DE202004014858U1 - Apparatus for mass spectrometry - Google Patents

Abstract

Device for identifying a modified protein with:
Means for digesting a modified protein to produce a number of peptides;
Means for ionizing at least one of the number of peptides to form one or more peptide ions;
Means for mass analyzing one or more peptidones to determine the mass or mass-to-charge ratio of at least one of the peptides or peptide ions; and
Means for identifying the modified protein by:
(i) establishing the theoretical unmodified mass or theoretical unmodified mass-to-charge ratio that would have a peptide or peptide ion, the protein from which the peptide or peptide ion is derived would not have been modified;
(ii) searching a database of peptides using the theoretical unmodified mass or the theoretical unmodified mass-to-charge ratio; and
(iii) Determination of peptides in the database having a mass or mass-to-charge ratio equal to the theoretical unmodified mass or theoretical unmodified mass.

Description

The The present invention relates to a device for identification a modified protein and a mass spectrometer with means to identify a modified protein.

The genetic information stored in the DNA defines the sequence of amino acids in polypeptides (proteins). The genetic code can be understood are defined as the set of rules by which DNA nucleotide base pair sequences translated or translated into a corresponding sequence of amino acids become. A codeword (codon) for an amino acid consists of a sequence of three nucleotide base pairs. The sequence of base pairs in DNA becomes first on information transferring Messenger RNA molecules (mRNA) in a transcription method. These then serve as a template for the Synthesis of polypeptides in translation mentioned method. Translation takes place in ribosomes in the Cytoplasm. The polypeptide chains thus formed leave the ribosome in an "unmodified" form, however, the polypeptide chains can then modified in different ways. Some, like those that are the amino acid Cysteine may contain modify themselves by folding into a particular shape, so that they can form disulfide bonds between the cysteines. This determines the shape of the protein, and often it is the shape of the protein Protein that determines its function. Others may be due to chemical derivatization modified by specific functional groups of specific amino acids become. For example, the functional group on the amino acid tyrosine be phosphorylated. This changed the polarity and the shape of the polypeptide and may enable it to be specific Perform functions, or prevent it from performing certain functions. The above mentioned are examples of post-translational modifications, which occur after the polypeptide has been synthesized and when the polypeptide freely mixes with other polypeptides or chemicals. There are many forms of post-translational modifications, almost all of them have some form of chemical derivatization include functional group. These include the methylation, Hydroxylation, oxidation, formylation, acetylation, carboxylation, Phosphorylation, sulphation, cysteinylation, glycosalation, farnesylation, Myristoylation, biotinylation, palmitoylation and stearoylation. Some of these modifications are more common than others.

The Ability, To identify proteins (commonly referred to as "protein characterization") in the field of life sciences or life sciences of particular Importance. Protein identification can be the recognition of a protein by comparing experimental data with data stored in a protein database to be held. Alternatively, the protein identification the ab-initio-working out the sequence of amino acids that to form a protein.

The Ability, To identify or characterize proteins that are post-translational have been modified is of particular interest. Mass spectrometry becomes common as a means of identifying proteins and post-translationally modified Used proteins.

It is known, experimental mass spectral data by mass analysis of peptide samples, with theoretical peptide data, that of protein sequence databases or expressed sequence tag databases (EST) were generated. Theoretical peptide data can be analyzed by Prediction of peptides that would be formed when proteins expressed in the protein database listed are to be digested become. Digest rules can to one or more proteins listed in the protein database are applied, according to which the mass-to-charge ratio of resulting peptide products can be determined.

In addition to Comparison of experimental mass spectral data with theoretical Peptide data it is also known fragmentation data (or MS / MS data) to use peptides and the protein from which these are derived or derived. Peptide ions become, though high-energy collisions in a collision or fragmentation cell a mass spectrometer, in a number of Fragment ions fragment, which are then mass analyzed can. The process by which peptide ions fragment can also be proceeding of peptides that are theoretically expected from the digestion of a protein listed in the protein database become. Theoretical fragmentation data obtained by prediction of the possible Fragmentation of peptides can then be obtained experimentally obtained fragmentation mass spectral data.

The Using fragmentation or MS / MS data is particularly effective or powerful in protein characterization (and especially for proteins, posttranslationally modified) because of fragmentation a peptide ion formed fragment ions from both the position (Location) as well as the nature of any post-translational modifications, which affects the protein have, depend becomes.

Early techniques to compare experimental mass spectral data with theoretical Peptide data included the direct comparison of experimental Mass spectral data with peptide data predicted in an indexed Be held peptide database. This approach was suitable for identification unmodified proteins, but did not allow identification of post-translationally modified proteins, as the peptide database Proteins that were post-translationally modified were unsupported.

Of the increasing need for support from Protein characterization and especially post-translationally modified Proteins meant that a different approach had to be taken to post-translationally to identify modified proteins. It has not been so far considered feasible, peptide data that digested from a derived posttranslational modified protein, with respect to conventional to search indexed predicted peptide databases to investigate the details of each unmodified peptide, the stored in the peptide database should be supplemented by numerous additional Entries Re of all possible Combinations of modifications and the different permutations the position of such modifications. A peptide database, the all potential Modifications and permutations of modifiable positions included, would be like that large, that their use would be impractical, since search times for searching such a database would be very slow or long.

In Considering the impracticality of trying to use a peptide database containing entries for all possible ones Modifications of a protein involves the conventional Approach to identifying post-translationally modified proteins in it, rather with a conventional protein database (as opposed to a predigested peptide database). Proteins listed in the protein database will become theoretical digested on the fly, d. H. the proteins become theoretical Digests every time experimental peptide data for analysis be entered.

According to the conventional Approach will be any specific modifications that a user in consideration want to pull, then completely or partially applied to the peptide products resulting from the theoretical Digestion or digestion of the protein result. The resulting theoretically modified peptide data can then match the actual experimental mass spectral data are compared. The conventional one Approach therefore allows searches to support any number potential (i.e., variable or random) modifications. one the disadvantages of the conventional Approach, however, is that it takes a relatively long time for proteins, which are listed in a protein database to theoretically digest. As well as the theoretical digestion of proteins in a protein database are listed, running or flying, each must thus produced peptide all Combinations or modifications that a user might consider wants to pull, to be analyzed. Furthermore, there are potentially several different ones Permutations of modifiers that need to be considered. For example must, if a peptide has different modifiable positions and a single one Modification is under consideration Any modifiable position is considered in turn are used in the generation of the theoretical peptide data and in the Looking for the best match or the best fit (combination and permutation) with respect to the experimental data. As a result, the conventional approach is relatively slow especially when considering a number of modifiers shall be.

It Therefore, the aim is a faster identification process or characterization of a protein that post-translationally modified is available to deliver.

• Mitteln zur Digestierung eines modifizierten Proteins zur Herstellung einer Anzahl von Peptiden; Mitteln zum Ionisieren wenigstens eines der Anzahl von Peptiden zur Bildung eines oder mehrerer Peptidionen; Mitteln zum Massenanalysieren eines oder mehrerer Peptidionen zur Bestimmung der Masse oder des Masse-Ladungs-Verhältnisses wenigstens eines der Peptide oder der Peptidionen; und Mitteln zum Identifizieren des modifizierten Proteins durch: (i) Bestimmen der theoretischen unmodifizierten Masse oder des theoretischen unmodifizierten Masse-Ladungs-Verhältnisses, die bzw. das ein Peptid oder ein Peptidion haben würde, wäre das Protein, von dem das Peptid oder Peptidion abgeleitet ist, nicht modifiziert worden; (ii) Durchsuchen einer Datenbank von Peptiden unter Verwendung der theoretischen unmodifizierten Masse oder des theoretischen unmodifizierten Masse-Ladungs-Verhältnisses; und (iii) Bestimmung von Peptiden in der Datenbank mit einer Masse oder einem Masse-Ladungs-Verhältinis, die bzw. das der theoretischen unmodifizierten Masse oder dem theoretischen unmodifizierten Masse-Ladungs-Verhältnis entspricht.

