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1.  Addressing Statistical Biases in Nucleotide-Derived Protein Databases for Proteogenomic Search Strategies 
Journal of Proteome Research  2012;11(11):5221-5234.
Proteogenomics has the potential to advance genome annotation through high quality peptide identifications derived from mass spectrometry experiments, which demonstrate a given gene or isoform is expressed and translated at the protein level. This can advance our understanding of genome function, discovering novel genes and gene structure that have not yet been identified or validated. Because of the high-throughput shotgun nature of most proteomics experiments, it is essential to carefully control for false positives and prevent any potential misannotation. A number of statistical procedures to deal with this are in wide use in proteomics, calculating false discovery rate (FDR) and posterior error probability (PEP) values for groups and individual peptide spectrum matches (PSMs). These methods control for multiple testing and exploit decoy databases to estimate statistical significance. Here, we show that database choice has a major effect on these confidence estimates leading to significant differences in the number of PSMs reported. We note that standard target:decoy approaches using six-frame translations of nucleotide sequences, such as assembled transcriptome data, apparently underestimate the confidence assigned to the PSMs. The source of this error stems from the inflated and unusual nature of the six-frame database, where for every target sequence there exists five “incorrect” targets that are unlikely to code for protein. The attendant FDR and PEP estimates lead to fewer accepted PSMs at fixed thresholds, and we show that this effect is a product of the database and statistical modeling and not the search engine. A variety of approaches to limit database size and remove noncoding target sequences are examined and discussed in terms of the altered statistical estimates generated and PSMs reported. These results are of importance to groups carrying out proteogenomics, aiming to maximize the validation and discovery of gene structure in sequenced genomes, while still controlling for false positives.
doi:10.1021/pr300411q
PMCID: PMC3703792  PMID: 23025403
proteogenomics; peptide spectrum match; false discovery rate; posterior error probability; expressed sequence tag
2.  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.
doi:10.1002/pmic.200800473
PMCID: PMC2899855  PMID: 19253293
proteomics; mass spectrometry; decoy database; search engine; scoring; false discovery rate
3.  Enhanced Peptide Identification by Electron Transfer Dissociation Using an Improved Mascot Percolator* 
Peptide identification using tandem mass spectrometry is a core technology in proteomics. Latest generations of mass spectrometry instruments enable the use of electron transfer dissociation (ETD) to complement collision induced dissociation (CID) for peptide fragmentation. However, a critical limitation to the use of ETD has been optimal database search software. Percolator is a post-search algorithm, which uses semi-supervised machine learning to improve the rate of peptide spectrum identifications (PSMs) together with providing reliable significance measures. We have previously interfaced the Mascot search engine with Percolator and demonstrated sensitivity and specificity benefits with CID data. Here, we report recent developments in the Mascot Percolator V2.0 software including an improved feature calculator and support for a wider range of ion series. The updated software is applied to the analysis of several CID and ETD fragmented peptide data sets. This version of Mascot Percolator increases the number of CID PSMs by up to 80% and ETD PSMs by up to 60% at a 0.01 q-value (1% false discovery rate) threshold over a standard Mascot search, notably recovering PSMs from high charge state precursor ions. The greatly increased number of PSMs and peptide coverage afforded by Mascot Percolator has enabled a fuller assessment of CID/ETD complementarity to be performed. Using a data set of CID and ETcaD spectral pairs, we find that at a 1% false discovery rate, the overlap in peptide identifications by CID and ETD is 83%, which is significantly higher than that obtained using either stand-alone Mascot (69%) or OMSSA (39%). We conclude that Mascot Percolator is a highly sensitive and accurate post-search algorithm for peptide identification and allows direct comparison of peptide identifications using multiple alternative fragmentation techniques.
doi:10.1074/mcp.O111.014522
PMCID: PMC3412976  PMID: 22493177
4.  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.
doi:10.1021/pr101157s
PMCID: PMC3707089  PMID: 21222473
bioinformatics; false discovery rate; multiple search engines; web server; data standards
5.  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.
doi:10.1021/pr8011107
PMCID: PMC2807930  PMID: 19275164
calibration; database search; peptide identification; tandem mass spectrometry
6.  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
7.  Decoy Methods for Assessing False Positives and False Discovery Rates in Shotgun Proteomics 
Analytical chemistry  2009;81(1):146-159.
