Dataset Information

Peptide-Spectrum Match Validation with Internal Standards (P-VIS): Internally-Controlled Validation of Mass Spectrometry-Based Peptide Identifications.

ABSTRACT: Liquid chromatography-tandem mass spectrometry is an increasingly powerful tool for studying proteins in the context of disease. As technological advances in instrumentation and data analysis have enabled deeper profiling of proteomes and peptidomes, the need for a rigorous, standardized approach to validate individual peptide-spectrum matches (PSMs) has emerged. To address this need, we developed a novel and broadly applicable workflow: PSM validation with internal standards (P-VIS). In this approach, the fragmentation spectrum and chromatographic retention time of a peptide within a biological sample are compared with those of a synthetic version of the putative peptide sequence match. Similarity measurements obtained for a panel of internal standard peptides are then used to calculate a prediction interval for valid matches. If the observed degree of similarity between the biological and the synthetic peptide falls within this prediction interval, then the match is considered valid. P-VIS enables systematic and objective assessment of the validity of individual PSMs, providing a measurable degree of confidence when identifying peptides by mass spectrometry.

SUBMITTER: Wiles TA

PROVIDER: S-EPMC7775876 | biostudies-literature | 2021 Jan

REPOSITORIES: biostudies-literature

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Peptide-Spectrum Match Validation with Internal Standards (P-VIS): Internally-Controlled Validation of Mass Spectrometry-Based Peptide Identifications.

Wiles Timothy Aaron TA Saba Laura M LM Delong Thomas T

Journal of proteome research 20200929 1

Liquid chromatography-tandem mass spectrometry is an increasingly powerful tool for studying proteins in the context of disease. As technological advances in instrumentation and data analysis have enabled deeper profiling of proteomes and peptidomes, the need for a rigorous, standardized approach to validate individual peptide-spectrum matches (PSMs) has emerged. To address this need, we developed a novel and broadly applicable workflow: PSM validation with internal standards (P-VIS). In this ap ...[more]

PMID: 32924495

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Project description:Accurate knowledge of retention time (RT) in liquid chromatography-based mass spectrometry data facilitates peptide identification, quantification, and multiplexing in targeted and discovery-based workflows. Retention time prediction is particularly important for peptide analysis in emerging data-independent acquisition (DIA) experiments such as SWATH-MS. The indexed RT approach, iRT, uses synthetic spiked-in peptide standards (SiRT) to set RT to a unit-less scale, allowing for normalization of peptide RT between different samples and chromatographic set-ups. The obligatory use of SiRTs can be costly and complicates comparisons and data integration if standards are not included in every sample. Reliance on SiRTs also prevents the inclusion of archived mass spectrometry data for generation of the peptide assay libraries central to targeted DIA-MS data analysis. We have identified a set of peptide sequences that are conserved across most eukaryotic species, termed Common internal Retention Time standards (CiRT). In a series of tests to support the appropriateness of the CiRT-based method, we show: (1) the CiRT peptides normalized RT in human, yeast, and mouse cell lysate derived peptide assay libraries and enabled merging of archived libraries for expanded DIA-MS quantitative applications; (2) CiRTs predicted RT in SWATH-MS data within a 2-min margin of error for the majority of peptides; and (3) normalization of RT using the CiRT peptides enabled the accurate SWATH-MS-based quantification of 340 synthetic isotopically labeled peptides that were spiked into either human or yeast cell lysate. To automate and facilitate the use of these CiRT peptide lists or other custom user-defined internal RT reference peptides in DIA workflows, an algorithm was designed to automatically select a high-quality subset of datapoints for robust linear alignment of RT for use. Implementations of this algorithm are available for the OpenSWATH and Skyline platforms. Thus, CiRT peptides can be used alone or as a complement to SiRTs for RT normalization across peptide spectral libraries and in quantitative DIA-MS studies.

Dataset Information

Peptide-Spectrum Match Validation with Internal Standards (P-VIS): Internally-Controlled Validation of Mass Spectrometry-Based Peptide Identifications.

Publications

Peptide-Spectrum Match Validation with Internal Standards (P-VIS): Internally-Controlled Validation of Mass Spectrometry-Based Peptide Identifications.

Similar Datasets

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