassXpert is executed, it automatically tries to read data configuration files. First, the user’s MassXpert configuration settings are located in the .config/MassXpert3 directory, in the configSettings.ini file. The excerpt below, for user Rusconi shows two important stanzas:

This excerpt shows that the system polymer chemistry definitions shipped with the software during install are located at /usr/share/massxpert3/data. Inside that directory two subdirectories contain the polymer chemistry definitions installed alongside the program and the polymer sequence examples, /usr/share/massxpert3/data/polChemDefs and /usr/share/massxpert3/data/polSeqs, respectively.

MassXpert loads all the available data from these two directories to make them available to the user upon terminating the program initialization.

Once this is done, MassXpert reads all the data configuration files in the installation directory (typically, on that would be the configuration data in the /usr/share/MassXpert/data directory or, on , the c:/Program Files/MassXpert/data directory).

We said above that MassXpert tries to read the data configuration files from the home directory. But upon its very first execution, right after installation, that directory does not exist, and in fact MassXpert creates that directory for us to populate it some day with interesting new data.

The $HOME/.MassXpert directory should have a structure mimicking the one that was created upon installation of the software, that is, it should contain the following two directories:

• polChemDefs
• polSeqs

Those are the directories where the user is invited to store their personal data. In order to start a new definition, one might simply copy in the polChemDefs one of the polymer chemistry definitions that are shipped with MassXpert. What should be copied? An entire polymer chemistry definition directory, like for example the following:

/usr/local/share/MassXpert/polChemDefs/protein-1-letter

or

C:/Program Files/MassXpert3/polChemDefs/protein-1-letter

Once that polymer chemistry definition is copied, one may start studying how it actually works. This directory contains the following kinds of files:

• protein-1-letter.xml: the polymer chemistry definition file. This is the file that is read upon selection of the corresponding polymer chemistry definition name in . If the polymer chemistry definition is not yet registered with the system (described later), then open that file by browsing to it by clicking Cancel (see Section 5);
• SVG files: scalar vector graphics files used to render graphically the sequence in the sequence editor. For example, arginine.svg contains the graphical representation of the arginine monomer. There are such graphics files also for the modifications (like, for example, the sulphation.svg contains the graphical representation of the sulphation modification. shows two examples of SVG files belonging to two distinct polymer chemistry definitions;
• chemPad.conf: configuration file for the chemical pad in the module;
• monomer_dictionary: file establishing the relationship between any monomer code of the polymer chemistry definition and the graphical SVG file to be used to render graphically that monomer in the sequence editor;
• modification_dictionary: file establishing the relationship between any monomer modification (see Section 6.8.1 – Selected Monomer(s) Modification) and the graphical SVG file to be used to render graphically that modification onto the modified monomer in the sequence editor;
• cross_linker_dictionary: file establishing the relationship between any cross-link (see Section 6.9 – Monomer Cross-linking) and the graphical SVG file to be used to render graphically that cross-link onto the cross-linked monomers in the sequence editor;
• pka_ph_pi.xml: file describing the acido-basic data (see Section 6.15 – pKa, pH, pI and Charges) pertaining to ionizable chemical groups in the different entities of the polymer chemistry definition;

The polymer sequence editor is not a classical editor. There is no font in this editor: when the user starts keying-in a polymer sequence in the editor, the small SVG graphics files are rendered into raster vignettes at both the proper resolution and screen size and displayed in the sequence editor. The user is totally in charge of designing the SVG graphics files for each of the monomers defined in the polymer sequence editor. Of course, reusing material is perfectly possible.

There is one constraint: that the monomer_dictionary file lists with precision “what monomer code goes with what SVG graphics file”. That file has the following contents, for example, for the “protein-1-letter” polymer chemistry definition, as shipped in the MassXpert package:

What one sees from the contents of the file is that each monomer code has an associated SVG file. For example, when the user has to key-in a valine monomer, they key-in the code and knows that the monomer vignette to show has to be rendered using the valine.svg file.

For the monomer modification graphical rendering, the situation is somewhat different, as seen in the modification_dictionary file:

There are two ways to render a chemical modification of a monomer:

• Opaque rendering: the initial monomer vignette is replaced using the one listed in the file for the modification. One example is the “AmidationGlu” modification:

  AmidationGlu%O%glutamine.svg

  Upon amidation of a glutamyl residue (“AmidationGlu” is the name of a modification in the current polymer chemistry definition), the graphical representation of the modification involves the replacement of the glutamyl residue vignette in the sequence editor with the new one, that happens to be in the glutamine.svg file. In other words, the process involves an “Opaque” overlay of the vignette for unmodified Glu with a vignette rendered by using the glutamine.svg file.

• Transparent rendering: the initial monomer vignette is overlaid with a new vignette that is read from an SVG file that has a transparent background. One example is the “Phosphorylation” modification:

  Phosphorylation%T%phospho.svg

  The monomer undergoing a phosphorylation has its vignette overlaid with a “Transparent” one showing a small red “P” that is read from the phospho.svg file.

When designing vignettes, the best thing to do is to convert the text to path, so that the rendering is absolutely perfect.

Once the new polymer chemistry has been correctly defined, it is time to register that new definition to the system. To recap: all the files for that definition should reside in a same directory, exactly the same way as the files pertaining to a given polymer chemistry definition are shipped in MassXpert altogether in one directory. The name of the new polymer chemistry definition should be unambiguous, with respect to other registered polymer chemistry definitions.

The way personal polymer chemistry definitions are registered is by creating a personal polymer chemistry definition catalogue file, which must comply with both following requirements:

• Be named xxxxx-polChemDefsCat, with “xxxxx” being a discretionary string (this might well be your login username). The requirement is that -polChemDefsCat be the last part of the filename.

• Be located in the $HOME/.MassXpert/data/polChemDefs directory and have the following format:

  dna=/path/to/definition/directory/dna/dna.xml

  In this example, the “dna” polymer chemistry definition is being registered as a file dna.xml located in the dna directory, itself located in the /path/to/definition/directory directory;

Note that if a new polymer chemistry definition should be made available system-wide, then it is logical that its directory be placed along the ones shipped with MassXpert and a new local catalogue file might be created to register the new polymer chemistry definition.

At this point the new polymer chemistry definition might be tested. Typically, that involves restarting the MassXpert program and creating a brand new polymer sequence of the new definition type. The first step is to check if the new definition is successfully registered with the system, that is, it should show up a an available definition upon creation of the new polymer sequence. If not, then that means that the catalogue file could not be found or parsed correctly.

When problems like this one occurs, the first thing to do is to ensure that the console window (on it is systematically started along with the program; on the way to have it is to start the program from the shell) so as to look with attention at the different messages that might help understanding what is failing.

Please, do not hesitate to submit bug reports ( See Feedback to the developers).

Appendix

GNU General Public License version 3

Version 3, 29 June 2007

Copyright ©︎ 2007 Free Software Foundation, Inc. https://fsf.org/

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When we speak of free software, we are referring to freedom, not price. Our General Public Licenses are designed to make sure that you have the freedom to distribute copies of free software (and charge for them if you wish), that you receive source code or can get it if you want it, that you can change the software or use pieces of it in new free programs, and that you know you can do these things.

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For example, if you distribute copies of such a program, whether gratis or for a fee, you must pass on to the recipients the same freedoms that you received. You must make sure that they, too, receive or can get the source code. And you must show them these terms so they know their rights.

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10. Automatic Licensing of Downstream Recipients.

Each time you convey a covered work, the recipient automatically receives a license from the original licensors, to run, modify and propagate that work, subject to this License. You are not responsible for enforcing compliance by third parties with this License.

An “entity transaction” is a transaction transferring control of an organization, or substantially all assets of one, or subdividing an organization, or merging organizations. If propagation of a covered work results from an entity transaction, each party to that transaction who receives a copy of the work also receives whatever licenses to the work the party’s predecessor in interest had or could give under the previous paragraph, plus a right to possession of the Corresponding Source of the work from the predecessor in interest, if the predecessor has it or can get it with reasonable efforts.

You may not impose any further restrictions on the exercise of the rights granted or affirmed under this License. For example, you may not impose a license fee, royalty, or other charge for exercise of rights granted under this License, and you may not initiate litigation (including a cross-claim or counterclaim in a lawsuit) alleging that any patent claim is infringed by making, using, selling, offering for sale, or importing the Program or any portion of it.

11. Patents.

A “contributor” is a copyright holder who authorizes use under this License of the Program or a work on which the Program is based. The work thus licensed is called the contributor’s “contributor version”.

