Patent Publication Number: US-8987662-B2

Title: System and method for performing tandem mass spectrometry analysis

Description:
BACKGROUND 
     Generally, mass spectrometers measure mass-to-charge ratios of charged samples, enabling contents of the samples to be identified. Use of mass spectrometers has been expanded to include identification of proteins and corresponding peptides. This requires ions of a protein in the sample to be volatilized, in accordance with a variety of volatilizing techniques, such as electrospray ionization (ESI) and matrix-assisted laser desorption and ionization (MALDI) and provided to a mass analyzer of the mass spectrometer. The proteins and peptides may then be identified, for example, by matching the measured mass-to-charge ratios to a database of mass-to-charge rations of known proteins and peptides. Tandem mass spectrometry (MS/MS) provides multiple stage measurements of a sample, for example, using separate analyzers corresponding to the multiple stages, or using a single analyzer to analyze the sample multiple times. 
     Currently, powerful computer processing and enhanced performance of bioinformatics tools that analyze mass spectrometry data make it possible to match results of an MS/MS scan of a sample to a peptide in real-time. That is, the peptide may be identified in a timescale comparable to the time between two successive acquisition events of a mass spectrometer (i.e., the time it takes to acquire one spectrum). 
     Bottom-up acquisition protocols for MS/MS have increased sample coverage by applying different rules on how to select the most intense precursor ions of a sample for further MS/MS acquisition. For example, rules on ion intensities have been used whereby a precursor ion exclusion list may be built based on information collected on the first of two consecutive runs of a sample, known as repetitive liquid chromatography (LC)-MS/MS. While repetitive LC-MS/MS extends coverage, it also doubles acquisition time, thus becoming impractical in high throughput workflows. 
    
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
       The representative embodiments are best understood from the following detailed description when read with the accompanying drawing figures. It is emphasized that the various features are not necessarily drawn to scale. In fact, the dimensions may be arbitrarily increased or decreased for clarity of discussion. Wherever applicable and practical, like reference numerals refer to like elements. 
         FIG. 1  is a flow diagram of a method for performing tandem mass spectrometry analysis using a watch list, according to a representative embodiment. 
         FIG. 2  is a functional block diagram illustrating a system for performing tandem mass spectrometry analysis, according to a representative embodiment. 
         FIG. 3  is a flow diagram of a method for performing tandem mass spectrometry analysis using a watch list, according to a representative embodiment. 
         FIG. 4  is a functional block diagram illustrating a system for performing tandem mass spectrometry analysis, according to a representative embodiment. 
     
    
    
     DETAILED DESCRIPTION 
     In the following detailed description, for purposes of explanation and not limitation, illustrative embodiments disclosing specific details are set forth in order to provide a thorough understanding of an embodiment according to the present teachings. However, it will be apparent that other embodiments according to the present teachings that depart from the specific details disclosed herein remain within the scope of the appended claims. Moreover, descriptions of well-known devices and methods may be omitted so as to not obscure the description of the example embodiments. Such methods and devices are clearly within the scope of the present teachings. 
     In the various embodiments, peptide sequence information is used in real-time, through matches of spectra to peptides, in a tandem mass spectrometry (MS/MS) acquisition process. More particularly, peptide sequence information can be used to increase otherwise limited sample coverage of tandem mass spectrometers (e.g., limiting protein coverage). 
     Generally, according to various embodiments, an initial dynamic watch list of proteins is established, for example, through prior knowledge of proteins expected to be present in the sample. The dynamic watch list includes masses of expected peptides corresponding to the proteins. As data acquisition continues, newly identified proteins and masses of their corresponding expected peptides (i.e., peptides belonging to the proteins, but not yet observed) are added to the dynamic watch list. Various embodiments use empirical knowledge of the sequence of proteins that have been confidently identified to populate the dynamic watch list. With knowledge of the sequence of proteins, exclusion criteria may be built, e.g., based on the expected peptides corresponding to the list of proteins. 
     Meanwhile, peptides which are confirmed to be present on the dynamic watch list, are not fully acquired, since doing so would be redundant. Results include more efficient identification of peptides on the dynamic watch list, as well as increased protein and/or peptide identifications. Also, data acquisition in proteomics workflows and/or in the analysis of complex protein samples, for example, may be improved. 
       FIG. 1  is a flow diagram of a method for performing tandem mass spectrometry analysis using a watch list, according to a representative embodiment. 
     In block  121 , an MS scan is performed on a sample to acquire a mass spectrum having peaks corresponding to masses of precursor ions. The precursor ion masses are compared to masses of expected peptides on a previously established dynamic watch list (e.g., the initial dynamic watch list, discussed above) at block  123 . As discussed above, the expected peptides correspond to proteins in the sample. 
     For each precursor ion mass that does not match an expected peptide mass on the dynamic watch list (block  123 : No), a full MS/MS scan is performed, including fragmentation of the respective precursor ion, to identify the corresponding peptide. For each precursor ion mass that does match an expected peptide mass on the dynamic watch list (block  123 : Yes), the match is scored at block  125  to assure adequacy or confidence of the match. The scoring process at block  125  may include a fast MS/MS scan of each precursor ion matching a mass of an expected peptide, discussed below. 
     In an embodiment, all of the precursor ion masses are sequentially compared to the expected peptide masses on the dynamic watch list before either a full MS/MS scan (block  142 ) or the scoring process (block  125 ) is performed on any of the precursor ions. In alternative embodiments, the precursor ion masses may be compared to the expected peptide masses in parallel, and/or the full MS/MS scan (block  142 ) or the scoring process (block  125 ) may be completed for one precursor ion before the mass of a subsequent precursor ion is compared to the expected peptide masses on the dynamic watch list. 
     When the scoring process indicates sufficient confidence in the match between masses of a precursor ion and an expected peptide on the dynamic watch list (block  125 : Pass), the expected peptide is removed from the dynamic watch list and no further MS/MS scans are performed on the corresponding precursor ion. When the scoring process indicates insufficient confidence in the match (block  125 : Fail), the full MS/MS scan is performed on the respective precursor ion. 
     The fast MS/MS scan, performed pursuant to the scoring process of block  125 , for example, quickly confirms the identity of a precursor ion, saving valuable acquisition time. The saved acquisition time may be used for scanning other ions, thus increasing sequence coverage within the time constraints imposed by the chromatography. The fast MS/MS scan differs from a regular, full MS/MS scan in that the detector of the mass spectrometer (e.g., detector  220  in  FIG. 2 , below) spends less time collecting transient signals. The time savings are particularly apparent while acquiring spectra of large proteins, for which several of the associated peptides ionize efficiently. In contrast, conventional methods needlessly perform full acquisition for all ions belonging to the large protein. 
     When deciding whether the fast MS/MS scan of a precursor ion matches a candidate peptide on the dynamic watch list, prior information about other peptides present in the sample may be used in order to establish the likelihood that the candidate peptide is present in the sample. The likelihood of the presence of the candidate peptide is then used to establish a peptide-specific threshold for scoring the quality of the match. Establishing the peptide-specific threshold, e.g., using prior information of the sample composition decreases the rate of false positive and false negative spectrum-to-peptide assignments, as compared to a situation in which the threshold for confirmation of precursor ions is constant. Thus, using prior information about the sample composition makes confirmation of the identity of the precursor ion more reliable than not assuming any prior knowledge. 
       FIG. 2  is a functional block diagram illustrating a tandem mass spectrometry system  200 , according to a representative embodiment. The tandem mass spectrometry system  200  collects, measures, processes and/or analyzes various samples for identification of the molecular contents, such as peptides, amino acids, proteins and the like. 
     In the depicted representative embodiment, the tandem mass spectrometry system  200  includes a tandem mass spectrometer  205  and a signal processor  230 . The tandem mass spectrometer  205  includes an ionizer  210 , mass analyzers  215  and  216 , and a detector  220 . The ionizer  210  receives samples that include proteins to be identified, each protein consisting of corresponding peptides. The ionizer  210  may be an ESI or MALDI source, for example, that ionizes the sample proteins to provide precursor ions to the mass analyzers  215  and  216 . During an MS/MS scan, the mass analyzer  215  selects and fragments precursor ions and the mass analyzer  216  sorts the precursor ions according to respective masses. Although two representative mass analyzers  215  and  216  are shown, the tandem mass spectrometer  200  may include additional mass analyzers. The sorted ions are provided to detector  220 , which measures the abundance of ions of the various masses in a mass range mass, to generate qualitative or quantitative data regarding the sample. 
     The signal processor  230  performs various processing operations relating to the MS/MS acquisition, including peptide and protein identification, in accordance with various embodiments discussed below with respect to  FIG. 3 . As stated above, although depicted separately, the signal processor  230  may be included within one or any combination of the ionizer  210 , the analyzer  215  and the detector  220 , in various embodiments. 
       FIG. 3  is a flow diagram illustrating a method of performing tandem mass spectrometry analysis using a watch list, according to a representative embodiment. More particularly,  FIG. 3  shows a process for comparing precursor ion masses, obtained by an initial MS scan, with expected peptide matches from a dynamic watch list, and excluding match precursor ions from further MS/MS scans. The various operations of the method may correspond to modules, realized by hard-wired logic circuits or customizable hardware, a program running on a processor, such as signal processor  230 , or any combination thereof. 
     Block  320  of  FIG. 3  indicates a process by which the initial dynamic watch list is provided. The process of block  320  is optional, since, in some embodiments, the dynamic watch list starts as an empty list. The initial dynamic watch list includes at least one protein which is known to be in the sample (each referred to as an “expected protein”) and peptides corresponding to each expected protein on the list and thus expected to be present in the sample (referred to as “expected peptides”), as well as corresponding masses, sequences and other properties of the expected peptides. In an embodiment, the initial dynamic watch list is provided before beginning acquisition of mass spectra and contains a list of expected proteins and corresponding expected peptides. For example, the initial dynamic watch list may include expected proteins known to be present in the sample, e.g., due to the nature of the sample or because the sample has been “spiked” with the expected proteins. In various embodiments, the expected proteins are manually entered on the initialized dynamic watch list by a user, for example, or obtained automatically from a database of expected proteins/expected peptides. 
     Alternatively, as stated above, the initial dynamic watch list of block  320  may simply be an empty list, which is incrementally populated through MS and MS/MS scans and corresponding peptide identification. That is, once a peptide or set of peptides has been successfully identified, the protein(s) which contains the peptide(s) can be identified. For example, the protein(s) may be immediately identified following an indexing scheme of the type used in peptide sequence databases. In order to establish confidently the presence of the protein in the sample, a minimum number of different peptides (e.g., two or three) may be required. Alternatively a single peptide with a very high score may be acceptable for protein identification. 
     Once a protein is identified, a list of the corresponding expected peptides, such as tryptic peptides or other proteolytic peptides depending on sample preparation, is generated. The expected peptides, as well as corresponding masses, sequences and other properties of the expected peptides are added to the dynamic watch list corresponding to the identified protein. 
     Referring again to  FIG. 3 , an MS scan takes place at block  321  to obtain an MS spectrum, which has peaks corresponding to precursor ions of the sample. More particularly, the MS scan produces a mass spectrum composed of a measured abundance at each of a number of discrete masses in a range of masses, and the mass spectrum exhibits peaks at certain masses. The masses of the observed peaks, corresponding to precursor ions, are compared to the masses of the expected peptides on the dynamic watch list at block  322 . It is determined whether the mass of each observed peak matches the mass of a respective one of the expected peptides (referred to as a “candidate peptides”) in the dynamic watch list at block  323 . When the mass of the observed peak matches the mass of one of the expected peptides in the dynamic watch list (block  323 : Yes), precursor ions of the corresponding mass may be excluded from subsequent full MS/MS acquisitions and processing, as discussed below. 
     In an embodiment, the precursor ions corresponding to masses found on the dynamic watch list are excluded from subsequent full MS/MS scans and processing performed after subsequent determinations have been made in blocks  324  through  327  of  FIG. 3 . More particularly, a scoring process is performed in blocks  324  through  327  to prevent observed peaks from being improperly matched with expected peptides in the dynamic watch list, and such precursor ions thus being improperly excluded from subsequent MS/MS acquisitions. For example, as stated above, the dynamic watch list grows as acquisition continues. When the dynamic watch list becomes too large, peptides from proteins unrelated to the proteins in the dynamic watch list may be improperly excluded because masses of the unrelated peptides match the masses of expected peptides in the dynamic watch list merely by chance. As the dynamic watch list grows, the probability of chance matches increases. The scoring process in blocks  324  through  327  is intended to prevent such false matches. 
     In a representative embodiment, a fast MS/MS scan is performed at block  324  to obtain an observed product ion mass spectrum of the observed peak. In an embodiment, the tandem mass spectrometer  205  is used to select precursor ions corresponding to an observed peak corresponding in mass to one of the expected peptides in the dynamic watch list and to fragment the precursor ions. The tandem mass spectrometer  205  is then used to measure an observed product ion mass spectrum, which is the mass spectrum of the product ions corresponding to the observed peak. The observed product ion mass spectrum is compared to the spectrum of the candidate peptide in block  325  to provide a score associated with the match of the mass spectrum to the observed peak. That is, the observed product ion mass spectrum from the fast MS/MS scan is compared to the known product ion mass spectrum of the candidate peptide at block  325 , resulting in a score that represents the similarity between the observed peak and the candidate peptide. Notably, the discussion above refers to a single precursor ion, a single observed peak and a single candidate peptide for the sake of clarity, although multiple precursor ions, observed peaks and/or candidate peptides may be processed simultaneously. For example, the observed product ion mass spectrum of the observed peak may be scored against the known product ion mass spectra of some or all of the expected peptides in the dynamic watch list, as discussed below. 
     Referring again to block  325 , any appropriate scoring functions may be used, without departing from the scope of the disclosure. For example, one representative probabilistic scoring method includes a hypothesis test, which compares between two competing hypotheses regarding the relationship between an observed product ion mass spectrum and a peptide sequence. In a first hypothesis, peaks appearing in the observed product ion mass spectrum are the result of ions resulting from true cleavages taking place in the fragmentation process induced by a mass spectrometer. In the first hypothesis, some combinations of peaks (mass fragments) and peak intensities occur more frequently than others. In a second hypothesis, the peaks are the result of ions generated by a random fragmentation process. The score for the comparison of the observed product ion mass spectrum, S (obtained in block  324 ) and the known product ion mass spectrum of each candidate peptide, P in the dynamic watch list can be defined as follows: 
     
       
         
           
             
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     Prob(S|P) is the probability of the observed product ion mass spectrum S being generated by fragmentation of peptide P and Prob(S|R) is the probability of observed product ion mass spectrum S being generated by a random fragmentation process. The higher the score of the comparison, the more likely that the observed product ion mass spectrum corresponds to the candidate peptide. 
     Continuing with  FIG. 3 , at block  326 , it is determined whether the score of only one candidate peptide is high enough to pass (block  326 : Yes), resulting in identification of the peak associated with the precursor ion and excluding the identified precursor ion from further MS/MS scan(s) and processing at block  327 . For example, in an embodiment, when the score is above a predetermined threshold (block  326 : Yes), the peak of the precursor ion is considered to correspond to an identified peptide and that peak is subject to no further analysis. Also, the mass of expected peptide matching the mass of the precursor ion is removed from the dynamic watch list at block  327 . 
     After the mass of the precursor ion is removed from the dynamic watch list at block  327 , a determination is made whether there are additional masses corresponding to peaks of the MS scan in block  321  that have not yet been compared to the masses of expected peptides on the dynamic watch list. When there are additional masses (block  360 : Yes), the process returns to block  322  to compare the next mass with the masses of expected peptides on the dynamic watch list. When there are no more masses (block  360 : No), the process ends. Notably, the order of the processing operations may vary. For example, in an embodiment, all of the precursor ion masses are sequentially compared to the expected peptide masses on the dynamic watch list (blocks  322  and  323 ) before the scoring process (blocks  324  to  327 ) or a full MS/MS scan (block  342 , discussed below) is performed on any of the precursor ions. Also, in alternative embodiments, the precursor ion masses may be compared to the expected peptide masses (blocks  322  and  323 ) in parallel. 
     The scoring process is relatively fast compared to standard peptide identification because there is no database search and acquiring the fast MS/MS spectrum for each new precursor ion causes only a slight decrease in acquisition performance (e.g., slight increase in time). If none of the candidate peptides provides a score above the threshold, or if more than one of the candidate peptides provides a score above the threshold (for that peptide), respectively, the decision at block  326  is declared inconclusive (block  326 : No) and a full MS/MS scan of the precursor ion is executed in block  342 , as discussed below. 
     Further, the threshold value to which the score is compared in block  326  may be determined and adjusted by a variety of techniques. For example, in a representative embodiment, the threshold value may be determined using the number of peptides (from the same protein to which the candidate peptide belongs) that have been previously identified. In other words, the threshold value used at block  326  becomes peptide-dependent. For example, it may be assumed that peptides P 1  and P 2  are the highest and second highest scoring peptides among peptides in the watch list that have the same mass as the observed peak. It is assumed that there are no previously identified peptides of the protein(s) to which peptide P 1  belongs, but there are several peptides previously identified of the protein(s) to which P 2  belongs. Thus, at block  326 , the threshold value for matching the fast MS/MS spectrum with peptide P 2  may be set lower than that of peptide P 1 , since there is a high likelihood that peptide P 2  is present in the sample, e.g., based on previously obtained information. However, there is no previously obtained information on whether peptide P 1  exists in the sample. A peptide dependent threshold value is possible through access to peptide sequence information of previously acquired mass spectra. 
     In another representative embodiment, observed peak intensities of peptides previously identified (which belong to the same protein as the candidate peptide) are used in support of lowering the threshold value for the score test performed at block  326 . For example, the score threshold value is lowered when intensities of the observed peaks of previously identified peptides from the same protein correlate well with the intensity of the observed peak being scored. In another representative embodiment, properties of the observed peptide being matched to the observed product ion mass spectrum are used to establish the peptide-dependent score threshold value. The peptide properties include, but are not limited to, predicted retention time of the peptide and how proteotypic the peptide is. 
     In the first example, retention time is the time at which a peptide with a particular sequence elutes from reverse phase chromatography. The retention time is predicted or estimated using models, such as a Sequence Specific Retention Calculator. In the Sequence Specific Retention Calculator model, properties like the retention coefficient assigned to each amino-acid, nearest neighbors effects, clusters of hydrophobic amino acids, proline content, isoelectric point of the peptide, peptide length, and propensity for helical structure, for example, are taken into account to estimate the predicted elution time of the peptide. In the second example, proteotypic peptides refer to peptides of a protein that are frequently observed in a mass spectrometry experiment. Not all peptides have the same likelihood of being detected by mass spectrometry. For instance, peptides may not be observed due to incomplete proteolytic digestion, poor binding or elution from the chromatography column, small size, and/or poor ionization. There are classification models that can determine how likely it is that a peak corresponding to a particular peptide will be observed in a mass spectrum, such as the classification model described by Sanders et al.,  Prediction of Peptides Observable by Mass Spectrometry Applied at the Experimental Set Level , BMC B IOINFORMATICS  (2007), 8 (Suppl 7): S23, the contents of which is hereby incorporated by reference. Typical features of the peptide used in the classification models include, for example, number of pralines, percentage of glycine, alanine, leucine, polar amino acids, hydrophobic amino acids and negative amino acids, size, amphiphilicity index, and propensity to form helices. It the representative embodiment, predicted retention time and proteotypic nature of a peptide can be predicted because the sequence of each previously identified peptide may be used. 
     Using sequence information of peptides previously identified at block  326  enables customization of score thresholds to take into account the likelihood of the observed peptide being present in the sample at that particular time in the chromatography. For example, the higher the number of expected peptides, corresponding to a given protein on the dynamic watch list, for which corresponding peaks have been previously identified, the higher the likelihood that a new observed product ion mass spectrum corresponds to another expected peptide of this protein from the dynamic watch list. The peptide dependent thresholds produce more accurate peptide assignments, decreasing the rate of false positive and false negative identifications, and thus increasing the quality of the identifications made in the remainder of the process. 
     In a related embodiment, information from curated protein-protein interactions databases/models or physiological/disease pathways are used to adjust peptide specific score thresholds in order to decrease false assignments and to increase scoring confidence. For example, assume that more than one peptide from protein A and more than one peptide from protein B have already been identified, and that a newly acquired observed product ion mass spectrum matches, with a certain score, a peptide from protein C. The threshold for accepting or rejecting this score at block  326  can be lowered based on whether proteins A, B and C work together along the same pathway or whether they are known to interact with each other, i.e., whether there is prior information indicating that proteins A, B and C can be expected in the same protein sample, together. A similar use of curated pathways for off-line (post-acquisition) processing of mass spectra is described, for example, by Freeman et al.,  Identification of Biochemical Pathway Activity using Mass Spectrometry and Probabilistic Methods,  56 TH  ASMS C ONFERENCE ON  M ASS  S PECTROMETRY  (June 2008), the contents of which is hereby incorporated by reference. 
     Referring again to block  326 , when all of the comparison scores are below the threshold value (block  326 : No), or when more than one candidate peptide provides a score above the threshold value, a full MS/MS scan of the precursor ion is performed to identify the observed peptide as a bona fide new peptide. Performing the full MS/MS scan and analyzing the resulting mass spectrum is accomplished through blocks  342  through  348 . 