According to the present invention, a device for identifying a modified protein is provided with.

• Means for digesting a modified protein to produce a number of peptides; Means for ionizing at least one of the number of peptides to form one or more peptide ions; Means for mass analyzing one or more peptide ions to determine the mass or mass-to-charge ratio of at least one of the peptides or the peptide ions; and means for identifying the modified protein by: (i) determining the theoretical unmodified mass or theoretical unmodified mass-to-charge ratio that a peptide or peptide ion would have would be the protein from which the peptide or peptide ion is derived is, has not been modified; (ii) searching a database of peptides using the theoretical unmodified mass or the theoretical unmodified mass-to-charge ratio; and (iii) determining, in the database, peptides having a mass or mass-to-charge ratio that corresponds to the theoretical unmodified mass or theoretical unmodified mass-to-charge ratio.

The modified protein was preferably post-translationally modified, for example by methylation or methylation, hydroxylation or Hydroxylation, oxidation or oxidation, formylation or formylation, rcetylation or acetylation, carboxylation or carboxylation, phosphorylation or phosphorylation, sulphating or sulphation, cysteinylation or cysteinylation, glycosalation or glycosalation, farnesylation or farnesylation, myristoylation or Myristoylation, biotinylation or biotinylation, palmitoylation or palmitoylation or stearoylation or stearoylation.

A or several modifications of interest are preferred determined, selected or otherwise considered drawn. The modified protein is preferably determined or considering drawn as by means of at least one modification of interest modified. At least one or more (preferably all) Combinations of modifications of interest are preferred enummeriert. A maximum number of modifications is preferred determined, selected or considering drawn. Preferably comprises the step of enumerating through combinations of modifications of interest the enumeration by combinations of modifications of Interest up to a maximum number of modifications.

Of the Step of determining the theoretical unmodified mass or of the theoretical unmodified mass-to-charge ratio preferably comprises the calculation or determination of mass or mass-to-charge ratio of a Combination of modifications of interest. The theoretical unmodified Mass or theoretical unmodified mass-to-charge ratio preferably obtained by change, Inversion, counteraction, negation, unmodification or de-modification or adjustment of mass or mass-to-charge ratio an experimentally observed peptide or peptide ion in consideration a combination of modifications of interest. Especially may this be the subtraction (or less preferably the addition) the mass or mass-to-charge ratio of the combination of Modifications of interest from (to) the mass or mass-to-charge ratio of the Peptides or peptide ions as determined by the mass analysis step was received.

The Device preferably comprises means for determining, selecting or Contemplate a permissible mass or mass-to-charge ratio tolerance between the theoretical unmodified mass or the theoretical unmodified Mass to charge ratio of the peptide or peptide ion and the mass or mass-to-charge ratio of Peptides listed in the database. The mass tolerance can be within an area selected from the group which consists of: (i) <0.01 There; (ii) 0.01-0.02 There; (iii) 0.02-0.03 There; (iv) 0.03-0.04 There; (v) 0.04 - 0.05 There; (vi) 0.05-0.06 There; (vii) 0.06-0.07 There; (viii) 0.07-0.08 There; (ix) 0.08-0.09 Da; (x) 0.09 - 0.10 Da; and (xi)> 0.10 Da.

The Mass-to-charge ratio tolerance can fall into an area selected from the group which consists of: (i) <0.1 Mass to charge ratio units; (ii) <0.01 mass-to-charge ratio units; (iii) <0.001 mass-to-charge ratio units; (iv) <0.0001 mass-to-charge ratio units; (v) <0.00001 mass-to-charge ratio units; (vi) <0.000001 Mass to charge ratio units.

Of the A step of searching a database of peptides preferably comprises the determination or selection of peptides in the database are listed which have a mass or mass-to-charge ratio, that of the theoretical unmodified mass or the theoretical one unmodified mass-to-charge ratio corresponds within the mass or mass-to-charge ratio tolerance.

The Device includes further preferably means for determining a peptide in the database of peptides that use CTCSS code. This one can a certain selection of the radio signals that are on the currently set Radio channel are received, achieve, or avoid, for example one to be monitored Person (baby) is heard from the neighborhood.

Of course they are instead of the selection described above or in addition to this other codings possible.

According to one particularly preferred embodiment comprises the inventively designed monitoring device as a base two conventional ones PMR or LPD handheld radios. PMR means Private Mobile Radio, 500 mW transmission power, 8 channels at 446 MHz, range up to 5 km. LPD means Low Power Device, 10 mW, 68 channels, 433 MHz, short range. These radios are approved for everyone.

• 1. Stummschalten des Lautsprechers (Baby soll nicht gestört werden),

These known conventional radios can be reconfigured in accordance with the invention to a monitoring device. Under "Setting the functions" of a suitably equipped radio, a sub-item "baby monitoring" with subsequent definition "stationary" (first radio) or "mobile" (second radio) could be included. These functions may include the following basic settings: on the stationary side (first radio):

• 1. Mute the speaker (baby should be not be disturbed),

at the preferred device determines whether the modifiable Positions of a peptide in the selection list of the possible Peptides exactly corresponds to a combination of modifications of interest. If the modifiable positions of a peptide in the selection list potential Peptides exactly or essentially a combination of modifications of interest, the peptide in the selection list is compared with or with regard to Fragmentation or MS / MS mass spectral data corresponding to the peptide ion correspond. The comparison or assessment includes the determination, Evaluation or calculation of the closeness of the fit between theoretical Data and experimental data. However, if the modifiable Positions of a peptide in the selection list of possible peptides are not exact or essentially a combination of modifications of interest correspond or agree with these, The modifications will be of interest according to the rest they concern, grouped. Combinations of modifiable positions then preferably enumerated, and modifications become of interest permuted under the modifiable positions, preferably on a group-by-group basis or a basis by group. Every permuted Peptide is then preferably compared or corrected for fragmentation or MS / MS mass spectral data, the correspond to the peptide ion.

The Database includes preferably a database of peptides derived from unmodified proteins are derived. The ones listed or listed in the database Peptides are preferably arranged according to their mass or mass-to-charge ratio. In particular, the Peptides arranged according to the monoisotopic mass-to-charge ratio be.

Of the The step of identifying the modified protein preferably comprises repeating steps (i), (ii) and (iii) for a number different peptides or peptide ions.

