Dataset Information

Assigning spectrum-specific P-values to protein identifications by mass spectrometry.

ABSTRACT:

Motivation

Although many methods and statistical approaches have been developed for protein identification by mass spectrometry, the problem of accurate assessment of statistical significance of protein identifications remains an open question. The main issues are as follows: (i) statistical significance of inferring peptide from experimental mass spectra must be platform independent and spectrum specific and (ii) individual spectrum matches at the peptide level must be combined into a single statistical measure at the protein level.

Results

We present a method and software to assign statistical significance to protein identifications from search engines for mass spectrometric data. The approach is based on asymptotic theory of order statistics. The parameters of the asymptotic distributions of identification scores are estimated for each spectrum individually. The method relies on new unbiased estimators for parameters of extreme value distribution. The estimated parameters are used to assign a spectrum-specific P-value to each peptide-spectrum match. The protein-level confidence measure combines P-values of peptide-to-spectrum matches.

Conclusion

We extensively tested the method using triplicate mouse and yeast high-throughput proteomic experiments. The proposed statistical approach improves the sensitivity of protein identifications without compromising specificity. While the method was primarily designed to work with Mascot, it is platform-independent and is applicable to any search engine which outputs a single score for a peptide-spectrum match. We demonstrate this by testing the method in conjunction with X!Tandem.

Availability

The software is available for download at ftp://genetics.bwh.harvard.edu/SSPV/.

Contact

ssunyaev@rics.bwh.harvard.edu

Supplementary information

Supplementary data are available at Bioinformatics online.

SUBMITTER: Spirin V

PROVIDER: S-EPMC3072553 | biostudies-literature | 2011 Apr

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Assigning spectrum-specific P-values to protein identifications by mass spectrometry.

Spirin Victor V Shpunt Alexander A Seebacher Jan J Gentzel Marc M Shevchenko Andrej A Gygi Steven S Sunyaev Shamil S

Bioinformatics (Oxford, England) 20110223 8

<h4>Motivation</h4>Although many methods and statistical approaches have been developed for protein identification by mass spectrometry, the problem of accurate assessment of statistical significance of protein identifications remains an open question. The main issues are as follows: (i) statistical significance of inferring peptide from experimental mass spectra must be platform independent and spectrum specific and (ii) individual spectrum matches at the peptide level must be combined into a s ...[more]

PMID: 21349864

Similar Datasets

Project description:The complexity of proteomic instrumentation for LC-MS/MS introduces many possible sources of variability. Data-dependent sampling of peptides constitutes a stochastic element at the heart of discovery proteomics. Although this variation impacts the identification of peptides, proteomic identifications are far from completely random. In this study, we analyzed interlaboratory data sets from the NCI Clinical Proteomic Technology Assessment for Cancer to examine repeatability and reproducibility in peptide and protein identifications. Included data spanned 144 LC-MS/MS experiments on four Thermo LTQ and four Orbitrap instruments. Samples included yeast lysate, the NCI-20 defined dynamic range protein mix, and the Sigma UPS 1 defined equimolar protein mix. Some of our findings reinforced conventional wisdom, such as repeatability and reproducibility being higher for proteins than for peptides. Most lessons from the data, however, were more subtle. Orbitraps proved capable of higher repeatability and reproducibility, but aberrant performance occasionally erased these gains. Even the simplest protein digestions yielded more peptide ions than LC-MS/MS could identify during a single experiment. We observed that peptide lists from pairs of technical replicates overlapped by 35-60%, giving a range for peptide-level repeatability in these experiments. Sample complexity did not appear to affect peptide identification repeatability, even as numbers of identified spectra changed by an order of magnitude. Statistical analysis of protein spectral counts revealed greater stability across technical replicates for Orbitraps, making them superior to LTQ instruments for biomarker candidate discovery. The most repeatable peptides were those corresponding to conventional tryptic cleavage sites, those that produced intense MS signals, and those that resulted from proteins generating many distinct peptides. Reproducibility among different instruments of the same type lagged behind repeatability of technical replicates on a single instrument by several percent. These findings reinforce the importance of evaluating repeatability as a fundamental characteristic of analytical technologies.

Project description:All large scale LC-MS/MS post-translational methylation site discovery experiments require methylpeptide spectrum matches (methyl-PSMs) to be identified at acceptably low false discovery rates (FDRs). To meet estimated methyl-PSM FDRs, methyl-PSM filtering criteria are often determined using the target-decoy approach. The efficacy of this methyl-PSM filtering approach has, however, yet to be thoroughly evaluated. Here, we conduct a systematic analysis of methyl-PSM FDRs across a range of sample preparation workflows (each differing in their exposure to the alcohols methanol and isopropyl alcohol) and mass spectrometric instrument platforms (each employing a different mode of MS/MS dissociation). Through (13)CD3-methionine labeling (heavy-methyl SILAC) of Saccharomyces cerevisiae cells and in-depth manual data inspection, accurate lists of true positive methyl-PSMs were determined, allowing methyl-PSM FDRs to be compared with target-decoy approach-derived methyl-PSM FDR estimates. These results show that global FDR estimates produce extremely unreliable methyl-PSM filtering criteria; we demonstrate that this is an unavoidable consequence of the high number of amino acid combinations capable of producing peptide sequences that are isobaric to methylated peptides of a different sequence. Separate methyl-PSM FDR estimates were also found to be unreliable due to prevalent sources of false positive methyl-PSMs that produce high peptide identity score distributions. Incorrect methylation site localizations, peptides containing cysteinyl-S-β-propionamide, and methylated glutamic or aspartic acid residues can partially, but not wholly, account for these false positive methyl-PSMs. Together, these results indicate that the target-decoy approach is an unreliable means of estimating methyl-PSM FDRs and methyl-PSM filtering criteria. We suggest that orthogonal methylpeptide validation (e.g. heavy-methyl SILAC or its offshoots) should be considered a prerequisite for obtaining high confidence methyl-PSMs in large scale LC-MS/MS methylation site discovery experiments and make recommendations on how to reduce methyl-PSM FDRs in samples not amenable to heavy isotope labeling. Data are available via ProteomeXchange with the data identifier PXD002857.

Dataset Information

Assigning spectrum-specific P-values to protein identifications by mass spectrometry.

Motivation

Results

Conclusion

Availability

Contact

Supplementary information

Publications

Assigning spectrum-specific P-values to protein identifications by mass spectrometry.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets