Fast Multi-blind Modification Search through Tandem Mass Spectrometry

Abstract

With great biological interest in post-translational modifications (PTMs), various approaches have been introduced to identify PTMs using MS/MS. Recent developments for PTM identification have focused on an unrestrictive approach that searches MS/MS spectra for all known and possibly even unknown types of PTMs at once. However, the resulting expanded search space requires much longer search time and also increases the number of false positives (incorrect identifications) and false negatives (missed true identifications), thus creating a bottleneck in high throughput analysis. Here we introduce MODa, a novel "multi-blind" spectral alignment algorithm that allows for fast unrestrictive PTM searches with no limitation on the number of modifications per peptide while featuring over an order of magnitude speedup in relation to existing approaches. We demonstrate the sensitivity of MODa on human shotgun proteomics data where it reveals multiple mutations, a wide range of modifications (including glycosylation), and evidence for several putative novel modifications. Based on the reported findings, we argue that the efficiency and sensitivity of MODa make it the first unrestrictive search tool with the potential to fully replace conventional restrictive identification of proteomics mass spectrometry data. Molecular & Cellular Proteomics 11: 10.1074/mcp.M111.010199, 1-13, 2012. Post-translational modifications (PTMs)1 regulate protein function, localization, and interactions inside a cell (1). Hundreds of PTM types are known so far, and yet a lot more may remain to be discovered (2, 3). The identification of PTMs is critical to gaining insight into biological functions but remains a formidable challenge. Tandem mass spectrometry (MS/MS) has emerged as a powerful tool for rapid identification of PTMs (4, 5), which can be detected by PTM-related diagnostic mass shifts of fragment ions in MS/MS spectra. However accurate computational identification of modified peptides remains a difficult problem often addressed with restrictive approaches that require "guessed" lists of possible PTMs to be provided in advance (6 -8). Such an approach may overlook potentially important PTMs if they are not guessed in advance. In recent PTM identification algorithms, peptide sequence tag approaches have been proposed to search for more types of PTMs and to speed up the search (9 -12). A small set of short sequence tags (2-4 amino acids long) are derived from an MS/MS spectrum and used to screen for matching peptides in a protein database; possible modifications are then inferred from the difference between the precursor ion mass of the experimental spectrum and the theoretically calculated mass of the matched peptide.In contrast with restrictive approaches, unrestrictive or blind approaches search MS/MS spectra for all known and even possibly unknown types of PTMs at once and derive the list of modifications directly from MS/MS data (13-22). OpenSea (13) and SPIDER (14) compared de novo sequencing results with peptide sequences from a protei...

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High Mobility Group Box 1 Protein Is Methylated and Transported to Cytoplasm in Clear Cell Renal Cell Carcinoma

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