     More particularly, at block  342  a full MS/MS scan is performed, involving fragmentation of on the precursor ion. At block  344 , it is determined whether the precursor ion can be identified, for example, through de novo peptide sequencing, database search, or other means of peptide identification, based on the MS/MS acquisition. For example, the identification may be implemented using InsPecT, described by Tanner et al,  InsPecT. Identification of Posttranslationally Modified Peptides from Tandem Mass Spectra , A NAL . C HEM ., Vol. 77, No. 14. (July 2005), pp. 4626-4639, the contents of which is hereby incorporated by reference. Generally, InsPecT provides a sequence tagging algorithm that uses an efficient trie technique, in order to index large sequence databases to filter and score candidate peptides against a mass spectrum of interest. Given the trend of multiprocessor architectures, computation on GPUs, FGPAs and computer clusters, peptide identification time may be further reduced, since the algorithm used in InsPecT has been successfully parallelized. 
     When the precursor ion can be identified from the observed product ion mass spectrum of the full MS/MS scan (block  344 : Yes), the process proceeds to block  346 , which determines whether there is enough protein coverage for each of the proteins to which the identified peptide belongs. In other words, a determination is made, e.g., through the peptide mass comparison scores previously discussed and/or through any previously obtained information regarding proteins and/or peptides in the sample (including all proteins in the sample identified so far), on the probability that a protein associated with the identified peptide is present in the sample and whether that probability is high enough to proceed to block  348 . For example, as discussed above, a protein is not determined to be in the sample at block  346  unless a minimum number of corresponding peptides (including the peptides identified at blocks  327  and  344 ) have been identified. 
     When there is sufficient protein coverage (block  346 : Yes), the protein is added to the dynamic watch list at block  348 , together with its associated expected peptides and corresponding masses, which include all peptides from the protein except the peptides previously identified at block  344  or  326 . The expected peptides and corresponding masses and sequences may be obtained, for example, from a previously populated/characterized database. The masses, sequences, and any other property of the expected peptides that is considered useful (e.g. predicted retention time and proteotypic index), now part of the dynamic watch list, are then included in comparisons of subsequently acquired scans of new precursor ions, for example, at block  324 . After the protein and peptides have been added to the dynamic watch list (block  348 ), or when there is insufficient protein coverage to decide to add the protein (block  346 : No), or when the precursor ion cannot be identified (block  244 : No), the process advances to block  260 , where it is determined whether the acquisition process is to continue. 
     As discussed above, a determination is made at block  360  whether there are additional masses corresponding to peaks of the MS scan of block  321  that have not yet been compared to the masses of expected peptides on the dynamic watch list. When there are additional masses (block  360 : Yes), the process returns to block  322  to compare the next mass with the masses of expected peptides on the dynamic watch list. When there are no more masses (block  360 : No), the process ends. Notably, the order of the processing operations may vary. For example, in an embodiment, all of the precursor ion masses are sequentially compared to the expected peptide masses on the dynamic watch list (blocks  322  and  323 ) before the scoring process (blocks  324  to  327 ) or the full MS/MS scan (block  342 ) is performed on any of the precursor ions. Also, in alternative embodiments, the precursor ion masses may be compared to the expected peptide masses (blocks  322  and  323 ) in parallel. 
     Using this aggressive exclusion strategy that predicts expected peptides based on previously identified peptides, more acquisition time can be spent searching for new peptides and proteins, thus increasing the peptide and protein sequence coverage (number of different peptides/proteins) of the sample. Notably, the new proteins include post-translationally modified versions of proteins in the dynamic watch list, so the strategy also increases the coverage of post-translational modifications (PTMs). 
     In an alternative embodiment, when it is determined that the precursor ion can not be identified (block  344 : No), subsequent MS/MS acquisition(s) of the precursor ion may be performed in an attempt to identify the new peptide. That is, the process may effectively enter a loop (not shown) between additional MS/MS acquisitions and determining whether the precursor ion can be identified based on additional information obtained in each additional MS/MS acquisition, until the precursor ion can be identified or the process times out. 
     The decision process at block  346  may be based on a determination of how many peptides are necessary to confidently include a new protein in the dynamic watch list, for example, at block  348 . Such a determination may be affected by the biological question being asked. By using available databases that classify protein localization, function or physiological/disease pathway, a user may indicate that a particular class of proteins of interest should not be included in the dynamic watch list until a large number of corresponding peptides has been identified. Alternatively, the user could indicate that a class of proteins be aggressively excluded from full MS/MS scans, and thus placed on the dynamic watch list when as few as one peptide of the protein has been identified. For example, if Endoplasmic Reticulum (ER) contamination of a Golgi isolation is believed to have occurred during sample preparation, ER proteins could be aggressively excluded to increase coverage of the Golgi fraction, or everything else that is not associated with the ER. 