The potential of getting a significant number of false positives (FPs) in peptide-spectrum matches (PSMs) obtained by proteomic database search has been well-recognized. Among the attempts to assess FPs, the concomitant use of target and decoy databases is widely practiced. By adjusting filtering criteria, FPs and false discovery rate (FDR) can be controlled at a desired level. Although the target-decoy approach is gaining in popularity, subtle differences in decoy construction (e.g., reversing vs. stochastic methods), rate calculation (e.g., total vs. unique PSMs), or searching (separate vs. composite) do exist among various implementations. In the present study, we evaluated the effects of these differences on FP and FDR estimations using a rat kidney protein sample and the SEQUEST search engine as an example. On the effects of decoy construction, we found that, when a single scoring filter (XCorr) was used, stochastic methods generated a higher estimation of FPs and FDR than sequence reversing methods, likely due to an increase in unique peptides. This higher estimation could largely be attenuated by creating decoy databases similar in effective size, but not by a simple normalization with a unique-peptide coefficient. When multiple filters were applied, the differences seen between reversing and stochastic methods significantly diminished, suggesting multiple filterings reduce the dependency on how a decoy is constructed. For a fixed set of filtering criteria, FDR and FPs estimated by using unique PSMs were almost twice those using total PSMs. The higher estimation seemed to be dependent on data acquisition setup. As to the differences between performing separate or composite searches, in general, FDR estimated from separate search was about three times that from composite search. The degree of difference gradually decreased as the filtering criteria became more stringent. Paradoxically, the estimated true positives in separate search were higher when multiple filters were used. By analyzing a standard protein mixture, we demonstrated that the higher estimation of FDR and FPs in separate search likely reflected an overestimation, which could be corrected with a simple merging procedure. Our study illustrates the relative merits of different implementations of the target-decoy strategy, which should be worth contemplating when large-scale proteomic biomarker discovery is to be attempted.
doi:10.1021/ac801664q
PMCID: PMC2653784  PMID: 19061407
True positives (TPs); False positives (FPs); False discovery rates (FDRs); Decoy database; Protein identification; Separate search; Composite search
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.
doi:10.1021/pr900153b
PMCID: PMC2730765  PMID: 19354237
9.  PeaksDB: New Software for Substantially Improved Peptide Identification from Orbitrap ETD Mass Spectrometry 
Objective: To substantially improve the peptide identification sensitivity and accuracy from the Orbitrap ETD data with computational methods. Method: The algorithm takes full advantage of the characteristics of the Orbitrap ETD data, including: (1) high mass resolution of the precursor ions, and (2) the distributions of different fragment ion types in the MS/MS scans. For the first characteristic, a pre-search step is conducted to determine the precursor mass error distribution. This does not only make the precursor mass more accurate by a software recalibration, but also allows the use of the mass error as an important feature in the peptide-spectrum matching score function. For the second characteristic, the frequencies of different fragment ion types at different precursor charge states are statistically learned, and used in the score calculation. Moreover, the precursor-related ions in the MS/MS spectra are removed. Additionally, the score function makes use of the similarity between a database peptide and the de novo sequencing result. Result: PeaksDB was compared against three other search engines: MSGF-DB, Mascot, and ZCore. The same shuffled decoy database was appended to the target database and searched together to estimate the false discovery rate (FDR) of each individual engine. The same search parameters were used for all engines except that MSGFDB does not support variable PTMs. If no variable PTM is allowed, the numbers of identified peptides of different engines at 1% FDR are: PeaksDB (2356) > MSGF-DB (2147) > Mascot (1459) > ZCore (1030). If a few common PTMs are allowed, the numbers change to PeaksDB (3501) > Mascot (2677) > MSGF-DB (2147) > ZCore(1125). Conclusion: PeaksDB substantially improved the sensitivity and accuracy of peptide identifications on Orbitrap ETD data. At 1% false discovery rate, PeaksDB identified 1.3 to 1.6 times as many peptides as Mascot 2.3.
PMCID: PMC3186472
10.  Learning from Decoys to Improve the Sensitivity and Specificity of Proteomics Database Search Results 
PLoS ONE  2012;7(11):e50651.
The statistical validation of database search results is a complex issue in bottom-up proteomics. The correct and incorrect peptide spectrum match (PSM) scores overlap significantly, making an accurate assessment of true peptide matches challenging. Since the complete separation between the true and false hits is practically never achieved, there is need for better methods and rescoring algorithms to improve upon the primary database search results. Here we describe the calibration and False Discovery Rate (FDR) estimation of database search scores through a dynamic FDR calculation method, FlexiFDR, which increases both the sensitivity and specificity of search results. Modelling a simple linear regression on the decoy hits for different charge states, the method maximized the number of true positives and reduced the number of false negatives in several standard datasets of varying complexity (18-mix, 49-mix, 200-mix) and few complex datasets (E. coli and Yeast) obtained from a wide variety of MS platforms. The net positive gain for correct spectral and peptide identifications was up to 14.81% and 6.2% respectively. The approach is applicable to different search methodologies- separate as well as concatenated database search, high mass accuracy, and semi-tryptic and modification searches. FlexiFDR was also applied to Mascot results and showed better performance than before. We have shown that appropriate threshold learnt from decoys, can be very effective in improving the database search results. FlexiFDR adapts itself to different instruments, data types and MS platforms. It learns from the decoy hits and sets a flexible threshold that automatically aligns itself to the underlying variables of data quality and size.
doi:10.1371/journal.pone.0050651
PMCID: PMC3506577  PMID: 23189209
11.  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.
doi:10.1021/pr200780j
PMCID: PMC3230778  PMID: 22010998
Mass spectrometry; target decoy strategy; false discovery rate; peptide identification; protein identification
12.  MUMAL: Multivariate analysis in shotgun proteomics using machine learning techniques 
BMC Genomics  2012;13(Suppl 5):S4.
Background
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.
Results
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.
Conclusion
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.
doi:10.1186/1471-2164-13-S5-S4
PMCID: PMC3477001  PMID: 23095859
Machine learning; Bioinformatics; Peptide/protein identification; Shotgun proteomics; Phosphoproteomics; Tandem mass spectrometry
13.  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.
doi:10.1021/pr300631t
PMCID: PMC3608465  PMID: 23402659
proteomics; mass spectrometry; bioinformatics; support vector machines; peptide spectrum match; database search engine; validation
14.  XGlycScan: An Open-source Software For N-linked Glycosite Assignment, Quantification and Quality Assessment of Data from Mass Spectrometry-based Glycoproteomic Analysis 
Mass spectrometry based glycoproteomics has become a major means of identifying and characterizing previously N-linked glycan attached loci (glycosites). In the bottom-up approach, several factors which include but not limited to sample preparation, mass spectrometry analyses, and protein sequence database searches result in previously N-linked peptide spectrum matches (PSMs) of varying lengths. Given that multiple PSM scan map to a glycosite, we reason that identified PSMs are varying length peptide species of a unique set of glycosites. Because associated spectra of these PSMs are typically summed separately, true glycosite associated spectra counts are lost or complicated. Also, these varying length peptide species complicate protein inference as smaller sized peptide sequences are more likely to map to more proteins than larger sized peptides or actual glycosite sequences. Here, we present XGlycScan. XGlycScan maps varying length peptide species to glycosites to facilitate an accurate quantification of glycosite associated spectra counts. We observed that this reduced the variability in reported identifications of mass spectrometry technical replicates of our sample dataset. We also observed that mapping identified peptides to glycosites provided an assessment of search-engine identification. Inherently, XGlycScan reported glycosites reduce the complexity in protein inference. We implemented XGlycScan in the platform independent Java programing language and have made it available as open source. XGlycScan's source code is freely available at https://bitbucket.org/paiyetan/xglycscan/src and its compiled binaries and documentation can be freely downloaded at https://bitbucket.org/paiyetan/xglycscan/downloads. The graphical user interface version can also be found at https://bitbucket.org/paiyetan/xglycscangui/src and https://bitbucket.org/paiyetan/xglycscangui/downloads respectively.