• einer Ionenquelle zur Ionisierung wenigstens eines einer Anzahl von Peptiden, die von einer Digestion eines modifizierten Proteins abgeleitet bzw. deriviert sind, zur Bildung eines oder mehrerer Peptidionen;
• einem Massenanalysator zum massenanalysieren eines oder mehrerer der Peptidionen zur Bestimmung der Masse oder des Masse-Ladungs-Verhältnisses wenigstens eines der Peptide oder der Peptidionen; und

In accordance with another aspect of the present invention, a mass spectrometer is provided with:

• an ion source for ionizing at least one of a number of peptides derived from a digest of a modified protein to form one or more peptide ions;
• a mass analyzer for mass analyzing one or more of the peptide ions to determine the mass or mass-to-charge ratio of at least one of the peptides or the peptide ions; and

• (i) die theoretische unmodifizierte Masse oder das theoretische unmodifizierte Masse-Ladungs-Verhältnis bestimmen, das ein Peptid oder Peptidion hätte, wäre das Protein, von dem das Peptid oder Peptidion abgeleitet ist, nicht modifiziert worden;
• (ii) die Datenbank von Peptiden unter Verwendung der theoretischen unmodifizierten Masse oder des theoretischen unmodifizierten Masse-Ladungs-Verhältnisses durchsuchen; und
• (iii) Peptide in der Datenbank bestimmen, die eine Masse oder ein Masse-Ladungs-Verhältnis aufweisen, die bzw. das der theoretischen unmodifizierten Masse oder dem theoretischen unmodifizierten Masse-Ladungs-Verhältnis entspricht.

Identification means for identifying the modified protein, wherein, in use, the identification means

• (i) determine the theoretical unmodified mass or theoretical unmodified mass to charge ratio that a peptide or peptide ion would have had if the protein from which the peptide or peptide ion is derived has not been modified;
• (ii) search the database of peptides using the theoretical unmodified mass or the theoretical unmodified mass-to-charge ratio; and
• (iii) determine peptides in the database which have a mass or mass-to-charge ratio equal to the theoretical unmodified mass or theoretical unmodified mass-to-charge ratio.

• (i) Mitteln zur Bestimmung der theoretischen unmodifizierten Masse oder des theoretischen unmodifizierten Masse-Ladungs-Verhältnisses, welches ein Peptid oder Peptidion hätte, wäre das Protein, von dem das Peptid oder das Peptidion abgeleitet ist, nicht modifiziert worden;
• (ii) Mitteln zur Durchsuchen einer Datenbank von Peptiden unter Verwendung der theoretischen unmodifizierten Masse oder des theoretischen unmodifizierten Masse-Ladungs-Verhältnisses; und
• (iii) Mitteln zur Bestimmung von Peptiden in der Datenbank, die eine Masse oder ein Masse-Ladungs-Verhältnis aufweisen, die bzw. das der theoretischen unmodifizierten Masse oder dem theoretischen unmodifizierten Masse-Ladungs-Verhältnis entspricht.

According to another aspect of the present invention there is provided an apparatus for identifying a modified protein comprising:

• (i) means for determining the theoretical unmodified mass or the theoretical unmodified mass-to-charge ratio which would have a peptide or peptide ion, the protein from which the peptide or peptide ion is derived would not have been modified;
• (ii) means for searching a database of peptides using the theoretical unmodified mass or the theoretical unmodified mass-to-charge ratio; and
• (iii) means for the determination of peptides in the database which have a mass or mass-to-charge ratio equal to the theoretical unmodified mass or the theoretical unmodified mass-to-charge ratio.

• (i) die theoretische unmodifizierte Masse oder das theoretischen unmodifizierten Masse-Ladungs-Verhältnis bestimmt, welche bzw. welches ein Peptid oder Peptidion hätte, wäre das Protein, von dem das Peptid oder Peptidion abgeleitet ist, nicht modifiziert worden;
• (ii) eine Datenbank von Peptiden unter Verwendung der theoretischen unmodifizierten Masse oder des theoretischen unmodifizierten Masse-Ladungs-Verhältnisses durchsucht; und
• (iii) Peptide in der Datenbank bestimmt, die eine Masse oder ein Masse-Ladungs-Verhältnis aufweisen, die bzw. das der theoretischen unmodifizierten Masse oder dem theoretischen unmodifizierten Masse-Ladungs-Verhältnis entspricht.

According to another aspect of the present invention, there is provided an apparatus for identifying a modified protein, wherein in use, the apparatus

• (i) the theoretical unmodified mass or theoretical unmodified mass-to-charge ratio determines what would have a peptide or peptide ion if the protein from which the peptide or peptide ion is derived were not modified;
• (ii) searching a database of peptides using the theoretical unmodified mass or the theoretical unmodified mass-to-charge ratio; and
• (iii) determining in the database peptides having a mass or mass-to-charge ratio equal to the theoretical unmodified mass or theoretical unmodified mass-to-charge ratio.

• (a) Mitteln zur Bestimmung, Auswählen und In-Erwägung-Ziehen einer oder mehrerer Modifikationen von Interesse;
• (b) Mitteln zur Enumerierung durch bzw. über eine oder mehrere Kombinationen von Modifikationen von Interesse;
• (c) Mitteln zum Berechnen der Masse oder des Masse-Ladungs-Verhältnisses einer oder mehrerer Kombinationen von Modifikationen von Interesse;
• (d) Mitteln zum Subtrahieren der berechneten Masse oder des berechneten Masse-Ladungs-Verhältnisses einer der Kombinationen von Modifikationen von Interesse von der experimentell bestimmten Masse oder dem experimentell bestimmten Masse-Ladungs-Verhältnisses eines Peptids oder Peptidions zur Bestimmung einer theoretischen unmodifizierten Masse oder eines theoretischen unmodifizierten Masse-Ladungs-Verhältnisses, die bzw. das ein Peptid oder Peptidion hätte, wäre das Protein, von dem das Peptidion abgeleitet ist, nicht modifiziert worden;
• (e) Mitteln zum Durchsuchen einer Datenbank von Peptiden unter Verwendung der theoretischen unmodifizierten Masse oder des theoretischen unmodifizierten Masse-Ladungs-Verhältnisses zur Ausgabe einer ersten Liste von Peptiden;
• (f) Mitteln zur Entfernung von Peptiden aus der ersten Liste von Peptiden, die die Kombination von Modifikationen von Interesse nicht unterstützen können, zur Erzeugung einer Auswahlliste von Peptiden; und
• (g) Mitteln zum Vergleichen bzw. Bewerten von Peptiden in der Auswahlliste von Peptiden bezüglich Fragmentations-Massenspektraldaten.

According to another aspect of the present According to the invention, a device for identifying a modified protein is provided with:

• (a) means for determining, selecting and considering one or more modifications of interest;
• (b) means for enumerating by or over one or more combinations of modifications of interest;
• (c) means for calculating the mass or mass-to-charge ratio of one or more combinations of modifications of interest;
• (d) means for subtracting the calculated mass or mass-to-charge ratio of one of the combinations of modifications of interest from the experimentally determined mass or the experimentally determined mass-to-charge ratio of a peptide or peptide ion to determine a theoretical unmodified mass or mass theoretical unmodified mass-to-charge ratio that would have a peptide or peptide ion, the protein from which the peptide ion is derived would not have been modified;
• (e) means for searching a database of peptides using the theoretical unmodified mass or the theoretical unmodified mass-to-charge ratio to output a first list of peptides;
• (f) means for removing peptides from the first list of peptides that are incapable of supporting the combination of modifications of interest to produce a selection list of peptides; and
• (g) means for comparing peptides in the selection list of peptides for fragmentation mass spectral data.

