# Fundamentals in Bottom-up Proteomics

This chapter is an optional chapter which the reader might be referred to upon reading other part of this manual.

## Protein Liquid and Gas Phase Chemistries

This section introduces the basics of protein polymer chemistry. The way this topic is going to be covered is admittedly biased towards mass spectrometry and proteins. 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 &xtpcpp; program. This manual is not a crash course in biochemistry.

The 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 the 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 another 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.

### Protein Biosynthesis

Proteins 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). fig_xtpcpp-peptide-bond-formation shows that the chemical reaction at the basis of protein synthesis is a condensation. A protein is the result of the condensation of amino acids with each other in an orderly polar fashion. A protein has a left end, called N-terminus; amino-terminal end and a right end, called C-terminus; carboxy-terminal end. The left end is an amino group (2HN–) corresponding to the non-reacted amino group of the very first amino acid of the protein sequence. Upon condensation of a new entering amino acid onto the first N-terminal one, the amino group of the entering amino acid reacts (nucleophilic attack) with the carboxyl group of the N-terminal amino acid. A water molecule is released, and the formation of an amide bond between the two amino acids yields a dipeptide. The right end of the dipeptide is a carboxyl group (–COOH) corresponding to the un-reacted carboxyl group of the last amino acid to have been polymerized in.

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 fig_xtpcpp-peptide-bond-formation, granted that the elongation always proceeds in the described direction (a new monomer arrives to the right end of the elongating polymer, and elongation is done from left to right).

#### Peptidic Bond Formation by Condensation.

The left end monomer R1 is condensed to the right end monomer R2 to yield a peptidic bond. A water molecule is lost during the process.

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 an amino acid is polymerized into a protein, it is no more called an amino acid , 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: —A protein is a chain of residues linked together in an orderly polar fashion, with the residues being numbered starting from 1 and ending at n, from the first residue on the left end to the last one on the right end. This definition is still partly inexact, however. Indeed, from what is shown in fig_xtpcpp-prot-polymer, there is still a problem with the extremities of the residual chain: what about the amino group on the left end of a protein (the amino group sits right onto the first amino acid of the protein), and what about the carboxyl group of the right end of a protein (the carboxyl group sits right onto the last amino acid of the protein)? Because these groups lie at the extremities of the residual chain, they remained unreacted during the polymerization process. But because we are simulating a residual chain using residues and not amino-acids, we still need to put the protein polymer molecule in its finished state: by capping the left end with a proton cap (so as to complete the amino group) and the right end with a hydroxyl cap (so as to complete the carboxyl group). The capping of the residual chain extremities ensures that the polymer is in its finished state, and that it cannot be elongated anymore. The proton is the left cap of the protein polymer and the hydroxyl is the right cap of the protein polymer.

#### End Capping Chemistry of the Protein Polymer

A protein is made of a chain of residues and of two caps. The left cap is the N-terminal proton and the right cap is the C-terminal hydroxyl. Altogether, the residual chain (enclosed here in the blue polygon) and both the H and OH red-colored caps do form a complete protein polymer in its finished state.

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, its sequence). Of course, proteins have three-dimensional structures, but this is of no interest to a program like &massXp;, 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)

### Protein Disrupting Chemistries

The polymer chain disrupting chemistry was mentioned earlier as a complex subject that was of enormous importance to the mass spectrometrist. 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; cleavages and fragmentations:

### Cleavages

These are chemical processes by which a cleaving agent will act directly on the protein residual chain making it fall into at least two separated pieces (the peptides).

### Fragmentations

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

#### Protein Cleavage

Upon cleavage of a protein, the cleaving molecule reacts with it, and by doing so directly or indirectly dissolves an inter-residue bond. A protein cleavage always occurs in such a way as to generate a set of true finished polymerization state proteins (smaller in size than the parent polymer, evidently, which is why they are called oligopeptides, or peptides). Indeed, let us take the example shown in fig_prot-cleavage, where a tripeptide (a very little protein, containing a methionyl residue at position 2) is submitted either to a water-mediated cleavage (hydrolysis, upper panel) or to a cyanogen bromide-mediated cleavage (lower panel). The two cases presented in this figure are similar in some respects and different in others:

- 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 homoseryl residue at the C-terminus of Oligomer 1, while hydrolysis generates a genuine methionyl residue. 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.

##### Protein cleavage by water and cyanogen bromide

A tripeptide is cleaved at position 1 either by hydrolysis (top) or by cyanogen bromide (bottom). Cyanogen bromide cleaves specifically on the right of a methionine monomer. Upon cleavage, the methionyl monomer gets converted into homoserine by the cyanogen bromide reagent

Nonetheless, the reader might have noted that—interestingly—all the four oligomers do effectively have their left cap (the proton, making the N-terminal amino group) and their right cap (the hydroxyl, making the C-terminal carboxyl group). 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 residues (modified or not) and 2) they are correctly capped (i.e. they are polymers in their finished polymerization 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, our definition of a peptide might be: a peptide is a protein (of at least one residue) in its finished polymerization state that was generated upon cleavage of a longer protein. Of course, when we use the term protein, above, we mean protein polymer, irrespective of its size.

When the protein cleavage reaction precisely reverses the reaction that was performed for the same protein's biosynthesis, there is no special difficulty. But when the cleavage reaction modifies the substrate, then this should be carefully taken into account when using &xtpcpp;. This is true for any chemical modification that happens onto a protein.

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 should one 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.

#### Protein Fragmentation

In a fragmentation process, the bond that is broken does not necessarily yield smaller-sized proteins because fragmentation does not necessarily break the inter-residue bond the same way that the hydrolysis does. Indeed, fragmentations are oft-times high energy chemical processes that can affect peptidic bonds at different locations, not necessarily between the CO-NH bond of the peptidic bond. This is one of the reasons why fragmentations do differ from cleavages.

Another peculiarity of fragmentations, compared with cleavages, is the fact that there is no cleaving molecule starting the process, like water or cyanogen bromide, for example. Indeed, in the gas phase, the peptidic ions are isolated: that is, very far one from each other. 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 obtained 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.

For the sake of completeness of this section, it must be noted that it is possible to have other chemical/physical entities intervene during the gas phase fragmentation process by enacting a chemical reaction, be these entities ions, electrons or photons. In bottom-up proteomics, the intervening molecules are gas molecules (nitrogen, most often, or helium) that act as physical entities imposing collisions to the peptidic ions with the effect that the ions acquire internal energy, eventually leading to dissociation (CID, for collisionally-activated dissociation).

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.

##### Protein fragmentation patterns most widely encountered.

An hexapeptide is fragmented in the seven most widely encountered manners, such as to generate a, b, c, x, y, z and immonium fragment ions. The figure illustrates the position of the cleavage for each kind of fragment (exemplified using the case of the smallest fragment possible) and the mass calculation method is described for each fragment kind; consider that each fragment bears only one positive charge.

As can be seen from fig_prot-fragmentation, the fragmentations do generate fragments of three categories: the ones that include the left end of the precursor polymer (a, b, c), the ones that include the right end of the precursor polymer (x, y, z), and finally the special case in which the fragment is an internal fragment, like the immonium ions. When looking at the fragmentations described in the figure, it becomes immediately clear why a fragmentation cannot be mistaken for a cleavage: the ionization of the fragment is not necessarily due to the captation of a proton by the fragment. Furthermore, we can also see that a fragmentation is not a cleavage because the fragment that is generated is absolutely not necessarily what we call a polymer, in the sense that the fragment might not be capped the same way as the precursor protein/peptide is (that is, the fragment is not in its finished polymerizaton state).

By looking at fig_prot-fragmentation, the reader should have noticed that the fragment naming scheme takes into consideration the fact that the fragment bears the left or the right end of the precursor polymer (or none, also). Indeed, the numbering of fragments holding the left end of the precursor polymer sequence begins at the left end, and for fragments that hold the right end, at the right end. Thus the third fragment of series a (a3) would involve monomers [1→] and the third fragment of series y (y3) would involve monomers [6→] (in the figure, these left-to-right and right-to-left directions are symbolized using lines).