PMCID: PMC4207213  PMID: 25346946
Bioinformatics; Peptide; Glycopeptide; Glycosite; Protein identification; Proteomics; Quality assessment
15.  Confident and sensitive phosphoproteomics using combinations of collision induced dissociation and electron transfer dissociation☆ 
Journal of Proteomics  2014;103(100):1-14.
We present a workflow using an ETD-optimised version of Mascot Percolator and a modified version of SLoMo (turbo-SLoMo) for analysis of phosphoproteomic data. We have benchmarked this against several database searching algorithms and phosphorylation site localisation tools and show that it offers highly sensitive and confident phosphopeptide identification and site assignment with PSM-level statistics, enabling rigorous comparison of data acquisition methods. We analysed the Plasmodium falciparum schizont phosphoproteome using for the first time, a data-dependent neutral loss-triggered-ETD (DDNL) strategy and a conventional decision-tree method. At a posterior error probability threshold of 0.01, similar numbers of PSMs were identified using both methods with a 73% overlap in phosphopeptide identifications. The false discovery rate associated with spectral pairs where DDNL CID/ETD identified the same phosphopeptide was < 1%. 72% of phosphorylation site assignments using turbo-SLoMo without any score filtering, were identical and 99.8% of these cases are associated with a false localisation rate of < 5%. We show that DDNL acquisition is a useful approach for phosphoproteomics and results in an increased confidence in phosphopeptide identification without compromising sensitivity or duty cycle. Furthermore, the combination of Mascot Percolator and turbo-SLoMo represents a robust workflow for phosphoproteomic data analysis using CID and ETD fragmentation.
Biological significance
Protein phosphorylation is a ubiquitous post-translational modification that regulates protein function. Mass spectrometry-based approaches have revolutionised its analysis on a large-scale but phosphorylation sites are often identified by single phosphopeptides and therefore require more rigorous data analysis to unsure that sites are identified with high confidence for follow-up experiments to investigate their biological significance. The coverage and confidence of phosphoproteomic experiments can be enhanced by the use of multiple complementary fragmentation methods. Here we have benchmarked a data analysis pipeline for analysis of phosphoproteomic data generated using CID and ETD fragmentation and used it to demonstrate the utility of a data-dependent neutral loss triggered ETD fragmentation strategy for high confidence phosphopeptide identification and phosphorylation site localisation.
Graphical abstract
Highlights
•We report and benchmark a data analysis pipeline for phosphoproteomic data analysis.•Combined use of Mascot Percolator and turbo-SLoMo to compare fragmentation methods•CID and ETD fragmentation for phosphorylation site identification•Demonstrate the utility of data-dependent neutral loss triggered ETD fragmentation•High confidence of phosphoproteomic analysis using ETD/CID spectral pairs
doi:10.1016/j.jprot.2014.03.010
PMCID: PMC4047622  PMID: 24657495
CID, collision induced dissociation; ECD, electron capture dissociation; ETD, electron transfer dissociation; ETcaD, ETD with (supplemental activation); FDR, false discovery rate; FLR, false localisation rate; IMAC, immobilised metal affinity chromatography; LC, liquid chromatography; MS, mass spectrometry; MS/MS, tandem mass spectrometry; PEP, posterior error probability; PSM, peptide spectrum match; SCX, strong cation exchange; Phosphoproteomics; Phosphorylation; Mass spectrometry; Post-translational modifications; Electron transfer dissociation; Plasmodium falciparum
16.  Peptide identification based on fuzzy classification and clustering 
Proteome Science  2013;11(Suppl 1):S10.