• (a) eine oder mehrere Modifikationen von Interesse bestimmt, auswählt oder in Erwägung zieht;
• (b) durch eine oder mehrere Kombinationen von Modifikationen von Interesse enumeriert;
• (c) die Masse oder das Masse-Ladungs-Verhältnis einer oder mehrerer Kombinationen von Modifikationen von Interesse berechnet;
• (d) die berechnete Masse oder das berechnete Masse-Ladungs-Verhältnis einer der Kombinationen von Modifikationen von Interesse von der experimentell bestimmten Masse oder dem experimentell bestimmten Masse-Ladungs-Verhältnis eines Peptids oder Peptidions subtrahiert zur Bestimmung einer theoretischen unmodifizierten Masse oder eines theoretischen unmodifizierten Masse-Ladungs-Verhältnisses, die bzw. das ein Peptid oder ein Peptidion hätte, wäre das Protein, von dem das Peptid oder Peptidion abgeleitet ist, nicht modifiziert worden;
• (e) die Datenbank von Peptiden unter Verwendung der theoretischen unmodifizierten Masse oder des theoretischen unmodifizierten Masse-Ladungs-Verhältnisses zur Ausgabe einer ersten Liste von Peptiden durchsucht;
• (f) aus der ersten Liste von Peptiden Peptide entfernt, die die Kombination von Modifikationen von Interesse nicht unterstützen können, zur Erzeugung einer Auswahlliste von Peptiden; und
• (g) Peptide in der Auswahlliste von Peptiden gegen Fragmentations-Massenspektraldaten vergleicht bzw.bewertet.

According to another aspect of the present invention, there is provided an apparatus for identifying a modified protein, wherein in use, the apparatus

• (a) determines, selects or considers one or more modifications of interest;
• (b) enumerated by one or more combinations of modifications of interest;
• (c) calculating the mass or mass-to-charge ratio of one or more combinations of modifications of interest;
• (d) the calculated mass or mass-to-charge ratio of one of the combinations of modifications of interest subtracted from the experimentally determined mass or the experimentally determined mass-to-charge ratio of a peptide or peptide ion to determine a theoretical unmodified mass or a theoretical unmodified mass Mass-to-charge ratio that would have a peptide or a peptide ion, the protein from which the peptide or peptide ion is derived would not have been modified;
• (e) searching the database of peptides using the theoretical unmodified mass or the theoretical unmodified mass-to-charge ratio to output a first list of peptides;
• (f) removing peptides from the first list of peptides that are incapable of supporting the combination of modifications of interest to produce a selection list of peptides; and
• (g) compares peptides in the shortlist of peptides against fragmentation mass spectral data.

The preferred device for identifying a modified protein involves direct searching for an indexed predicted one unmodified peptide database or a database for indexed predigested unmodified peptides. Traditionally, it was not considered possible viewed, peptide data re of a posttranslational modified protein directly against a Peptide database re to screen unmodified proteins. The preferred embodiment shows, however, that this assumption is incorrect.

advantageously, allows the preferred embodiment to consider any number of variable (random) modifications or in consideration pull and is thus just as powerful as conventional Approaches. However, since the preferred embodiment not the ongoing theoretical digesting of proteins that occur in a protein database, includes, and then a Indexing these proteins, it is much faster.

One important aspect of the preferred embodiment is that the mass or the mass-to-charge ratio altered or otherwise "de-modified" to the effect of an experimentally observed peptide. the one particular post-translational modification to the mass of the peptide has to counteract. The majority of post translational Modifications of interest have the effect of increasing or Magnification of the Mass or mass-to-charge ratio of the protein and thus is the mass or mass-to-charge ratio of different peptides, derived from the protein are also increased. An exception to this is dehydration, which has the effect of reducing mass or mass-to-charge ratio of a protein.

The step of counteracting, reversing or negating the effect of a post-translational modification includes the Be If the mass or mass-to-charge ratio that an experimentally measured peptide would have, the corresponding protein from which the peptide is derived would not have undergone post-translational modification. The preferred embodiment therefore assumes that variable or random modifications have occurred to the protein (and thus affected the resulting peptides), and searches a conventional indexed predicted peptide database of unmodified peptides using the calculated mass or calculated mass Charge ratio of the unmodified peptide, ie, reversed with the effects of possible modifications.

to Reduction of the total search times as far as possible filters the preferred one embodiment possible Peptides (i.e., peptides listed in the peptide database, which have a mass or mass-to-charge ratio that of the mass or the mass-to-charge ratio of the experimentally observed peptides as subsequently de-modified corresponds) at high speed. This allows for peptides that are in of the peptide database are listed and the correct mass or that have correct mass-to-charge ratio, the but not actually which may support modifications of interest, excluded or removed become. This ensures those contained in the selection list peptides containing the Filtering processes happen, suitable modifiable positions exhibited by the modifications of interest can. In this way, the preferred embodiment can quickly do those Peptides are then determined as possible matches or mappings for the experimental peptide data should be considered.

As soon as Peptides are filtered to produce a selection list of possible peptides, become these possible Analyzed peptides in a second characterization stage and compared or compared with the experimental data rated. traditionally, was for every experimentally observed peptide to analyze a theoretical digest of one or more proteins in one Protein database are listed performed. The time for one theoretical digest of proteins in a protein database can contribute much to the overall search time, though efficient algorithms are known for determining the digestion products a protein. Furthermore, each must Protein, by the theoretical digestion of a protein then by using combinations of user-selected ones Modifiers (if any of them can be applied) a given peptide is processed, and then the modification (s) among all different possible ones modifiable positions on the peptide core in question become. This is computer time consuming and contributes to slow overall search times at, which are characteristic for conventional Method for identifying post-translationally modified proteins.

The preferred device for identifying a modified protein has two important advantages the conventional ones approaches on. First, it is according to the preferred one Embodiment not necessary, one contained or listed in a protein database To theoretically digest protein or index the peptide list, whereby these relatively time-consuming steps are avoided can. Second, the number of possible Peptides that need to be characterized are reduced in a rapid manner or otherwise limited become.

Even though not essential for the present invention is the preferred embodiment particularly advantageous when using accurate mass spectral data become. This is for example by using a hybrid quadrupole time-of-flight mass spectrometer possible, and allows significantly faster overall search times. For example, typical Search times according to the preferred embodiment be at least eight times faster than conventional searches, if accurate Mass spectral data can be used. The use of accurate mass spectral data allows that peptide filtering is particularly effective at reducing the number of possible Peptides that must then be considered or characterized.

Various embodiments The invention will now be described, by way of example only, and with reference on the attached Drawings, described.

1 Figure 4 shows a number of different possible post-translational modifications which may involve a protein, the mass change resulting from such modification and the peptide position to which such modifications are applicable;

2 Figure 3 shows the steps taken in a preferred embodiment, according to the preferred embodiment, of unmodifying or de-modifying the mass or mass-to-charge ratio of experimentally observed peptides, screening a peptide database for peptides, or the mass or mass-to-charge ratio of the peptide correspond to unmodified or de-modified peptides to a predetermined tolerance, and then to filter for possible peptides to exclude peptides that are unable to support the modifications of interest a selection list of possible peptides having both the correct mass or mass-to-charge ratio and which also theoretically support the modification (s) of interest;

3 Figure 12 shows the subsequent steps taken in a second stage according to a preferred embodiment wherein peptides taken in a selection list in a previous filtering stage are compared with experimental fragmentation data;

4 Figure 4 shows a number of combinations and binary searches performed when 168 peptides were analyzed and different numbers of modifications and modifiable positions were considered;

5 shows in greater detail the number of peptides filtered and subsequently characterized and the associated times needed to filter, permute and compare the experimental data peptides when considering two modifications;

6 shows in greater detail the number of peptides that were filtered and subsequently characterized, and the associated time required for filtering, permuting, and comparing the peptides with experimental data when considering four modifications; and

7 shows comparison results for conventional seek times with seek times achieved in accordance with the preferred embodiment and illustrates the improvement in overall search time achievable in accordance with the preferred embodiment.