A contributor’s “essential patent claims” are all patent claims owned or controlled by the contributor, whether already acquired or hereafter acquired, that would be infringed by some manner, permitted by this License, of making, using, or selling its contributor version, but do not include claims that would be infringed only as a consequence of further modification of the contributor version. For purposes of this definition, “control” includes the right to grant patent sublicenses in a manner consistent with the requirements of this License.

Each contributor grants you a non-exclusive, worldwide, royalty-free patent license under the contributor’s essential patent claims, to make, use, sell, offer for sale, import and otherwise run, modify and propagate the contents of its contributor version.

In the following three paragraphs, a “patent license” is any express agreement or commitment, however denominated, not to enforce a patent (such as an express permission to practice a patent or covenant not to sue for patent infringement). To “grant” such a patent license to a party means to make such an agreement or commitment not to enforce a patent against the party.

If you convey a covered work, knowingly relying on a patent license, and the Corresponding Source of the work is not available for anyone to copy, free of charge and under the terms of this License, through a publicly available network server or other readily accessible means, then you must either (1) cause the Corresponding Source to be so available, or (2) arrange to deprive yourself of the benefit of the patent license for this particular work, or (3) arrange, in a manner consistent with the requirements of this License, to extend the patent license to downstream recipients. “Knowingly relying” means you have actual knowledge that, but for the patent license, your conveying the covered work in a country, or your recipient’s use of the covered work in a country, would infringe one or more identifiable patents in that country that you have reason to believe are valid.

If, pursuant to or in connection with a single transaction or arrangement, you convey, or propagate by procuring conveyance of, a covered work, and grant a patent license to some of the parties receiving the covered work authorizing them to use, propagate, modify or convey a specific copy of the covered work, then the patent license you grant is automatically extended to all recipients of the covered work and works based on it.

A patent license is “discriminatory” if it does not include within the scope of its coverage, prohibits the exercise of, or is conditioned on the non-exercise of one or more of the rights that are specifically granted under this License. You may not convey a covered work if you are a party to an arrangement with a third party that is in the business of distributing software, under which you make payment to the third party based on the extent of your activity of conveying the work, and under which the third party grants, to any of the parties who would receive the covered work from you, a discriminatory patent license (a) in connection with copies of the covered work conveyed by you (or copies made from those copies), or (b) primarily for and in connection with specific products or compilations that contain the covered work, unless you entered into that arrangement, or that patent license was granted, prior to 28 March 2007.

Nothing in this License shall be construed as excluding or limiting any implied license or other defenses to infringement that may otherwise be available to you under applicable patent law.

12. No Surrender of Other's Freedom.

If conditions are imposed on you (whether by court order, agreement or otherwise) that contradict the conditions of this License, they do not excuse you from the conditions of this License. If you cannot convey a covered work so as to satisfy simultaneously your obligations under this License and any other pertinent obligations, then as a consequence you may not convey it at all. For example, if you agree to terms that obligate you to collect a royalty for further conveying from those to whom you convey the Program, the only way you could satisfy both those terms and this License would be to refrain entirely from conveying the Program.

13. Use with the #acronym(GNU) Affero General Public License.

Notwithstanding any other provision of this License, you have permission to link or combine any covered work with a work licensed under version 3 of the GNU Affero General Public License into a single combined work, and to convey the resulting work. The terms of this License will continue to apply to the part which is the covered work, but the special requirements of the GNU Affero General Public License, section 13, concerning interaction through a network will apply to the combination as such.

14. Revised Versions of this License.

The Free Software Foundation may publish revised and/or new versions of the GNU General Public License from time to time. Such new versions will be similar in spirit to the present version, but may differ in detail to address new problems or concerns.

Each version is given a distinguishing version number. If the Program specifies that a certain numbered version of the GNU General Public License “or any later version” applies to it, you have the option of following the terms and conditions either of that numbered version or of any later version published by the Free Software Foundation. If the Program does not specify a version number of the GNU General Public License, you may choose any version ever published by the Free Software Foundation.

If the Program specifies that a proxy can decide which future versions of the GNU General Public License can be used, that proxy’s public statement of acceptance of a version permanently authorizes you to choose that version for the Program.

Later license versions may give you additional or different permissions. However, no additional obligations are imposed on any author or copyright holder as a result of your choosing to follow a later version.

15. Disclaimer of Warranty.

THERE IS NO WARRANTY FOR THE PROGRAM, TO THE EXTENT PERMITTED BY APPLICABLE LAW. EXCEPT WHEN OTHERWISE STATED IN WRITING THE COPYRIGHT HOLDERS AND/OR OTHER PARTIES PROVIDE THE PROGRAM “AS IS” WITHOUT WARRANTY OF ANY KIND, EITHER EXPRESSED OR IMPLIED, INCLUDING, BUT NOT LIMITED TO, THE IMPLIED WARRANTIES OF MERCHANTABILITY AND FITNESS FOR A PARTICULAR PURPOSE. THE ENTIRE RISK AS TO THE QUALITY AND PERFORMANCE OF THE PROGRAM IS WITH YOU. SHOULD THE PROGRAM PROVE DEFECTIVE, YOU ASSUME THE COST OF ALL NECESSARY SERVICING, REPAIR OR CORRECTION.

16. Limitation of Liability.

IN NO EVENT UNLESS REQUIRED BY APPLICABLE LAW OR AGREED TO IN WRITING WILL ANY COPYRIGHT HOLDER, OR ANY OTHER PARTY WHO MODIFIES AND/OR CONVEYS THE PROGRAM AS PERMITTED ABOVE, BE LIABLE TO YOU FOR DAMAGES, INCLUDING ANY GENERAL, SPECIAL, INCIDENTAL OR CONSEQUENTIAL DAMAGES ARISING OUT OF THE USE OR INABILITY TO USE THE PROGRAM (INCLUDING BUT NOT LIMITED TO LOSS OF DATA OR DATA BEING RENDERED INACCURATE OR LOSSES SUSTAINED BY YOU OR THIRD PARTIES OR A FAILURE OF THE PROGRAM TO OPERATE WITH ANY OTHER PROGRAMS), EVEN IF SUCH HOLDER OR OTHER PARTY HAS BEEN ADVISED OF THE POSSIBILITY OF SUCH DAMAGES.

17. Interpretation of Sections 15 and 16.

If the disclaimer of warranty and limitation of liability provided above cannot be given local legal effect according to their terms, reviewing courts shall apply local law that most closely approximates an absolute waiver of all civil liability in connection with the Program, unless a warranty or assumption of liability accompanies a copy of the Program in return for a fee.

END OF TERMS AND CONDITIONS

How to Apply These Terms to Your New Programs

If you develop a new program, and you want it to be of the greatest possible use to the public, the best way to achieve this is to make it free software which everyone can redistribute and change under these terms.

To do so, attach the following notices to the program. It is safest to attach them to the start of each source file to most effectively state the exclusion of warranty; and each file should have at least the “copyright” line and a pointer to where the full notice is found.

Also add information on how to contact you by electronic and paper mail.

If the program does terminal interaction, make it output a short notice like this when it starts in an interactive mode:

The hypothetical commands “show w” and “show c” should show the appropriate parts of the General Public License. Of course, your program’s commands might be different; for a GUI interface, you would use an “about box”.

You should also get your employer (if you work as a programmer) or school, if any, to sign a “copyright disclaimer” for the program, if necessary. For more information on this, and how to apply and follow the GNU GPL, see https://www.gnu.org/licenses/.

The GNU General Public License does not permit incorporating your program into proprietary programs. If your program is a subroutine library, you may consider it more useful to permit linking proprietary applications with the library. If this is what you want to do, use the GNU Lesser General Public License instead of this License. But first, please read https://www.gnu.org/licenses/why-not-lgpl.html.

Colophon

About the authors

Filippo Rusconi is a senior research scientist at the French national research council (Centre national de la Recherche scientifique, CNRS). Filippo has a background in biochemistry and organic chemistry and was trained during his Ph.D. as a bioanalytical chemist. He has extensive knowledge of analytical techniques involved in the study of biopolymers.

Filippo Rusconi is the author of a handbook about mass spectrometry for biochemists (French). The book was published by the French sci/tech publisher Lavoisier Tech&Doc.

Book preparation

The look of this book (PDF file) is the result of me having read many books from the O’Reilly publisher.

The typesetting of the book has been done on a Debian GNU/Linux computer using only Free Software. The application was used for rendering the document code.

The main font used is EBGaramond and the symbol/mathematical font was from the STIX project (font: STIX2Math).

The screen shots were taken with Spectacle, the screen capture program shipped along with my KDE desktop environment and resampled either with (the GNU image manipulation program) or . Illustrations were done in Inkscape, a vectorial drawing software.

Index