Background
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.
Results
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.
Conclusions
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.
doi:10.1186/1477-5956-11-S1-S10
PMCID: PMC3908838  PMID: 24564935
Peptide identification; Peptide spectrum matches (PSMs); Fuzzy support vector machine (SVM); Fuzzy silhouette
17.  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 http://fenchurch.mc.vanderbilt.edu/.
doi:10.1021/pr900360j
PMCID: PMC2810655  PMID: 19522537
bioinformatics; parsimony; protein assembly; protein inference; false discovery rate
18.  Practical and Efficient Searching in Proteomics: A Cross Engine Comparison 
WebmedCentral  2013;4(10):WMCPLS0052.
Background
Analysis of large datasets produced by mass spectrometry-based proteomics relies on database search algorithms to sequence peptides and identify proteins. Several such scoring methods are available, each based on different statistical foundations and thereby not producing identical results. Here, the aim is to compare peptide and protein identifications using multiple search engines and examine the additional proteins gained by increasing the number of technical replicate analyses.
Methods
A HeLa whole cell lysate was analyzed on an Orbitrap mass spectrometer for 10 technical replicates. The data were combined and searched using Mascot, SEQUEST, and Andromeda. Comparisons were made of peptide and protein identifications among the search engines. In addition, searches using each engine were performed with incrementing number of technical replicates.
Results
The number and identity of peptides and proteins differed across search engines. For all three search engines, the differences in proteins identifications were greater than the differences in peptide identifications indicating that the major source of the disparity may be at the protein inference grouping level. The data also revealed that analysis of 2 technical replicates can increase protein identifications by up to 10-15%, while a third replicate results in an additional 4-5%.
Conclusions
The data emphasize two practical methods of increasing the robustness of mass spectrometry data analysis. The data show that 1) using multiple search engines can expand the number of identified proteins (union) and validate protein identifications (intersection), and 2) analysis of 2 or 3 technical replicates can substantially expand protein identifications. Moreover, information can be extracted from a dataset by performing database searching with different engines and performing technical repeats, which requires no additional sample preparation and effectively utilizes research time and effort.
doi:10.9754/journal.wplus.2013.0052
PMCID: PMC4208621  PMID: 25346847
Mass spectrometry; proteomics; search engine
19.  Improved LC-MS/MS Spectral Counting Statistics by Recovering Low Scoring Spectra Matched to Confidently Identified Peptide Sequences 
Journal of proteome research  2010;9(11):5698-5704.
Spectral counting has become a popular method for LC-MS/MS based proteome quantification; however, this methodology is often not reliable when proteins are identified by a small number of spectra. Here we present a simple strategy to improve spectral counting based quantification for low abundance proteins by recovering low quality or low scoring spectra for confidently identified peptides. In this approach, stringent data filtering criteria were initially applied to achieve confident peptide identifications with low false discovery rate (e.g., < 1% at peptide level) after LC-MS/MS analysis and database search by SEQUEST. Then, all low scoring MS/MS spectra that match to this set of confidently identified peptides were recovered, leading to more than 20% increase of total identified spectra. The validity of these recovered spectra was assessed by the parent ion mass measurement error distribution, retention time distribution, and by comparing the individual low score and high score spectra that correspond to the same peptides. The results support that the recovered low scoring spectra have similar confidence levels in peptide identifications as the spectra passing the initial stringent filter. The application of this strategy of recovering low scoring spectra significantly improved the spectral count quantification statistics for low abundance proteins, as illustrated in the identification of mouse brain region specific proteins.
doi:10.1021/pr100508p
PMCID: PMC2974764  PMID: 20812748
Spectral count; LC-MS/MS; false negative; quantification
20.  The Effects of Mass Accuracy on the Validation of MS/MS Data 
Journal of Biomolecular Techniques : JBT  2010;21(3 Suppl):S22-S23.