The rapid identification of proteins using mass spectral data and protein databases is now a standard proteomic technique Work. With the development of this research area is the Ability, To characterize proteins, and in particular the ability to the presence and position of any post-translational Modifying the protein has become increasingly important. The preferred embodiment relates to a method for identifying post-translational modified Proteins in a faster way than was possible with conventional techniques.

1 Figure 12 shows a list of post-translational modifications that may affect a protein, as well as the peptide positions or residues to which such modifications are applicable. The figure also shows the resulting change (normally increase) in the mass of the protein caused by the modification.

It It is known to derive peptides derived from the digestion of a protein are, a fragmentation in a collision or fragmentation cell to undergo a mass spectrometer. The resulting fragmentation or MS / MS mass spectral data then analyzed by searching or searching for data, that are contained in a database, or data that is contained in a database Database data are derived. One or more variable Modifications can while selected such a search and considering to be pulled.

It however, was not considered practicable after modified Peptides using a conventional predicted peptide database, which lists only unmodified peptides. To enable the implementation Such a search was assumed to be the peptide database all potential potential combinations of modifications and their permutations enable on a peptide or must include. As a result, it was considered that the peptide database greatly expanded would have to, with The result is that the database will be so large and impractical when searching would, d. H. Search times would be very slow or long.

Out For this reason, the conventional Approach not to try pre-established peptide databases but rather with the use of a protein database to start and then run proteins in the protein database are listed, to digest theoretically. Subsequently, variable Modifications to the peptides that are determined to result from the digestion process, be applied. The resulting theoretically modified Peptides can then compared to the real experimental peptide data. In particular, you can selected Peptides fragmented in a fragmentation cell of a mass spectrometer and the resulting fragmentation mass spectral data theoretical fragmentation data obtained by theoretical fragmentation of peptides resulting from the theoretical digestion of a Protein listed in the protein database become. According to this conventional Approach, each protein in the protein database is sequentially digested, and the process is then repeated.

In contrast to the conventional approach, the preferred embodiment uses an indexed predigested unmodified peptide database in the search for peptides derived from a protein from which it is believed will be modified post-translationally. Such an approach therefore avoids the need for ongoing digestion of proteins listed in a protein database. Variable modification searches can be performed relatively quickly after we have done so. In accordance with the preferred method, accurate mass spectral data obtained, for example, from a quadrupole time-of-flight mass spectrometer, enables the implementation of a high speed peptide filter. This allows a significant reduction in search times and faster protein characterization opportunities.

The unmodified peptides listed in the peptide database are against whom is being sought, or compared with, are preferably arranged in the order of the monoisotopic mass, to facilitate the database search. It is important that the preferred embodiment the search directly against or with respect to unmodified peptides, which are stored in the peptide database includes. thats why it is not necessary to try multiple entries per peptide in the peptide database to address many different possible modifications, which are to be considered to cover. The use of a database predigested unmodified peptides simplifies the search process significant.

The Searching or referring the database of unmodified peptides preferably comprises two major ones subsequent or sequential stages - a first filtering stage followed by a second characterization stage.

In In the first stage, a first list of possible peptides is generated. All Peptides in this first list have a mass or mass-to-charge ratio that or that of or that of the experimentally observed Peptides within a custom tolerance match (such as unmodified or de-modified according to the preferred embodiment). This first list is then preferably filtered for generation a selection list of possible Candidate peptides. This filtering process involves the removal all peptides from the first list, who are unable to make the modifications of interest. All Peptides in the resulting selection list are therefore preferred Having peptide positions, the considered or considered Support modifications.

peptides in the selection list of possible peptides are then in a second stage, focusing on the peptide characterization relates (identification and localization of modifications) edited, and each peptide included in the selection list is scanned with fragmentation data referring to the experimentally observed peptide, the is analyzed.

The first high speed peptide filtering stage as implemented according to the preferred embodiment is described in greater detail with reference to FIG 2 shown and described.

According to the preferred Embodiment will be successively different combinations of user selected modifiers taken or used and on the data, an experimental observed peptide, which is analyzed, applied, applied.

In the preferred embodiment, it is believed that an experimentally observed peptide is derived from a protein that has been post-translationally modified. Accordingly, the mass or mass-to-charge ratio of the experimentally observed peptide is adjusted or "de-modified" to take into account the one or more assumed modifications, for example, when a phosphorylation of a serine is one of the modified or one of the modifications of As a result, a single phosphoryl modifier (H ₂ PO ₃ ) enumerated by different combinations is considered, assuming that an experimentally observed peptide carries a single phosphorolated serine, its mass will have been increased by 79.9663 as a result. Accordingly, to search for the database of predicted unmodified peptides, the mass of the experimentally observed peptide is adjusted or de-modified to account for the phosphorylation of the protein by subtracting the value of 79.9663 from the experimentally determined mass.

The approach described above of adapting or de-modifying the experimentally determined mass or the experimentally determined mass-to-charge ratio and the search against the peptide database using the matched or unmodified peptide mass or the adapted or unmodified mass-to-charge ratio is an important aspect of the preferred embodiment. This approach then allows a search to be performed against a conventional indexed predigested peptide database, taking into account variable modifications. After changing the mass or mass-to-charge ratio of the experimentally observed peptide to account for one or more assumed modifications, the indexed peptide database can now be modeled using the new (unmodified) theoretical mass of the Peptides are searched. The output of such a peptide database search will include a first list of possible peptides having a mass or mass-to-charge ratio that corresponds to that of the experimentally observed peptide within certain tolerances (as de-modified according to the preferred embodiment). This first list of peptides is then preferably filtered to provide a selection list of candidate peptides. All peptides in this selection list will support the desired combination of modifiers. In other words, the candidate peptide selection list will comprise a list of peptides, all of which have at least the correct number of modifiable positions, and wherein the modifiable positions will be of the correct type. To proceed with the above example relating to a single phosphoryl modifier, after the filtering step, the peptides in the selection list of possible peptides will all have at least one serine residue, and will also have a mass or mass-to-charge ratio which that substantially corresponds to that of the observed peptide (unmodified or de-modified according to the preferred embodiment).

There preferably sorts the peptide data in the peptide database or are arranged according to their mass, all possible peptides, the similar Masses or mass-to-charge ratios to the mass or mass-to-charge ratio of a experimentally observed peptide (unmodified or de-modified according to the preferred embodiment) obtained using two binary searches from the peptide database become. The two binary searches correspond to the mass or the mass-to-charge ratio of the unmodified Peptides plus or less the user-defined tolerance in mass or mass-to-charge ratio units. Such a binary search will therefore give upper and lower border peptides between which all Peptides listed in the peptide database, bulk or the mass-to-charge ratio of the experimentally observed peptide (as in the preferred embodiment unmodified or de-modified) within a certain tolerance.

By accurate measurement of the mass-to-charge ratio of ions using, for example of a quadrupole time-of-flight mass spectrometer can the number of possible Peptides coming from the two binary ones Search results are advantageously kept as small as possible as the custom tolerance can be kept small. This first List can then be shortened immediately to a selection list, as many of the peptides that the mass or mass mass-to-charge ratio of experimentally observed peptide (unmodified or de-modified according to the preferred Out of execution form) will not actually support the current combination of modifiers, d. H. the peptides become the appropriate (s) modifiable Do not include position (s).

The indexed peptide database against which or preferred to be sought is, consists of a linear array of records. each Record preferably consists of a number of fields, the protein index information (i.e., information that the peptide with a corresponding protein in a protein database bring) together with details of the exact monoisotonic Mass or monoisotonic mass-to-charge ratio. Everyone Includes peptide entry furthermore, information regarding the length and position of the peptide is preferred within the particular protein sequence.

