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1.  FDRAnalysis: A tool for the integrated analysis of tandem mass spectrometry identification results from multiple search engines 
Journal of proteome research  2011;10(4):2088-2094.
Confident identification of peptides via tandem mass spectrometry underpins modern high-throughput proteomics. This has motivated considerable recent interest in the post-processing of search engine results to increase confidence and calculate robust statistical measures, for example through the use of decoy databases to calculate false discovery rates (FDR). FDR-based analyses allow for multiple testing and can assign a single confidence value for both sets and individual peptide spectrum matches (PSMs). We recently developed an algorithm for combining the results from multiple search engines, integrating FDRs for sets of PSMs made by different search engine combinations. Here we describe a web-server, and a downloadable application, which makes this routinely available to the proteomics community. The web server offers a range of outputs including informative graphics to assess the confidence of the PSMs and any potential biases. The underlying pipeline provides a basic protein inference step, integrating PSMs into protein ambiguity groups where peptides can be matched to more than one protein. Importantly, we have also implemented full support for the mzIdentML data standard, recently released by the Proteomics Standards Initiative, providing users with the ability to convert native formats to mzIdentML files, which are available to download.
PMCID: PMC3707089  PMID: 21222473
bioinformatics; false discovery rate; multiple search engines; web server; data standards
2.  MyriMatch: highly accurate tandem mass spectral peptide identification by multivariate hypergeometric analysis 
Journal of proteome research  2007;6(2):654-661.
Shotgun proteomics experiments are dependent upon database search engines to identify peptides from tandem mass spectra. Many of these algorithms score potential identifications by evaluating the number of fragment ions matched between each peptide sequence and an observed spectrum. These systems, however, generally do not distinguish between matching an intense peak and matching a minor peak. We have developed a statistical model to score peptide matches that is based upon the multivariate hypergeometric distribution. This scorer, part of the “MyriMatch” database search engine, places greater emphasis on matching intense peaks. The probability that the best match for each spectrum has occurred by random chance can be employed to separate correct matches from random ones. We evaluated this software on data sets from three different laboratories employing three different ion trap instruments. Employing a novel system for testing discrimination, we demonstrate that stratifying peaks into multiple intensity classes improves the discrimination of scoring. We compare MyriMatch results to those of Sequest and X!Tandem, revealing that it is capable of higher discrimination than either of these algorithms. When minimal peak filtering is employed, performance plummets for a scoring model that does not stratify matched peaks by intensity. On the other hand, we find that MyriMatch discrimination improves as more peaks are retained in each spectrum. MyriMatch also scales well to tandem mass spectra from high-resolution mass analyzers. These findings may indicate limitations for existing database search scorers that count matched peaks without differentiating them by intensity. This software and source code is available under Mozilla Public License at this URL:
PMCID: PMC2525619  PMID: 17269722
Proteomics; Identification; Statistical Distribution; Reversed Database; Peak Filtering
3.  P17-M Improving Sensitivity by Combining Results from Multiple MS/MS Search Methodologies with the Scaffold Computer Algorithm 
Database-searching programs generally identify only a fraction of the spectra acquired in a standard LC/MS/MS study of digested proteins. Subtle variations in database-searching algorithms of MS/MS spectra have been known to provide different identification results. To leverage this variation, we developed Scaffold to probabilistically combine the results of multiple search engines, including Sequest, Mascot, and X!Tandem. Here we present a “tell all” explanation of the specific methodology behind Scaffold that converts scores into search engine independent peptide probabilities. These probabilities can be readily combined across search engines using Bayesian rules and the Expectation Maximization learning algorithm. We demonstrate how we normally gain 20% to 100% more highly confident (>95%) MS/MS spectrum identifications with each additional search engine, which is primarily due to increased confidence in low-scoring matches. We also show that this method works reliably across a variety of search engines and instrumentation platforms without re-tuning.
Figure 1
PMCID: PMC2291941
4.  Peptide identification based on fuzzy classification and clustering 
Proteome Science  2013;11(Suppl 1):S10.
The sequence database searching has been the dominant method for peptide identification, in which a large number of peptide spectra generated from LC/MS/MS experiments are searched using a search engine against theoretical fragmentation spectra derived from a protein sequences database or a spectral library. Selecting trustworthy peptide spectrum matches (PSMs) remains a challenge.