RP-9
Objective: Analyze how precursor and fragment mass tolerance affect the number of true positives and false positives. Introduction: Mass spectrometry coupled to database searching is a powerful and popular protein identification tool. A typical shotgun proteomics experiment begins with degrading intact proteins into peptides. The peptide mixture then undergoes LC-MS/MS analysis, and the resulting experimental spectra are compared to theoretical spectra derived from protein, cDNA, or EST databases. Successful database searching is dependent on database size, post-translational modifications, and precursor and fragment ion m/z tolerance. Method: A standard protein set was made containing 62 verified T. cruzi recombinant proteins spiked into an E. coli lysate. This mixture was digested then analyzed by LC-MS/MS using an LTQ-Orbitrap. Resulting spectra were searched against forward, reverse, and concatenated databases using Sequest, Mascot, and X!Tandem. Peptide probabilities were calculated using ProteinProphet, and peptide false discovery rates (FDR's) were calculated by using ProteoIQ. It is necessary to use a standardized protein mixture to determine the number of true positives (T. cruzi proteins) and false positives (random proteins) found as a function of m/z search tolerance. Preliminary Results: At a 95% probability, more true positives are discovered as ion precursor mass accuracy is increased; however, more false positives are also discovered and at a higher rate. For example, as mass accuracy is increased from +/−1000ppm to +/−20ppm, the number of spectra corresponding to true positives increases by 50% while the number for false positives increases by 380%. Using a 5% FDR filter with the same mass accuracy change yields a 37% increase in true positive matches, while leaving the number of false positives unchanged. Conclusions: FDR filtering can result in more successful data validation than probability filtering when performing high resolution mass spectrometry.
PMCID: PMC2918171
21.  Identification of alternative splice variants in Aspergillus flavus through comparison of multiple tandem MS search algorithms 
BMC Genomics  2011;12:358.
Background
Database searching is the most frequently used approach for automated peptide assignment and protein inference of tandem mass spectra. The results, however, depend on the sequences in target databases and on search algorithms. Recently by using an alternative splicing database, we identified more proteins than with the annotated proteins in Aspergillus flavus. In this study, we aimed at finding a greater number of eligible splice variants based on newly available transcript sequences and the latest genome annotation. The improved database was then used to compare four search algorithms: Mascot, OMSSA, X! Tandem, and InsPecT.
Results
The updated alternative splicing database predicted 15833 putative protein variants, 61% more than the previous results. There was transcript evidence for 50% of the updated genes compared to the previous 35% coverage. Database searches were conducted using the same set of spectral data, search parameters, and protein database but with different algorithms. The false discovery rates of the peptide-spectrum matches were estimated < 2%. The numbers of the total identified proteins varied from 765 to 867 between algorithms. Whereas 42% (1651/3891) of peptide assignments were unanimous, the comparison showed that 51% (568/1114) of the RefSeq proteins and 15% (11/72) of the putative splice variants were inferred by all algorithms. 12 plausible isoforms were discovered by focusing on the consensus peptides which were detected by at least three different algorithms. The analysis found different conserved domains in two putative isoforms of UDP-galactose 4-epimerase.