The Peptide records stored in the peptide database, are preferred in the order of mass or mass-to-charge ratio sorted and arranged. Therefore, it is for finding peptides, the one particular mass or mass-to-charge ratio within have a custom tolerance, only necessary perform a binary search to find upper and lower bounds within the peptide database.

If it may be necessary to support a number of digests an additional Integer field may be included in each peptide record. Every bit in the additional Integer field can be used as a flag to indicate that a given Peptide is a product of a given digest. additional Fields can also a digest probability that in a protein evaluation or. -Vergleichsschema is included.

The Preferred search methods can be implemented using a software code, for example, using a C / C ++ programming language is written and running on one Microsoft Windows XP (RTM) platform using Microsoft Visual Studio (RTM) version 6.0 is compiled. According to the preferred embodiment can be an indexed peptide database based on a tryptic Digestes of a protein are generated and stored in the main memory of a computer getting charged. The peptide database can then be searched being possible, being possible Peptides first filtered and then included in a selection list peptides according to the preferred embodiment be characterized.

According to one another embodiment a slightly different indexing scheme can be used, with two Index files are generated. One of the index files contains only Details or details of the exact monoisotopic mass or the exact monoisotopic mass-to-charge ratio the peptides together with a peptide index information based on a corresponding or corresponding second file shows. The second File may contain further information regarding the peptides, d. H. Protein index information and information regarding the Length and Position of the peptides within certain protein sequences.

The preferred embodiment treats individually tryptic peptides that occur multiple times (in the same or different protein entries) that the indexed peptide database many copies of the same peptide (especially for small peptides). Preferably, a common Memory model or shared memory model be used so that only a single copy of the database in the memory of the processing system is maintained.

According to one embodiment, a mechanism for handling a number of digests or multiple digests may be implemented. For example, the peptide record may contain a flag containing the number of missed or disregarded cleavage products or cleavages associated with the peptide. With reference to 2 For example, the process of peptide filtering, as preferably implemented, will now be described in more detail. First, all combinations of user-selected modifiers are preferably enumerated. A standard algorithm for enumerating through all combinations can be used, such as that described by Tucker in Applied Combinatorics (John Wiley and Sons Inc., 2001). This is a process for obtaining all combinations (of different sizes) with repetitions. For a given number of modifiers n and a fixed number of modifiable positions k, the total number of combinations (ie, size k) is given by C k = (N + k-1)! / (N-1)! K! Since the number of positions is unknown, it is necessary to consider the range of modifiable positions from 0 to any user-specified maximum number of modifiable positions. Thus, the total number of combinations to be considered per search against the experimental data is given by:

For each of these combinations, a modified mass or a modified mass to charge ratio is calculated, and preferably subtracted from the experimentally determined mass or the experimentally determined mass to charge ratio of each peptide to be analyzed. The number of binary searches B _{S of} the indexed peptide database to be performed is therefore: B S = 2T q C where Tq is the total number of queries. A factor of 2 is taken into account because both a search for an upper bound and a search for a lower bound are necessary. Searching the database preferably uses an iterative binary search to obtain the upper and lower peptide boundaries. These mass limited peptides are then filtered.

The Ensures filtering level preferably that all possible Peptides, which are then added be characterized, at least the correct number of residues and that these residues are of the correct type. The Filtering stage includes therefore the counting the remains in the possible Peptides and comparing the possible peptides with the current one Combination of modifiers. It is important that modifier concern only a single remainder, or be applied to this. Certain modifiers that can be applied to multiple residues (degenerate Modifiers) were transformed into several non-degenerate modifiers refactored.

Corresponding allows indexing peptides in the database along with the exact ones Measurement of the mass-to-charge ratio that the first stage of processing as a high-speed filter serves to substantially reduce the number of peptides that then have to be characterized in a second stage.

When a selection list of peptides is formed, a second characterization step is then performed on the selection list of possible peptides. This characterization step is described in more detail with reference to 3 shown and described. The characterization step implements possible permutations of the modifiers under modifiable positions of the peptide and evaluates each permutation for experimentally observed fragment mass spectral data (MS / MS).

The characterization stage includes a number of fonts. First, for each in the off The selected or filtered peptide determines whether the modifiable positions on the peptide in question correspond exactly to the current selection of modifiers. If this is the case, the peptide is derived from the experimentally derived fragmentation data or MS / MS data, after which the next peptide included in the selection list is considered.

If however, the modifiable positions on the peptide are not accurate corresponding to the current selection of modifiers, becomes the selected combination of modifiers preferably regrouped according to the remainder, to which the modifiers refer. Then all combinations become more modifiable Items enumerated or counted. Modifier among the Positions are then preferably with modified positions, which are permuted, permuted on a group-by-group basis. This is because there are more modifiable positions on the potential Peptide as it is necessary to modify. With a given number of positions that can be modified and the number of modifiers applied will be all Combinations of positions, the modifications applied can have selected.

To the For purposes of illustration, the peptide PGPCCKDKCECAEGGCKT considered become. For example, it can be determined that C and K are modified can be and a restriction on or from 4 modifiable positions can be used. The 8 positions, thus, with their appearance in order can be modified are thus CCKKCCCK. The different possibilities can thus Numbered or enumerated as follows: (i) CCKK_____ (shown in software by a size 4 array as 0123); (ii) CCK__C____ (shown in software by a size 4 array as 0124); (iii) CCK___C____ (represented in software by a size 4 array as 0125); (iv) CCK____ C__ (represented in software by a size 4 array as 0126); (v) CCK____ K (represented in software by a size 4 array as 0127); (vi) CC__ KC____ (represented in software by an array the size 4 as 0134) ... and so on However, it may be that only a K-modification and three C modifications are allowed, which means that some of the above enumerated ways such as (i) can be discarded.

For every valid modifiable Position combination then the modifiers are preferably under the positions to which the modifiers apply are permuted (on a group-by-group basis). So if M1 up C holds, M2 applies to C, M3 applies to C, and M4 applies to K, then have to M1, M2 and M3 are applied in all three positions, and for each of them Positions must be M4 be applied to K. This is then repeated for the next enumeration. Algorithms for implementation such processes can standard lexicographic algorithms such as those in Knuth "The Art of Computer Programming ", volume 4, (Pre-fascicle 2b: Generating all permutations) are disclosed.

For every permutation of modifiers, the permuted peptide becomes experimental obtained fragmentation or MS / MS data is compared, after which the next permutation is considered becomes. After all Permutations are considered, the next one added to the selection list Peptide considered.

experimental Results will now be presented be that the improvement in the total search time, which can be achieved according to the invention is, illustrate. Two sentences of results are presented. One Sentence was obtained by considering only two modifiers, the other sentence was considered considering 4 modifiers receive.

Five at random or arbitrarily selected Proteins were considered. These five Proteins included TCMO MEDSA; SNC2 YEAST, DAK2 SCHPO, CSPB BACGO, and FMT BOVIN. The Proteins were theoretically synthesized using trypsin and a omitted or missed Apolysis theoretically digested. Resulting peptides with one Length smaller as 8 residues or larger than 24 residues were removed from further consideration or discarded, reducing a total of 168 to be considered Peptides remained.