A novel scoring method named FC-Ranker is developed to assign a nonnegative weight to each target PSM based on the possibility of its being correct. Particularly, the scores of PSMs are updated by using a fuzzy SVM classification model and a fuzzy silhouette index iteratively. Trustworthy PSMs will be assigned high scores when the algorithm stops.
Our experimental studies show that FC-Ranker outperforms other post-database search algorithms over a variety of datasets, and it can be extended to solve a general classification problem with uncertain labels.
PMCID: PMC3908838  PMID: 24564935
Peptide identification; Peptide spectrum matches (PSMs); Fuzzy support vector machine (SVM); Fuzzy silhouette
5.  ScanRanker: Quality Assessment of Tandem Mass Spectra via Sequence Tagging 
Journal of proteome research  2011;10(7):2896-2904.
In shotgun proteomics, protein identification by tandem mass spectrometry relies on bioinformatics tools. Despite recent improvements in identification algorithms, a significant number of high quality spectra remain unidentified for various reasons. Here we present ScanRanker, an open-source tool that evaluates the quality of tandem mass spectra via sequence tagging with reliable performance in data from different instruments. The superior performance of ScanRanker enables it not only to find unassigned high quality spectra that evade identification through database search, but also to select spectra for de novo sequencing and cross-linking analysis. In addition, we demonstrate that the distribution of ScanRanker scores predicts the richness of identifiable spectra among multiple LC-MS/MS runs in an experiment, and ScanRanker scores assist the process of peptide assignment validation to increase confident spectrum identifications. The source code and executable versions of ScanRanker are available from
PMCID: PMC3128668  PMID: 21520941
spectral quality; sequence tagging; bioinformatics; tandem mass spectrometry; cross-linking
6.  Improvements to the Percolator algorithm for peptide identification from shotgun proteomics data sets 
Journal of proteome research  2009;8(7):3737-3745.
Shotgun proteomics coupled with database search software allows the identification of a large number of peptides in a single experiment. However, some existing search algorithms, such as SEQUEST, use score functions that are designed primarily to identify the best peptide for a given spectrum. Consequently, when comparing identifications across spectra, the SEQUEST score function Xcorr fails to discriminate accurately between correct and incorrect peptide identifications. Several machine learning methods have been proposed to address the resulting classification task of distinguishing between correct and incorrect peptide-spectrum matches (PSMs). A recent example is Percolator, which uses semi-supervised learning and a decoy database search strategy to learn to distinguish between correct and incorrect PSMs identified by a database search algorithm. The current work describes three improvements to Percolator. (1) Percolator’s heuristic optimization is replaced with a clear objective function, with intuitive reasons behind its choice. (2) Tractable nonlinear models are used instead of linear models, leading to improved accuracy over the original Percolator. (3) A method, Q-ranker, for directly optimizing the number of identified spectra at a specified q value is proposed, which achieves further gains.
PMCID: PMC2710313  PMID: 19385687
shotgun proteomics; tandem mass spectrometry; machine learning; peptide identification
7.  Adaptive Discriminant Function Analysis and Re-ranking of MS/MS Database Search Results for Improved Peptide Identification in Shotgun Proteomics 
Journal of proteome research  2008;7(11):4878-4889.
Robust statistical validation of peptide identifications obtained by tandem mass spectrometry and sequence database searching is an important task in shotgun proteomics. PeptideProphet is a commonly used computational tool that computes confidence measures for peptide identifications. In this paper, we investigate several limitations of the PeptideProphet modeling approach, including the use of fixed coefficients in computing the discriminant search score and selection of the top scoring peptide assignment per spectrum only. To address these limitations, we describe an adaptive method in which a new discriminant function is learned from the data in an iterative fashion. We extend the modeling framework to go beyond the top scoring peptide assignment per spectrum. We also investigate the effect of clustering the spectra according to their spectrum quality score followed by cluster-specific mixture modeling. The analysis is carried out using data acquired from a mixture of purified proteins on four different types of mass spectrometers, as well as using a complex human serum dataset. A special emphasis is placed on the analysis of data generated on high mass accuracy instruments.
PMCID: PMC3744223  PMID: 18788775
Tandem Mass Spectrometry; Database searching; Peptide Identification; Statistical Modeling; Adaptive Discriminant Analysis; Mass Accuracy; Decoy Sequences
8.  A New Probabilistic Database Search Algorithm for ETD Spectra 
Journal of proteome research  2009;8(6):3198-3205.