#### a fragment series

If we take the a fragment series, the fig_prot-fragmentation indicates that the fragments include the left end and that their last monomer lacks its carbonyl group (see, on top of fig_prot-fragmentation, that the a1 arrow goes between the CαH and the CO of monomer 1). So we would say that each fragment of the a series should be challenged with the following chemical treatments: 1) addition of the mass corresponding to the left cap (proton), 2) removal of the mass corresponding to the lacking CO group. This way we have the mass of fragment a1. If we were interested in the fragment a4 we would have summed the masses of monomers 1 to 4, added the mass of the left cap, and finally removed the mass of a CO. The mass calculation is thus mathematically expressed a i = LC + ∑ i=1 i M i - CO

#### b fragment series

Similarly, the mass calculation is mathematically expressed b i = LC + ∑ i=1 i M i

#### c fragment series

The mass calculation is mathematically expressed c i = LC + ∑ i=1 i M i + NH 3

#### x fragment series

For this 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. The mass calculation is mathematically expressed x i = RC + ∑ i=1 i M i - CO

#### y fragment series

The calculation is mathematically expressed y i = RC + ∑ i=1 i M i + H 2

#### z fragment series

In low energy CID, the z fragments are expressed this way: z i = RC + ∑ i=1 i M i - NH

#### z fragment series

In higher energy CID, the z fragments are expressed this way (one more proton is often measured): z i = RC + ∑ i=1 i M i - NH + H

#### immonium fragment series

These fragments are internal fragments in the sense that they do not hold neither of the two precursor polymer's ends. &massXp; 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 sect_protein-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. The mass calculation for these special internal fragments is expressed imm i = M i - CO + H

## General overview of bottom-up proteomics

Bottom-up proteomics is a field of endeavour where the ultimate aim is to identify the greatest number of proteins in a given sample. This aim might also, depending on the project at hand, be doubled with another aim: characterize at the finest level possible the nature and the position of post-translational/chemical modifications beared by the proteins.

To achieve the best results, proteomics has developed over the years a set of methods and techniques that, taken together, have allowed scientists to obtain impressive results of protein identification on pretty complex samples. These are listed below:

- Mass spectrometers: The development of mass spectrometers of ever-greater resolution power has allowed to attain at ever-lower false discovery rates over the years. In particular, the development of the Orbitrap analyzers, along with the huge improvements of the time-of-flight (TOF) mass analyzer technology, have strongly increased the identification results reliability by allowing the downstream data processing step to be more stringent in the protein identification task (see below);

- Chromatography: The development of highly resolutive chromatography resins along with the elaboration of hardware (columns, chromatography setups) that yields sensitivity improvements has its part in the way proteomics has evolved over the years;

- Bioinformatics: The development and refinement of software that can cope with extremely large data sets (think metaproteomics) is one major field that enabled significant advances in proteomics. Also, refinement of algorithms related to the simulation of isotopic clusters and comparison with experimental data have had their part. Likewise so for algorithms that detect the charge of ions based on the analysis of the isotopic cluster peaks. Being able to single out without error the monoisotopic peak of an isotopic cluster (whater the ion charge or m/z ratio) is a big part of the challenges at the root of successful proteomics data processing.

In this section, we will review the bioinformatics-based mass spectrometry data processing, as it is the core subject of this user manual. In particular, we will provide an outline of how the major software packages on the market perform protein identification on the basis of mass spectrometric analyses of biological samples.

### From the sample to the protein identifies

This section will outline in very rough terms how bottom-up proteomics works from the protein sample to the protein identification list.

#### The first step: digestion of the sample's proteins

The very first step in the bottom-up protemics workflow is to digest all the proteins in the initial biological sample with a site-specific endoprotease: typically tryspin.