     The various embodiments described herein may be used for proteomics applications, although improving confidence in scores, in particular, may also be applied to metabolomics workflows. For example, the process for improving confidence in scores at blocks  324  through  327  may be applied to known metabolite distribution patterns (or fingerprints) of certain physiological states instead of pathways. Also, the watch list preciously mentioned would involve metabolites instead of proteins. 
     The various embodiments are intended to increase peptide and protein coverage. The dynamic watch list process generally enables protein quantitation via intensities of the peaks corresponding to precursor ions, for example. Notably, though, if spectral counting is to be implemented for quantitation, e.g., to quantify an amount of each protein or peptide, modifications may be made to take into account the fact that MS/MS spectra of some peptides will be obtained in a shorter amount of time due to block  324 , in which expected peptides are identified using the fast MS/MS scan. 
     Regardless of the origin and contents of the dynamic watch list, the dynamic watch list grows throughout the acquisition process of a sample as more proteins are identified. Further, because use of the dynamic watch list is generally more beneficial the larger the size of the list, the benefits of the dynamic watch list are higher at later stages of the acquisition, creating an increased performance gradient over time. 
     However, in an embodiment, this performance gradient may be neutralized to a degree through a repetitive LC/MS strategy that uses the watch list previously mentioned. The first run is used to build a complete list of expected peptides and proteins for the dynamic watch list, this time the dynamic watch list contains all peptides from proteins observed, plus all expected, but unidentified, peptides. Unlike standard exclusion lists, which contain masses of the ions to include, the watch list of the embodiment also contains sequence information of the peptides, as well as sequence-related properties of these peptides, such as predicted (and/or actual) retention times and proteotypicity. Furthermore, the watch list contains peptides from proteins that have been confidently identified in the previous run, regardless of whether the peptides were seen in the previous run. Then, in the second run, the dynamic watch list built from the first run may be used as a starting point to which additional entries are added. This solution is comprehensive and aggressive, since it excludes not just previously observed masses from further MS/MS acquisitions, but also predicted masses (via predicted peptides of observed proteins), regardless of their ion intensities (abundance in the sample). 
     The various embodiments are independent of the manner in which MS/MS spectra are acquired. Most MS/MS acquisition methods select precursor ions to scan according to predetermined criteria and then, for each precursor ion selected, acquire a full MS/MS spectrum. Any MS/MS acquisition modification which improves the probability of being able to match an MS/MS spectrum to a peptide (e.g., at blocks  342  through  344 ) has a synergistic (additive) effect with the embodiments described above. An example of an MS/MS acquisition modification for improving the probability of matches is described in U.S. patent application Ser. No. 12/465,001 by Satulovsky, entitled Data Dependent Acquisition System and Method, filed as patent application Ser. No. 12/465,001 on May 13, 2009, and published as U.S. patent application publication No. 2010/0288917 on Nov. 18, 2010, the contents of which is hereby incorporated by reference. 
       FIG. 4  is a functional block diagram illustrating a tandem mass spectrometry system  400 , according to a representative embodiment. The tandem mass spectrometry system  400  may be part of an LC/MS/MS system, for example, which collects, measures, processes and/or analyzes various samples for identification of the molecular contents, such as peptides, amino acids, proteins and the like. 
     In the depicted representative embodiment, the tandem mass spectrometry system  400  includes a tandem mass spectrometer  405  and a signal processor  430 . The tandem mass spectrometer includes an ionizer  410 , mass analyzers  415  and  416 , and a detector  420 . The ionizer  410  receives samples that include proteins to be identified, each protein consisting of corresponding peptides. The ionizer  410  may be an ESI or MALDI source, for example, that ionizes the sample proteins to provide precursor ions to the mass analyzers  415  and  416 . 
     During an MS/MS scan, the mass analyzer  415  selects and fragments precursor ions and the mass analyzer  416  sores the precursor ions according to respective masses. Although two representative mass analyzers  415  and  416  are shown, the tandem mass spectrometer  400  may include additional mass analyzers. The multiple mass analyzers  415  and  416  may be the same type, such as quadrupole/quadrupole mass spectrum analyzers, or different types, such as quadrupole/time-of-flight (Q-TOF) mass spectrum analyzers, for example. The sorted ions are provided to detector  420 , which measures the abundance of ions of the various masses in a mass range, to generate qualitative or quantitative data regarding the sample, as would be apparent. 