Peptide characterization using electron transfer dissociation (ETD) is an important analytical tool for protein identification. The fragmentation observed in ETD spectra is complementary to that seen when using the traditional dissociation method, collision activated dissociation (CAD). Applications of ETD enhance the scope and complexity of the peptides that can be studied by mass spectrometry-based methods. For example, ETD is shown to be particularly useful for the study of post-translationally modified peptides.
To take advantage of the power provided by ETD, it is important to have an ETD-specific database search engine - an integral tool of mass spectrometry-based analytical proteomics. In this paper, we report on our development of a database search engine using ETD spectra and protein sequence databases to identify peptides. The search engine is based on the probabilistic modeling of shared peaks count and shared peaks intensity between the spectra and the peptide sequences. The shared peaks count accounts for the cumulative variations from amino acid sequences, while shared peaks intensity models the variations between the candidate sequence and product ion intensities. To demonstrate the utility of this algorithm for searching real-world data, we present the results of applications of this model to two high throughput data sets. Both data sets were obtained from yeast whole cell lysates. The first data set was obtained from a sample digested by Lys-C and the second data set was obtained by a digestion using trypsin. We searched the data sets against a combined forward and reversed yeast protein database to estimate false discovery rates. We compare the search results from the new methods with the results from a search engine often employed for ETD spectra, OMSSA. Our findings show that overall the new model performs comparably to OMSSA for low false discovery rates. At the same time, we demonstrate that there are substantial differences with OMSSA for results on subsets of data. Therefore, we conclude the new model can be considered as being complementary to previously developed models.
We also analyze the effect of the precursor mass accuracy on the false discovery rates of peptide identifications. It is shown that a substantial (30%) improvement on false discovery rates is achieved by the use of the mass accuracy information in combination with the database search results.
PMCID: PMC2730765  PMID: 19354237
9.  IDPicker 2.0: Improved Protein Assembly with High Discrimination Peptide Identification Filtering 
Journal of proteome research  2009;8(8):3872-3881.
Tandem mass spectrometry-based shotgun proteomics has become a widespread technology for analyzing complex protein mixtures. A number of database searching algorithms have been developed to assign peptide sequences to tandem mass spectra. Assembling the peptide identifications to proteins, however, is a challenging issue because many peptides are shared among multiple proteins. IDPicker is an open-source protein assembly tool that derives a minimum protein list from peptide identifications filtered to a specified False Discovery Rate. Here, we update IDPicker to increase confident peptide identifications by combining multiple scores produced by database search tools. By segregating peptide identifications for thresholding using both the precursor charge state and the number of tryptic termini, IDPicker retrieves more peptides for protein assembly. The new version is more robust against false positive proteins, especially in searches using multispecies databases, by requiring additional novel peptides in the parsimony process. IDPicker has been designed for incorporation in many identification workflows by the addition of a graphical user interface and the ability to read identifications from the pepXML format. These advances position IDPicker for high peptide discrimination and reliable protein assembly in large-scale proteomics studies. The source code and binaries for the latest version of IDPicker are available from
PMCID: PMC2810655  PMID: 19522537
bioinformatics; parsimony; protein assembly; protein inference; false discovery rate
10.  Statistical calibration of the SEQUEST XCorr function 
Obtaining accurate peptide identifications from shotgun proteomics liquid chromatography tandem mass spectrometry (LC-MS/MS) experiments requires a score function that consistently ranks correct peptide-spectrum matches (PSMs) above incorrect matches. We have observed that, for the Sequest score function X corr, the inability to discriminate between correct and incorrect PSMs is due in part to spectrum-specific properties of the score distribution. In other words, some spectra score well regardless of which peptides they are scored against, and other spectra score well because they are scored against a large number of peptides. We describe a protocol for calibrating PSM score functions, and we demonstrate its application to X corr and the preliminary Sequest score function Sp. The protocol accounts for spectrum- and peptide-specific effects by calculating p values for each spectrum individually, using only that spectrum’s score distribution. We demonstrate that these calculated p values are uniform under a null distribution and therefore accurately measure significance. These p values can be used to estimate the false discovery rate, therefore eliminating the need for an extra search against a decoy database. In addition, we show that the p values are better calibrated than their underlying scores; consequently, when ranking top-scoring PSMs from multiple spectra, p values are better at discriminating between correct and incorrect PSMs. The calibration protocol is generally applicable to any PSM score function for which an appopriate parametric family can be identified.
PMCID: PMC2807930  PMID: 19275164
calibration; database search; peptide identification; tandem mass spectrometry
11.  ScanRanker: Quality Assessment of Tandem Mass Spectra via Sequence Tagging 
Tandem mass spectrometry has become a remarkably powerful technology to identify proteins in proteomics. Bioinformatics tools, especially database searching tools, are essential for the interpretation of large quantities of proteomics data. Despite recent improvements in database searching algorithms, only a relatively small fraction of spectra can be confidently assigned to peptide sequences in a typical proteomics analysis. The remaining unassigned spectra often consist of low quality spectra that cause a significant amount of computational overhead but that contribute little to protein identification. On the other hand, many high quality spectra remain unassigned due to modifications, mutations, and the deficiencies of the scoring methods implemented in database searching tools. Here we present ScanRanker, an open-source algorithm that offers a robust method for spectral quality assessment. Unlike existing tools that require training software for each type of instrument to be employed, ScanRanker evaluates quality of tandem mass spectra via sequence tagging, providing reliable performance in data sets from different instruments. The superior performance of ScanRanker enables it not only to filter low quality spectra prior to database searching, but also to find unassigned high quality spectra that evade identification through database search.
PMCID: PMC2918183
12.  Two-Dimensional Target Decoy Strategy for Shotgun Proteomics 
Journal of proteome research  2011;10(12):5296-5301.
The target-decoy approach to estimating and controlling false discovery rate (FDR) has become a de facto standard in shotgun proteomics, and it has been applied at both the peptide-to-spectrum match (PSM) and protein levels. Current bioinformatics methods control either the PSM- or the protein-level FDR, but not both. In order to obtain the most reliable information from their data, users must employ one method when the number of tandem mass spectra exceeds the number of proteins in the database and another method when the reverse is true. Here we propose a simple variation of the standard target-decoy strategy that estimates and controls PSM and protein FDRs simultaneously, regardless of the relative numbers of spectra and proteins. We demonstrate that even if the final goal is a list of PSMs with a fixed low FDR and not a list of protein identifications, the proposed two-dimensional strategy offers advantages over a pure PSM-level strategy.
PMCID: PMC3230778  PMID: 22010998
Mass spectrometry; target decoy strategy; false discovery rate; peptide identification; protein identification
13.  Non-parametric estimation of posterior error probabilities associated with peptides identified by tandem mass spectrometry 
Bioinformatics  2008;24(16):i42-i48.
Motivation: A mass spectrum produced via tandem mass spectrometry can be tentatively matched to a peptide sequence via database search. Here, we address the problem of assigning a posterior error probability (PEP) to a given peptide-spectrum match (PSM). This problem is considerably more difficult than the related problem of estimating the error rate associated with a large collection of PSMs. Existing methods for estimating PEPs rely on a parametric or semiparametric model of the underlying score distribution.
Results: We demonstrate how to apply non-parametric logistic regression to this problem. The method makes no explicit assumptions about the form of the underlying score distribution; instead, the method relies upon decoy PSMs, produced by searching the spectra against a decoy sequence database, to provide a model of the null score distribution. We show that our non-parametric logistic regression method produces accurate PEP estimates for six different commonly used PSM score functions. In particular, the estimates produced by our method are comparable in accuracy to those of PeptideProphet, which uses a parametric or semiparametric model designed specifically to work with SEQUEST. The advantage of the non-parametric approach is applicability and robustness to new score functions and new types of data.
Availability: C++ code implementing the method as well as supplementary information is available at
PMCID: PMC2732210  PMID: 18689838
14.  Calibrating E-values for MS2 database search methods 
Biology Direct  2007;2:26.
The key to mass-spectrometry-based proteomics is peptide identification, which relies on software analysis of tandem mass spectra. Although each search engine has its strength, combining the strengths of various search engines is not yet realizable largely due to the lack of a unified statistical framework that is applicable to any method.