The sample is subjected to proteolysis with all its proteins unresolved. This produces a highly complex mixture of peptides, each having a constant characteristic: each peptide has one predictable end (unless it is either the protein's N-terminal or the C-terminal peptide, as detailed below), either N-terminal or C-terminal:

- Predictable N-terminus: when the protease cuts at the N-terminal end of the target residue. For example, EndAspN cleaves left of Asp residues, thus producing peptides that always have Asp as their N-terminal residue. The only exception is when the peptide is the protein's N-terminal peptide and first residue is not Asp);

- Predictable C-terminus: when the protease cuts at the C-terminal end of the target residue. For example, the most used enzyme, trypsin, cuts right of the basic residues Lys and Arg. The generated peptides thus necessarily end with one of these two residues. The only exception is when the peptide is the protein's C-terminal peptide and last residue is not Lys nor Arg.

  One interesting feature of trypsinolyis is that it generates peptides that&emdash;for their major part&emdash;will most probably be protonated twice: on their N-terminal end (the primary NH2 amine group

  If not either converted to an amide group by acetylation or formylation or cyclised.

  and on the basic residual chain of the basic residue found at their C-terminal position (the &lnepsilon;-amine group for Lys and the guanidium group for Arg). Upon fragmentation of the peptide's precursor ion, both the left hand side fragment and the right hand side fragment will bear a proton and will thus be detected, thus potentially providing a better coverage of the peptide's sequence during the MS/MS experiment.

#### Chromatographic Separation of the Peptidic Mixture

One major analytic step in bottom-up proteomics is the separation of the peptides obtained by endoproteolysis of all the proteins in the sample. Indeed, analyzing all the peptides in one single injection without prior chromatographic resolution would yield catastrophic results similar to having injected nothing in the mass spectrometer.

The typical method for resolving peptides is by separating them on a chromatographic column functionalized with a hydrophobic group (for peptides, that would be a C18 reversed phase column).

The chromatographic gradient that will elute the peptides progressively according to their increasing hydrophobicity will be developed over the 5&endash;95 % of acetonitrile (a non-protic organic solvent).

Using acetonitrile as the non-protic organic solvent has the huge benefit of not injecting protons inside the mass spectrometer as the chromatographic gradient develops.

The effluent of the chromatographic column is directly injected into the mass spectrometer's source. The role of the mass spectrometer's source device is to ensure that the analytes are desolvated and ionized upon their entering in the core part of the mass spectrometer. Most often, that source is an electrospray source that is fed a liquid (typically, the effluent from the column) and that evaporates the solvent and&emdash;having an electric potential applied to it&emdash; ionizes the eluted analytes. The electrically charged analytes in the gas phase are thus ions the &mz; (mass-to-charge) ratio of which can be measured by the mass spectrometer analyzer.

There are two main sources used in the mass spectrometry for biology specialty: the matrix-assisted laser desorption ionization (MALDI) source and the electrospray ionization (ESI) source. One important difference between the two is that the MALDI process mostly produces mono-charged ions (&mh;, while the ESI process mostly produces multi-charged ions (&mnh;). This has huge implications in the mass data analysis.

The source that is mainly used in bottom-up proteomics is the ESI source.

#### Mass spectrometric analysis of the peptides

Upon elution off the chromatographic column, the peptides are desolvated, ionized and drawn into the mass spectrometer using an electrical field. Once they have entered the mass spectrometer they are analyzed in the mass analyzer of the instrument.

There are a variety of mass analyzers commonly used in bottom-up proteomics. In fact, one single instrument might have as many as 4 or 5 mass analyzers. However, not all the analyzers in the instrument are responsible for the &mz; measurement.

Sometimes, during the whole cycle of the analysis, two different mass analyzers are used at different steps of the cycle. This will be described later when the gas fragmentation of the peptides is explained.

In bottom-up proteomics, two different kinds of mass spectrometric data are required&emdash;ideally, for each peptide eluted from the column&emdash; in order to effectively identify the proteins in the initial sample:

- The mass-to-charge ratio (&mz;) of the peptide ion;

- The &mz; values of the fragments (the product ions) of the peptidic precursor ion that has undergone and MS/MS gas phase fragmentation

  Most often, that fragmentation step is performed using collisionally-activated dissociation (CID). In this process, the peptidic precursor ion is first isolated in the gas phase on the basis of its &mz; value and then is accelerated against a gas fog inside of the collision cell of the instrument. The ion hits gas molecules multiple times, acquires a lot of energy and finally breaks.