     The signal processor  430  performs various processing operations relating to the MS/MS acquisition, including peptide and protein identification, in accordance with various embodiments discussed above. The signal processor  430  includes central processing unit (CPU)  431 , internal memory  432 , bus  439  and interfaces  435 - 438 , and is configured to receive data from the detector  420  through detector interface  421 . In various embodiments, the signal processor  430  also interfaces with the ionizer  410  and the mass analyzers  415  and  416 , as needed, through respective interfaces (not shown). As stated above, it is understood that, although depicted separately, the signal processor  430  may be included within the detector  420 , or any combination of the ionizer  410 , the mass analyzers  415  and  416 , and the detector  420 , in various embodiments. 
     With respect to the signal processor  430 , the internal memory  432  includes at least nonvolatile read only memory (ROM)  433  and volatile random access memory (RAM)  434 , although it is understood that internal memory  432  may be implemented as any number, type and combination of ROM and RAM, and may provide look-up tables and/or other relational functionality. In various embodiments, the internal memory  432  may include a disk drive or flash memory, for example. Further, the internal memory  432  may store program instructions and results of calculations or summaries performed by CPU  431 . 
     The CPU  431  is configured to execute one or more software algorithms, including the peptide detection process using a dynamic watch list of the embodiments described herein, in conjunction with the internal memory  432 . In various embodiments, the CPU  431  may also execute software algorithms to control the basic functionality of the system  300 . The CPU  431  may include its own memory (e.g., nonvolatile memory) for storing executable software code that allows it to perform the various functions. Alternatively, the executable code may be stored in designated memory locations within internal memory  432 . The CPU  431  executes an operating system, such as Windows® operating systems available from Microsoft Corporation, Linux operating systems, Unix operating systems (e.g., Solaris™ available from Sun Microsystems, Inc.), or NetWare® operating systems available from Novell, Inc. The operating system may control execution of other programs, including collection and separation of samples, mass analysis and detection, e.g., by the ionizer  410 , the mass analyzer  415  and the detector  420 . 
     In an embodiment, a user and/or other computers may interact with the signal processor  430  using input device(s)  445  through I/O interface  435 . The input device(s)  445  may include any type of input device, for example, a keyboard, a track ball, a mouse, a touch pad or touch-sensitive display, and the like. Also, information may be displayed by the signal processor  430  on display  446  through display interface  436 , which may include any type of graphical user interface (GUI), for example. The displayed information includes the processing results obtained by the CPU  431  executing the method of peptide, described herein. 
     The processing results of the CPU  431  may also be stored in the database  348  through memory interface  438 . The database  448  may include any type and combination of volatile and/or nonvolatile storage medium and corresponding interface, including hard disk, compact disc (e.g., CD-R/CD/RW), universal serial bus (USB), flash memory, or the like. The stored processing results may be viewed, e.g., on the display  446 , and/or further processed at a later time. Also, the processing results may be provided to other computer systems connected to network  447  through network interface  437 . The network  447  may be any network capable of transporting electronic data, such as the Internet, a local area network (LAN), a wireless LAN, and the like. The network interface  437  may include, for example, a transceiver (not shown), including a receiver and a transmitter, that provides functionality for the tandem mass spectrometry system  400  to communicate wirelessly over the data network through an antenna system (not shown), according to appropriate standard protocols. However, it is understood that the network interface  437  may include any type of interface (wired or wireless) with the communications network, including various types of digital modems, for example. 
     The various “parts” shown in the signal processor  430  may be physically implemented using a software-controlled microprocessor, hard-wired logic circuits, or a combination thereof. Also, while the parts are functionally segregated in the signal processor  430  for explanation purposes, they may be combined variously in any physical implementation. 
     In accordance with various embodiments, protein and post-translational modifications coverage of a proteomics sample is increased, effectively extending the protein coverage of the tandem mass spectrometer. Increased coverage is achieved by predicting, based on information from previously acquired spectra, precursor ion masses that should only be confirmed and not analyzed during future acquisition events. 
     While specific embodiments are disclosed herein, many variations are possible, which remain within the concept and scope of the invention. Such variations would become apparent after inspection of the specification, drawings and claims herein. The invention therefore is not to be restricted except within the scope of the appended claims.