We have developed a universal scheme for statistical calibration of peptide identifications. The protocol can be used for both de novo approaches as well as database search methods. We demonstrate the protocol using only the database search methods. Among seven methods -SEQUEST (v27 rev12), ProbID (v1.0), InsPecT (v20060505), Mascot (v2.1), X!Tandem (v1.0), OMSSA (v2.0) and RAId_DbS – calibrated, except for X!Tandem and RAId_DbS most methods require a rescaling according to the database size searched. We demonstrate that our calibration protocol indeed produces unified statistics both in terms of average number of false positives and in terms of the probability for a peptide hit to be a true positive. Although both the protocols for calibration and the statistics thus calibrated are universal, the calibration formulas obtained from one laboratory with data collected using either centroid or profile format may not be directly usable by the other laboratories. Thus each laboratory is encouraged to calibrate the search methods it intends to use. We also address the importance of using spectrum-specific statistics and possible improvement on the current calibration protocol. The spectra used for statistical (E-value) calibration are freely available upon request.
Open peer review
Reviewed by Dongxiao Zhu (nominated by Arcady Mushegian), Alexey Nesvizhskii (nominated by King Jordan) and Vineet Bafna. For the full reviews, please go to the Reviewers' comments section.
PMCID: PMC2206012  PMID: 17983478
15.  Pepitome: evaluating improved spectral library search for identification complementarity and quality assessment 
Journal of Proteome Research  2012;11(3):1686-1695.
Spectral libraries have emerged as a viable alternative to protein sequence databases for peptide identification. These libraries contain previously detected peptide sequences and their corresponding tandem mass spectra (MS/MS). Search engines can then identify peptides by comparing experimental MS/MS scans to those in the library. Many of these algorithms employ the dot product score for measuring the quality of a spectrum-spectrum match (SSM). This scoring system does not offer a clear statistical interpretation and ignores fragment ion m/z discrepancies in the scoring. We developed a new spectral library search engine, Pepitome, which employs statistical systems for scoring SSMs. Pepitome outperformed the leading library search tool, SpectraST, when analyzing data sets acquired on three different mass spectrometry platforms. We characterized the reliability of spectral library searches by confirming shotgun proteomics identifications through RNA-Seq data. Applying spectral library and database searches on the same sample revealed their complementary nature. Pepitome identifications enabled the automation of quality analysis and quality control (QA/QC) for shotgun proteomics data acquisition pipelines.
PMCID: PMC3292681  PMID: 22217208
16.  Improving sensitivity in proteome studies by analysis of false discovery rates for multiple search engines 
Proteomics  2009;9(5):1220-1229.
Tandem mass spectrometry, run in combination with liquid chromatography (LC-MS/MS), can generate large numbers of peptide and protein identifications, for which a variety of database search engines are available. Distinguishing correct identifications from false positives is far from trivial because all data sets are noisy, and tend to be too large for manual inspection, therefore probabilistic methods must be employed to balance the trade-off between sensitivity and specificity. Decoy databases are becoming widely used to place statistical confidence in results sets, allowing the false discovery rate (FDR) to be estimated. It has previously been demonstrated that different MS search engines produce different peptide identification sets, and as such, employing more than one search engine could result in an increased number of peptides being identified. However, such efforts are hindered by the lack of a single scoring framework employed by all search engines.
We have developed a search engine independent scoring framework based on FDR which allows peptide identifications from different search engines to be combined, called the FDRScore. We observe that peptide identifications made by three search engines are infrequently false positives, and identifications made by only a single search engine, even with a strong score from the source search engine, are significantly more likely to be false positives. We have developed a second score based on the FDR within peptide identifications grouped according to the set of search engines that have made the identification, called the combined FDRScore. We demonstrate by searching large publicly available data sets that the combined FDRScore can differentiate between between correct and incorrect peptide identifications with high accuracy, allowing on average 35% more peptide identifications to be made at a fixed FDR than using a single search engine.
PMCID: PMC2899855  PMID: 19253293
proteomics; mass spectrometry; decoy database; search engine; scoring; false discovery rate
17.  Spectral Probabilities and Generating Functions of Tandem Mass Spectra: a Strike Against Decoy Databases 
Journal of proteome research  2008;7(8):3354-3363.