  .

These two kinds of data are necessary because the protein identification process is based on searches in protein databases using the precursion ions' &mz; value and the &mz; values of that ion's fragment when it is fragmented. The way the protein databases are used in order to be the substrated of these searches is described in the next section.

#### The protein databases and their use

The previous section ended on the idea that the protein identification process, that is based on the analysis of all the peptides of a peptidic mixture resulting from the endoproteolysis of a sample containing many proteins, requires searches into protein databases.

A bottom-up proteomics experiment typically needs at least one protein database: a database listing all the known proteins of the organism from which the initial sample of proteins was prepared. That organism might be a bacterium, a Eucaryote like a fungus, a protist, a mammalian&ellipsis; Optional databases might be used, like contaminant protein databases, for example.

The protein databases have to store the protein data in files having the following FASTA format:

              >GRMZM2G009506_P01 NP_001149383 serine/threonine-protein kinase receptor
              MEEQHMAGPPYRYRLQHRRLMDIAPASASDDDSGHHGSNGMAIMVSILVVVIVCTLFYCV
              YCWRWRKRNAVRRAQIERLRPMSSSDLPLMDLSSIHEATNSFSKENKLGEGGFGPVYRGV
              MGGGAEIAVKRLSARSRQGAAEFRNEVELIAKLQHRNLVRLLGCCVERDEKMLVYEYLPN
              RSLDSFLFDSRKSGQLDWKTRQSIVLGIARGMLYLHEDSCLKVIHRDLKASNVLLDNRMN
              PKISDFGMAKIFEEEGNEPNTGPVVGTYGYMAPEYAMEGVFSVKSDVFSFGVLVLEILSG
              QRNGSMYLQEHQHTLIQDAWKLWNEDRAAEFMDAALAGSYPRDEAWRCFHVGLLCVQESP
              DLRPTMSSVVLMLISDQTAQQMPAPAQPPLFASSRLGRKASASDLSLAMKTETTKTQSVN
              EVSISMMEPRFWADPGTSNGAATSHPATGACKKRGGQGGDRNVKDGLAARTPTHQPVARW
              HHDRRIVD

This format is really simple, because it only contains three information pieces in as many stanzas as there are proteins in the database:

- The protein's accession id in the database (`GRMZM2G009506_P01`) that comes right after the '>' prompt that signals a new protein stanza in the file;

- The protein description (`NP_001149383 serine/threonine-protein kinase receptor`) that provides some functional data bits for the protein at hand;

- The protein sequence (the rest of the stanza above).

The first (id) and second (description) information bits are used in various places in the &xtpcpp; program.

The protein databases are used by the protein identification software as the very first step in a bottom-up proteomics data analysis process: the proteins in the database are digested in silico in order to produce the following data bits in memory (for each protein):

- Digestion using a site-specific protease. This step produces a list of peptides for each protein. For each of these peptides: the following data bits are generated:

  - sequence: This peptide's sequence;

  - &mz; value: This peptide's &mz; value, often computed for the mono-protonated (&mh;) ion;

  - MS/MS spectrum: This peptide's fragmentation spectrum is nothing but an array of &mz; values corresponding to the set of calculated fragments (b and y ion series). These product ions &mz; values are crucial for the database search algorithm;

#### Matching fragmentation spectra with theoretical spectra

The mass spectrometric data acquisition yields a huge set of the following pairs:

- The &mz; value of the peptidic precursor ion;

- The list of peptidic fragments (product ions' &mz;).

The protein database is processed to have exactly the same set of data, but in theory: for each protein in the database, it is digested in silico into a list of peptides. Each peptide (with its &mz; value) is fragmented and the list of fragments is associated to that &mz; value.

The peptide vs spectrum matches are performed by looking&emdash;for each acquired MS/MS spectrum&emdash;into that huge list of theoretical ions which ions match the ones that were effectively measured. When matches are found and that these matches respect some constraints, the software reports the peptide sequence, and the name of the protein that it comes from, associated to the precursor ion's &mz; value.