Depending on these choices, the resulting tandem mass (MS/MS) spectra vary in fragmentation propensities and precision. Empowered by these changes, MS researchers now have diverse choices with respect to the questions: ‘what fragmentation method to use?’, ‘how accurate should be the measurements of the mass-to-charge ( m/z) ratios?’, ‘what proteases to use?’ and ‘what post-translational modification (PTM) to focus on (for example, phosphorylation)?’. Although trypsin remains a dominant protease in proteomics studies, digesting proteins with diverse proteases is becoming popular 1. New fragmentation technologies have emerged and high-precision mass spectrometers like Orbitrap have become widely available. Mass spectrometry (MS) instruments and experimental protocols have greatly advanced over the last decade. We emphasize that although MS-GF+ is not specifically designed for any particular experimental set-up, it improves on the performance of tools specifically designed for these applications (for example, specialized tools for phosphoproteomics). For all these data sets, MS-GF+ significantly increases the number of identified peptides compared with commonly used methods for peptide identifications. We benchmark MS-GF+ using diverse spectral data sets: (i) spectra of varying fragmentation methods (ii) spectra of multiple enzyme digests (iii) spectra of phosphorylated peptides and (iv) spectra of peptides with unusual fragmentation propensities produced by a novel alpha-lytic protease. We present a database search tool MS-GF+ that is sensitive (it identifies more peptides than most other database search tools) and universal (it works well for diverse types of spectra, different configurations of MS instruments and different experimental protocols). Mass spectrometry (MS) instruments and experimental protocols are rapidly advancing, but the software tools to analyse tandem mass spectra are lagging behind.
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