A key problem in computational proteomics is distinguishing between correct and false peptide identifications. We argue that evaluating the error rates of peptide identifications is not unlike computing generating functions in combinatorics. We show that the generating functions and their derivatives (spectral energy and spectral probability) represent new features of tandem mass spectra that, similarly to Δ-scores, significantly improve peptide identifications. Furthermore, the spectral probability provides a rigorous solution to the problem of computing statistical significance of spectral identifications. The spectral energy/probability approach improves the sensitivity-specificity trade-off of existing MS/MS search tools, addresses the notoriously difficult problem of “one-hit-wonders” in mass spectrometry, and often eliminates the need for decoy database searches. We therefore argue that the generating function approach has the potential to increase the number of peptide identifications in MS/MS searches.
PMCID: PMC2689316  PMID: 18597511
18.  Accelerating the scoring module of mass spectrometry-based peptide identification using GPUs 
BMC Bioinformatics  2014;15:121.
Tandem mass spectrometry-based database searching is currently the main method for protein identification in shotgun proteomics. The explosive growth of protein and peptide databases, which is a result of genome translations, enzymatic digestions, and post-translational modifications (PTMs), is making computational efficiency in database searching a serious challenge. Profile analysis shows that most search engines spend 50%-90% of their total time on the scoring module, and that the spectrum dot product (SDP) based scoring module is the most widely used. As a general purpose and high performance parallel hardware, graphics processing units (GPUs) are promising platforms for speeding up database searches in the protein identification process.
We designed and implemented a parallel SDP-based scoring module on GPUs that exploits the efficient use of GPU registers, constant memory and shared memory. Compared with the CPU-based version, we achieved a 30 to 60 times speedup using a single GPU. We also implemented our algorithm on a GPU cluster and achieved an approximately favorable speedup.
Our GPU-based SDP algorithm can significantly improve the speed of the scoring module in mass spectrometry-based protein identification. The algorithm can be easily implemented in many database search engines such as X!Tandem, SEQUEST, and pFind. A software tool implementing this algorithm is available at
PMCID: PMC4049470  PMID: 24773593
19.  A Novel Algorithm for Validating Peptide Identification from a Shotgun Proteomics Search Engine 
Journal of proteome research  2013;12(3):1108-1119.
Liquid chromatography coupled with tandem mass spectrometry has revolutionized the proteomics analysis of complexes, cells, and tissues. In a typical proteomic analysis, the tandem mass spectra from a LC/MS/MS experiment are assigned to a peptide by a search engine that compares the experimental MS/MS peptide data to theoretical peptide sequences in a protein database. The peptide spectra matches are then used to infer a list of identified proteins in the original sample. However, the search engines often fail to distinguish between correct and incorrect peptides assignments. In this study, we designed and implemented a novel algorithm called De-Noise to reduce the number of incorrect peptide matches and maximize the number of correct peptides at a fixed false discovery rate using a minimal number of scoring outputs from the SEQUEST search engine. The novel algorithm uses a three step process: data cleaning, data refining through a SVM-based decision function, and a final data refining step based on proteolytic peptide patterns. Using proteomics data generated on different types of mass spectrometers, we optimized the De-Noise algorithm based on the resolution and mass accuracy of the mass spectrometer employed in the LC/MS/MS experiment. Our results demonstrate De-Noise improves peptide identification compared to other methods used to process the peptide sequence matches assigned by SEQUEST. Because De-Noise uses a limited number of scoring attributes, it can be easily implemented with other search engines.
PMCID: PMC3608465  PMID: 23402659
proteomics; mass spectrometry; bioinformatics; support vector machines; peptide spectrum match; database search engine; validation
20.  Integrating de Novo Sequencing and Database Search for Peptide Identification 
Introduction: Peptide identification with high sensitivity and accuracy is vital in mass spectrometry-based proteomics. One approach to increase confidence of peptide identification is through high resolution tandem mass spectrometry on both precursor and fragment steps. A workflow is resented to combine de novo sequencing and database search for peptide identification with high resolution data. METHODS: The workflow integrates de novo sequencing and database searching for peptide identification. It contains 3 steps. 1. Perform database search with all MS/MS spectra against protein sequence database. Database peptides were selected with 1% FDR. 2. For unidentified spectra in step 1, perform modification search using confident de novo tags and turning on all modifications in Unimod database. Peptides containing un-suspected modifications were selected with 1% FDR. 3. Select the spectra with high confident de novo sequences but not identified in above steps. RESULTS: The workflow was implemented in PEAKS. A high resolution MS dataset published by D.S. Kelkar on MCP was tested, in which 253394 MS/MS spectra were obtained from cell lysates of Mycobacterium tuberculosis with strong cation exchange chromatography on LTQ-Orbitrap Velos. 112480 peptide-spectrum matches (PSMs) were identified by database sequence searching in step 1, with 5 ppm precursor mass errors and 40 ppm of fragment mass errors. 28120 of 112480 spectra have de novo confidence scores (ALC) great than 70%. Compared with database peptides, the percentage of consistent amino acids for de novo sequences is 94%. 5706 PSMs were identified by modification search in step 2, with 5 ppm precursor mass errors and 45 ppm of fragment mass errors. In addition, 3976 PSMs with ALC great than 70% were selected in step 3, with 5 ppm precursor mass errors and 42 ppm of fragment mass errors. CONCLUSION: Integrating de novo sequencing and database search improves peptide identification.