Conclusions
We were able to detect dozens of new peptides using the improved alternative splicing database with the recently updated annotation of the A. flavus genome. Unlike the identifications of the peptides and the RefSeq proteins, large variations existed between the putative splice variants identified by different algorithms. 12 candidates of putative isoforms were reported based on the consensus peptide-spectrum matches. This suggests that applications of multiple search engines effectively reduced the possible false positive results and validated the protein identifications from tandem mass spectra using an alternative splicing database.
doi:10.1186/1471-2164-12-358
PMCID: PMC3146456  PMID: 21745387
22.  PepArML: A Meta-Search Peptide Identification Platform 
The PepArML meta-search peptide identification platform provides a unified search interface to seven search engines; a robust cluster, grid, and cloud computing scheduler for large-scale searches; and an unsupervised, model-free, machine-learning-based result combiner, which selects the best peptide identification for each spectrum, estimates false-discovery rates, and outputs pepXML format identifications. The meta-search platform supports Mascot; Tandem with native, k-score, and s-score scoring; OMSSA; MyriMatch; and InsPecT with MS-GF spectral probability scores — reformatting spectral data and constructing search configurations for each search engine on the fly. The combiner selects the best peptide identification for each spectrum based on search engine results and features that model enzymatic digestion, retention time, precursor isotope clusters, mass accuracy, and proteotypic peptide properties, requiring no prior knowledge of feature utility or weighting. The PepArML meta-search peptide identification platform often identifies 2–3 times more spectra than individual search engines at 10% FDR.
doi:10.1002/0471250953.bi1323s44
PMCID: PMC4317344
Proteomics; Mass-Spectrometry; Machine-Learning; Cloud-Computing
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.  Fast and accurate database searches with MS-GF+Percolator 
Journal of proteome research  2013;13(2):890-897.
One can interpret fragmentation spectra stemming from peptides in mass spectrometry-based proteomics experiments using so called database search engines. Frequently, one also runs post-processors such as Percolator to assess the confidence, infer unique peptides and increase the number of identifications. A recent search engine, MS-GF+, has shown promising results, due to a new and efficient scoring algorithm. However, MS-GF+ provides few statistical estimates about the peptide-spectrum matches, hence limiting the biological interpretation. Here, we enabled Percolator-processing for MS-GF+ output, and observed an increased number of identified peptides for a wide variety of datasets. In addition, Percolator directly reports p values and false discovery rate estimates, such as q values and posterior error probabilities, for peptide-spectrum matches, peptides and proteins, functions that are useful for the whole proteomics community.
doi:10.1021/pr400937n
PMCID: PMC3975676  PMID: 24344789
25.  Open MS/MS spectral library search to identify unanticipated post-translational modifications and increase spectral identification rate 
Bioinformatics  2010;26(12):i399-i406.
Motivation: Identification of post-translationally modified proteins has become one of the central issues of current proteomics. Spectral library search is a new and promising computational approach to mass spectrometry-based protein identification. However, its potential in identification of unanticipated post-translational modifications has rarely been explored. The existing spectral library search tools are designed to match the query spectrum to the reference library spectra with the same peptide mass. Thus, spectra of peptides with unanticipated modifications cannot be identified.
Results: In this article, we present an open spectral library search tool, named pMatch. It extends the existing library search algorithms in at least three aspects to support the identification of unanticipated modifications. First, the spectra in library are optimized with the full peptide sequence information to better tolerate the peptide fragmentation pattern variations caused by some modification(s). Second, a new scoring system is devised, which uses charge-dependent mass shifts for peak matching and combines a probability-based model with the general spectral dot-product for scoring. Third, a target-decoy strategy is used for false discovery rate control. To demonstrate the effectiveness of pMatch, a library search experiment was conducted on a public dataset with over 40 000 spectra in comparison with SpectraST, the most popular library search engine. Additional validations were done on four published datasets including over 150 000 spectra. The results showed that pMatch can effectively identify unanticipated modifications and significantly increase spectral identification rate.
Availability: http://pfind.ict.ac.cn/pmatch/
Contact: yfu@ict.ac.cn; rxsun@ict.ac.cn
Supplementary information: Supplementary data are available at Bioinformatics online.
doi:10.1093/bioinformatics/btq185
PMCID: PMC2881370  PMID: 20529934

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