These remaining 168 peptides were then modified. The sequences the five fortuitously chosen Proteins were read, and then either two or four Modifications applied to appropriate or corresponding residues in the sequence. For the introduction Another random effect was just the leftovers that one straight Position within the peptide sequence had modified. The four potential Modifications that were approved were palmitoylation applicable for or on Threonine, phosphoryl, applicable to serine, carboxymethyl applicable for cysteine, and hydroxyl applicable to aspartic acid.

4 Figure 12 shows the total number of combinations and binary searches needed to search for the 168 peptides for different numbers of modifications and modifiable positions. 5 shows in more detail the results for two modifiers and 6 shows in size Details of the results for four modifiers.

The 5 and 6 show separately the time taken for the first stage of the first filtering of possible peptides and also the second stage of the subsequent permutation and evaluation or comparison of peptides. The figures also show corresponding results in which different mass tolerances and a maximum number of modifiable positions have been taken into account. The times were obtained by searching against a peptide database derived from the Swiss Prot database with 105224 entries indexed using a tryptic digest. The code was run on a Compaq Evo W6000 (RTM) running at 1.7 GHz in single processor mode.

The 5 and 6 show the time taken to filter the peptides and the time taken to permute and evaluate the final peptides, ie all potential combinations. It can be seen that with an increase in the number of modifiers and modifiable locations, significantly more peptides are initially returned which must then be filtered. Essentially, however, the number of peptides that pass through the filtering process does not increase at the same rate as increases the total number of peptides. As the total number of peptides to be considered increases, the filter time begins to account for a significant portion of the total search time. This is due to the fact that as the number of combinations to be considered increases, the same peptides are repeatedly evaluated to determine whether or not they can support the current combination. Likewise, the same peptides will repeatedly fail this test, making evaluation unnecessary for them.

The Extension of the mass tolerance results in the consideration additional Peptides and their passage through the filter. If, for example the mass tolerance of 0, 01 As is increased to 0, 10 Da, the total number increases to be considered Peptides by a factor of about 10, and this results in a corresponding increase in filter time. It can therefore be seen be that it is particularly advantageous, the mass or the mass-charge ratio of To accurately determine peptides to be analyzed, as this is significant reduces the total number of peptides that need to be considered. Further improvements can be obtained are stored by storing information regarding the number and type the residues having a peptide. For example, a memory cache or cache memory used to make a repeated bill to prevent and thus the filter time and thus the entire search time to reduce.

7 shows comparison results for conventional search times with respect to search times according to the preferred embodiment. It will be appreciated that the preferred embodiment allows the overall search time to be significantly shortened compared to conventional search times. Traditional search times were obtained using the Proteinlynx (RTM) Global Server 2.05B19. The search times according to the preferred embodiment are taken from some of the highlighted results that appear in the 5 and 6 are presented. In the 7 listed search times relating to the preferred embodiment include about 2 seconds to read the peptide data to be analyzed.

From the in 7 As shown, with an increase in mass tolerance, a corresponding decrease in the acceleration factor (or improvement) in search time according to the preferred embodiment is associated with the search time required using conventional techniques. The reason for this decrease in the improvement is not fully understood at the present time, as presumably the conventional "on-going" implementation will have to cope with a correspondingly increased number of peptides with increased mass tolerance, however, the results show that the total search time associated with Analysis of peptides according to the preferred embodiment can be significantly reduced compared to conventional seek times, especially if the mass or mass-to-charge ratio of the peptides is accurately determined, ie the mass tolerance of the precursor ion (s) or the Thus, the present invention represents a significant improvement in the art.

The preferred embodiment is also able to identify proteins when the number of Modifications of interest is set to zero.

Even though the present invention with reference to preferred embodiments experts will understand that different changes executed in form and detail can be without the scope of the invention as indicated in the appended claims is to leave.

Claims (40)

Device for identifying a modifi encoded protein with: means for digesting a modified protein to produce a number of peptides; Means for ionizing at least one of the number of peptides to form one or more peptide ions; Means for mass analyzing one or more peptidones to determine the mass or mass-to-charge ratio of at least one of the peptides or peptide ions; and means for identifying the modified protein by: (i) determining the theoretical unmodified mass or theoretical unmodified mass-to-charge ratio that a peptide or peptide ion would have would be the protein from which the peptide or peptide ion is derived , not modified; (ii) searching a database of peptides using the theoretical unmodified mass or the theoretical unmodified mass-to-charge ratio; and (iii) determining, in the database, peptides having a mass or mass-to-charge ratio equal to the theoretical unmodified mass or theoretical unmodified mass-to-charge ratio. Apparatus according to claim 1, wherein the modified Protein was posttranslationally modified. Device according to one of claims 1 or 2, wherein the modified Protein was modified by methylation, hydroxylation, Oxidation or oxidation, formylation, acetylation, carboxylation, Phosphorylation, sulphation, cysteinylation, glycosylation, Farnesylation, myristoylation, biotinylation, palmitoylation, Stearoylation or stearoylation. Apparatus according to any one of the preceding claims, further comprising the determination, selection and consideration of one or more Modifications of interest. Apparatus according to claim 4, wherein the determined or considered or it is assumed that the modified protein by at least one of the modifications of interest is modified. Device according to one of claims 4 or 5, further comprising Enumeration means by at least one or more combinations en) of modifications of interest. The device of claim 6, further comprising means for enumeration by all combinations of modifications of Interest. Device according to one of claims 6 or 7, further comprising Means for determining, selecting or considering a maximum Number of modifications. Apparatus according to claim 8, wherein the means for Enumerate by combinations of modifications of interest through combinations of modifications from interest to Enumerate the maximum number of modifications. Device according to one of claims 4 to 9, wherein the means for the determination of the theoretical unmodified mass or theoretical unmodified mass-to-charge ratio the mass or mass-to-charge ratio of a combination of Calculate or determine modifications of interest. Apparatus according to claim 10, wherein the means for the determination of the theoretical unmodified mass or theoretical mass unmodified mass-to-mass ratio or mass Mass-to-charge ratio of the peptide or peptide ion with respect to the combination of modifications of interest change, reverse, counteract, negate or unmodify or demodify or discontinue. Apparatus according to claim 11, wherein the means for the determination of the theoretical unmodified mass or theoretical mass unmodified mass-to-mass ratio or mass Mass-to-charge ratio the combination of modifications of interest from the mass or the Mass-to-charge ratio of the peptide or peptide ion as determined by the mass analysis step is determined, subtract. The method of claim 11, wherein the means for Determination of theoretical unmodified mass or theoretic unmodified mass-to-charge ratio the mass or mass-to-charge ratio of the Combination of modifications of interest to the mass or mass-to-charge ratio of the Peptides or peptide ions as determined by the means of mass analysis is, add. Apparatus according to any one of the preceding claims, further comprising means for determination, selection or consideration a permissible mass tolerance or mass-to-charge ratio tolerance between the theoretical unmodified mass or the theoretical one unmodified mass-to-charge ratio of the peptide or peptide ion and the mass or mass-to-charge ratio of the peptides used in the Database are listed. Apparatus according to claim 14, wherein the Mass tolerance falls within a range selected from the group consisting of: (i) <0.01 Da; (ii) 0.01-0.02 Da; (iii) 0, 02 - 0, 03 Da; (iv) 0, 03 -0, 04 Da; (v) 0, 04 - 0.05 Da; (vi) 0.05-0.06 Da; (vii) 0.06-0.07 Da; (viii) 0.07-0.08 Da; (ix) 0.08-0.09 Da; (x) 0.09 - 0.10 Da; and (xi)> 0.10 Da. Apparatus according to claim 14, wherein the mass-to-charge ratio tolerance falling into an area the selected is selected from the group consisting of: (i) <0.1 mass-to-charge ratio units; (ii) <0.01 mass-to-charge ratio units; (iii) <0.001 mass-to-charge ratio units; (iv) <0.0001 mass-to-charge ratio units; (v) <0.00001 mass-to-charge ratio units; (vi) <0.000001 Mass to charge ratio units. Device according to one of claims 14, 15 or 16, in which the means of searching or searching a database of peptides Peptides listed in the database, and a mass or have a mass-to-charge ratio, that of the theoretical unmodified mass or the theoretical one unmodified mass-to-charge ratio within corresponds to a mass tolerance or a mass-to-charge ratio tolerance, determine or select. The device of claim 17, further comprising means for determining a peptide in the database of peptides, the has a mass or mass-to-charge ratio that is theoretical unmodified mass or the theoretical unmodified mass-to-charge ratio within an upper limit of the mass tolerance or the mass-to-charge ratio tolerance equivalent. Device according to one of claims 17 or 18, further comprising Means for determining a peptide in the database of peptides, which has a mass or a mass-to-charge ratio, the the theoretical unmodified mass or the theoretical unmodified Mass-to-charge ratio within a lower limit of mass tolerance or mass-to-charge ratio tolerance equivalent. Device according to one of claims 18 or 19, further comprising Agent for selecting peptides in the database of peptides with Mass or mass-to-charge ratios between the upper limit and the lower limit. The device of claim 20, wherein peptides in the database of peptides with masses or mass-to-charge ratios between the upper limit and the lower limit a first list of peptides. The device of claim 21, further comprising means for rejection or rejection of peptides from the first list of peptides, not at least one of the combinations of modifications of Support interest. The device of claim 22, further comprising means to form a selection list of possible peptides. Apparatus according to claim 23, wherein at least a plurality of peptides in the selection list of peptides comprises: (i) a mass or mass-to-charge ratio, that of the theoretical unmodified mass or the theoretical unmodified mass-to-charge ratio of the Peptides or Peptidions within a mass tolerance or a Mass-to-charge ratio tolerance corresponds; and (ii) at least the same number and type of modifiable Positions or sites supporting at least one of Combinations of modifications of interest. Device according to one of claims 23 or 24, further comprising Means for determining whether the modifiable positions of a peptide in the selection list possible Peptides of exactly a combination of modifications of interest correspond. Apparatus according to claim 25, wherein, if the modifiable positions of a peptide in the selection list of potential Peptides exactly or essentially a combination of modifications of Interest, the peptide in this shortlist for fragmentation or MS / MS mass spectral data, which correspond to the Peptidion, is assessed or compared. Apparatus according to claim 25, wherein, if the modifiable positions of a peptide in the selection list of Peptides are not exactly or essentially a combination of Modifications of interest correspond to the modifications of Interest is grouped according to the rest to which they are applicable become. The device of claim 27, further comprising means for enumeration by combinations of modifiable positions. The device of claim 28, further comprising means for the permutation of modifications of interest among the modifiable ones Positions. Apparatus according to claim 29, wherein the modifications of interest be permuted on a group by group basis. Apparatus according to claim 29 or 30, further comprising means for evaluating each permuted peptide against fragmentation or MS / MS mass spectral data corresponding to the peptide ion. Device according to one of the preceding claims, wherein the database is a database of peptides that are unmodified Proteins are derived. Apparatus according to claim 32, wherein in the database listed peptides in order of mass or mass-to-charge ratio are arranged. The device of claim 33, wherein the peptides in the order of their monoisotopic mass or their monoisotopic Mass to charge ratio are arranged. Device according to one of the preceding claims, wherein the means of identification of the modified protein further of steps (i), (ii) and (iii) for a number of different ones Repeat peptides or peptide ions. Mass spectrometer with an ion source for Ionizing at least a number of peptides resulting from digestion derived from a modified protein, to form a or more peptide ions; a mass analyzer for mass analyzing a or more of the peptide ions for the determination of the mass or the Mass to charge ratio at least one of the peptides or peptide ions; and identifying means for the identification of the modified protein, the identification means in use (i) the theoretical unmodified mass or determine the theoretical unmodified mass-to-charge ratio, that would have a peptide or peptide ion, would be the protein, from which the peptide or peptide ion is derived is not modified been; (ii) a database of peptides using the theoretical unmodified mass or the theoretical unmodified Mass to charge ratio search; and (iii) determine peptides in the database which have a mass or a mass-to-charge ratio, the or the theoretical unmodified mass or the theoretical unmodified Mass-to-charge ratio equivalent. Device for identifying a modified Protein with: (i) means for determining the theoretical unmodified mass or theoretical unmodified mass-to-charge ratio, that would have a peptide or peptide ion, would be that Protein from which the peptide or Peptidion is derived, not modified; (ii) means for searching a database of peptides using the theoretical unmodified Mass or theoretical unmodified mass-to-charge ratio; and (iii) means for determining peptides in the database, which have a mass or a mass-to-charge ratio, the or the theoretical unmodified mass or the theoretical unmodified Mass to charge ratio corresponds. Device for identifying a modified Protein, wherein in use the device (i) the theoretical unmodified mass or the theoretical unmodified Mass-to-charge ratio which had a peptide or peptide ion, would be the protein from which the peptide or peptide ion is derived is not modified been; (ii) searching a database of peptides using the theoretical unmodified mass or the theoretical unmodified Mass to charge ratio; and (iii) Peptides in the database that determines a mass or a mass-to-charge ratio have, that of the theoretical unmodified mass or the theoretical unmodified mass-to-charge ratio. Apparatus for identifying a modified protein comprising: (a) means for determining, selecting or considering one or more modifications of interest; (b) means for enumerating by one or more combinations of modifications of interest; (c) means for calculating the mass or mass-to-charge ratio of the one or more combinations of modifications of interest; (d) means for subtracting the calculated mass or mass-to-charge ratio of one of the combinations of modifications of interest from the experimentally determined mass or the experimentally determined mass-to-charge ratio of a peptide or peptide ion to determine a theoretical unmodified mass or mass theoretical unmodified mass-to-charge ratio that would have a peptide or peptide ion, the protein from which the peptide or peptide ion is derived would not have been modified; (e) means for searching a database of peptides using the theoretical unmodified mass or the theoretical unmodified mass-to-charge ratio to output a first list of peptides; (f) means for removing from the first list of peptides peptides which can not support the combination of modifications of interest Generation of a selection list of peptides; and (g) means for evaluating peptides in the selection list of peptides for fragmentation mass spectral data. Device for identifying a modified Protein, wherein in use the device (a) one or multiple modifications of interest, selects or considered; (B) by one or more combinations of modifications of interest enumerated; (c) the mass or mass-to-charge ratio of a or multiple combinations of modifications of interest; (D) a calculated mass or the calculated mass-to-charge ratio of one of the combinations of Modifications of interest from the experimentally determined mass or the experimentally determined mass-to-charge ratio of a peptide or peptide ion, to determine a theoretical unmodified mass or a theoretical unmodified mass Mass to charge ratio, which would have a peptide, would be the protein from which the peptide or peptide ion is derived is not modified been; (d) searching a database of peptides using the theoretical unmodified mass or the theoretical unmodified Mass to charge ratio for issuing a first list of peptides; (f) from the first List of peptides peptides removed, showing the combination of modifications of interest do not support can, and thus produces a selection list of peptides; and (g) peptides in the selection list of peptides for fragmentation mass spectral data rated or compared.