PMCID: PMC3630621
21.  MUMAL: Multivariate analysis in shotgun proteomics using machine learning techniques 
BMC Genomics  2012;13(Suppl 5):S4.
The shotgun strategy (liquid chromatography coupled with tandem mass spectrometry) is widely applied for identification of proteins in complex mixtures. This method gives rise to thousands of spectra in a single run, which are interpreted by computational tools. Such tools normally use a protein database from which peptide sequences are extracted for matching with experimentally derived mass spectral data. After the database search, the correctness of obtained peptide-spectrum matches (PSMs) needs to be evaluated also by algorithms, as a manual curation of these huge datasets would be impractical. The target-decoy database strategy is largely used to perform spectrum evaluation. Nonetheless, this method has been applied without considering sensitivity, i.e., only error estimation is taken into account. A recently proposed method termed MUDE treats the target-decoy analysis as an optimization problem, where sensitivity is maximized. This method demonstrates a significant increase in the retrieved number of PSMs for a fixed error rate. However, the MUDE model is constructed in such a way that linear decision boundaries are established to separate correct from incorrect PSMs. Besides, the described heuristic for solving the optimization problem has to be executed many times to achieve a significant augmentation in sensitivity.
Here, we propose a new method, termed MUMAL, for PSM assessment that is based on machine learning techniques. Our method can establish nonlinear decision boundaries, leading to a higher chance to retrieve more true positives. Furthermore, we need few iterations to achieve high sensitivities, strikingly shortening the running time of the whole process. Experiments show that our method achieves a considerably higher number of PSMs compared with standard tools such as MUDE, PeptideProphet, and typical target-decoy approaches.
Our approach not only enhances the computational performance, and thus the turn around time of MS-based experiments in proteomics, but also improves the information content with benefits of a higher proteome coverage. This improvement, for instance, increases the chance to identify important drug targets or biomarkers for drug development or molecular diagnostics.
PMCID: PMC3477001  PMID: 23095859
Machine learning; Bioinformatics; Peptide/protein identification; Shotgun proteomics; Phosphoproteomics; Tandem mass spectrometry
22.  Improving Peptide Identification Sensitivity in Shotgun Proteomics by Stratification of Search Space 
Journal of proteome research  2013;12(6):2571-2581.
Due to its high specificity, trypsin is the enzyme of choice in shotgun proteomics. Nonetheless, several publications do report the identification of semi-tryptic and non-tryptic peptides. Many of these peptides are conjectured to be signaling peptides or to have formed during sample preparation. It is known that only a small fraction of tandem mass spectra from a trypsin-digested protein mixture can be confidently matched to tryptic peptides. Leaving aside other possibilities such as post-translational modifications and single amino acid polymorphisms, this suggests that many unidentified spectra originate from semi-tryptic and non-tryptic peptides. To include them in database searches, however, may not improve overall peptide identification due to possible sensitivity reduction from search space expansion. To circumvent this issue for E-value based search methods, we have designed a scheme that categorizes qualified peptides ( i.e., peptides whose molecular weight differences from the parent ion are within a specified error tolerance) into three tiers: tryptic, semi-tryptic and non-tryptic. This classification allows peptides belonging to different tiers to have different Bonferroni correction factors. Our results show that this scheme can significantly improve retrieval performance when compared to search strategies that assign equal Bonferroni correction factors to all qualified peptides.
PMCID: PMC3753091  PMID: 23668635
23.  IDSieve: Protein Identification Using Peptide pI Filtering of MS/MS Data for Improved Confidence in Identifications 
The main challenge of tandem mass spectrometry based proteomic analysis is to correctly match the tandem mass spectra produced to the correct peptides. However, the large number of protein sequences in a database increases the chances of a false positive identification for any given peptide match. Here we present an automated algorithm called IDSieve that utilizes target-decoy database search strategy in combination with pI filtering to allow greater confidence for peptide identifications. IDSieve considers the SEQUEST parameters Xcorr and äCn to assign statistical confidence (false discovery rates) to the peptide matches. The distribution of predicted pI values for peptide spectrum matches (PSMs) is considered separately for each immobilized pH gradient isoelectric focusing fraction, and matches with pI values within 1.5 times inter-quartile range (within pI range) are analyzed independently of matches outside the pI ranges. We tested the performance of IDSieve and Peptide/Protein Prophet on the SEQUEST outputs from 60 immobilized pH gradient isoelectric focusing fractions derived from mouse intestinal epithelial cell protein extracts. Our results demonstrated that IDSieve produced 1355 more peptide spectrum matches (or 330 more peptides) than Peptide Prophet using comparable false positive rate cutoffs. Therefore, combining pI filtering with the appropriate statistical significance measurements allows for a higher number of protein identifications without adversely affecting the false positive rate. We further tested the performance of pI filtering using ID Sieve when samples were prefractionated using either pH range 3.5–4.5 or 3–10, and either 24cm or 7cm IPG strips.
PMCID: PMC3186675
24.  xComb: a cross-linked peptide database approach to protein-protein interaction analysis 
Journal of proteome research  2010;9(5):2508-2515.
We developed an informatic method to identify tandem mass spectra composed of chemically cross-linked peptides from those of linear peptides and to assign sequence to each of the two unique peptide sequences. For a given set of proteins the key software tool, xComb, combs through all theoretically feasible cross-linked peptides to create a database consisting of a subset of all combinations represented as peptide FASTA files. The xComb library of select theoretical cross-linked peptides may then be used as a database that is examined by a standard proteomic search engine to match tandem mass spectral datasets to identify cross-linked peptides. The database search may be conducted against as many as 50 proteins with a number of common proteomic search engines, e.g. Phenyx, Sequest, OMSSA, Mascot and X!Tandem. By searching against a peptide library of linearized, cross-linked peptides, rather than a linearized protein library, search times are decreased and the process is decoupled from any specific search engine. A further benefit of decoupling from the search engine is that protein cross-linking studies may be conducted with readily available informatics tools for which scoring routines already exist within the proteomic community.
PMCID: PMC2884221  PMID: 20302351
25.  Quality assessment of peptide tandem mass spectra 
BMC Bioinformatics  2008;9(Suppl 6):S13.
Tandem mass spectrometry has emerged as a cornerstone of high throughput proteomic studies owing in part to various high throughput search engines which are used to interpret these tandem mass spectra. However, majority of experimental tandem mass spectra cannot be interpreted by any existing methods. There are many reasons why this happens. However, one of the most important reasons is that majority of experimental spectra are of too poor quality to be interpretable. It wastes time to interpret these "uninterpretable" spectra by any methods. On the other hand, some spectra of high quality are not able to get a score high enough to be interpreted by existing search engines because there are many similar peptides in the searched database. However, such spectra may be good enough to be interpreted by de novo methods or manually verifying methods. Therefore, it is worth in developing a method for assessing spectral quality, which can used for filtering the spectra of poor quality before any interpretation attempts or for finding the most potential candidates for de novo methods or manually verifying methods.
This paper develops a novel method to assess the quality of tandem mass spectra, which can eliminate majority of poor quality spectra while losing very minority of high quality spectra. First, a number of features are proposed to describe the quality of tandem mass spectra. The proposed method maps each tandem spectrum into a feature vector. Then Fisher linear discriminant analysis (FLDA) is employed to construct the classifier (the filter) which discriminates the high quality spectra from the poor quality ones. The proposed method has been tested on two tandem mass spectra datasets acquired by ion trap mass spectrometers.
Computational experiments illustrate that the proposed method outperforms the existing ones. The proposed method is generic, and is expected to be applicable to assessing the quality of spectra acquired by instruments other than ion trap mass spectrometers.
PMCID: PMC2423436  PMID: 18541048

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