Conserved peptide fragmentation as a benchmarking tool for mass spectrometers and a discriminating feature for targeted proteomics.
Bottom Line: In both cases, confidence in peptide identification is directly related to the quality of spectral matches.Altogether, this study validates the use of the normalized spectral contrast angle as a sensitive spectral similarity measure for targeted proteomics, and more generally provides a methodology to assess the performance of spectral comparisons and to support the rational selection of the most appropriate similarity measure.The algorithms used in this study are made publicly available as an open source toolset with a graphical user interface.
Affiliation: From the ‡Department of Biology, Institute of Molecular Systems Biology, ETH Zurich, 8093 Zurich, Switzerland;Show MeSH
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Mentions: The shotgun LTQ Orbitrap XL, Orbitrap Elite (.raw) and TripleTOF (.wiff) data files were converted to centroided mzXML using the Proteowizard converter (35) and searched with Sorcerer-SEQUEST (TurboSequest v4.0.3rev11 running on Sage-N Sorcerer v4.0.4) using the Saccharomyces cerevisiae yeast SGD database (release 03 Febr. 2011, containing 6717 yeast protein entries, concatenated with 6717 corresponding “tryptic peptide pseudo-reverse” decoy protein sequences). For the search, we allowed for semi-tryptic digests and up to two missed cleavages per peptide, and we used carbamidomethylation as fixed modification on cysteine and oxidation as variable modification on methionine. The Sequest search results were converted to pep.xml format and further processed using PeptideProphet (TPP version 4.5.2) (36). The search results were sorted by decreasing PeptideProphet probability and filtered at 1% false discovery rate (FDR) by decoy counting at the peptide spectrum matches (PSM) level. The complete list of peptide identifications for the various instrument modes can be found in the supplemental Tables S1. Consensus spectra libraries were built independently for each file with SpectraST (v. 4, included in TPP version 4.5.2) using the peptides identified above 1% FDR for the replicate experiment (for the intrinsic comparison, for example, Fig. 2 and 3) or for the instrument mode (for the portability analysis, Fig. 4 and 6). For the high mass accuracy MS/MS measurements (e.g. Elite HCD and TripleTOF), the CID-QTOF option was used when running SpectraST to improve the quality of consensus generation. For the TripleTOF and Elite HCD shotgun results, the peptide library files were further converted into an input assay list using an in-house developed python script. The script used the SpectraST .sptxt library as input and retrieved the top 6–100 most intense (singly or doubly charged) y or b fragment ions for each spectra with m/z above 400 and outside of the range of the 25 Da swath/precursor fragmentation window and whose m/z matched the theoretical fragment ion masses within 0.05 Da tolerance. For the phospho-peptide libraries, y and b fragment ions with neutral losses (-80,-98) were also exported, when those obeyed the same selection rules as above. For the dephosphorylated peptide assays used to query for phospho-modified peptides counterparts, we used only the transitions until the modification site, with the same relative intensities and retention time and we modified the mass of the precursors accordingly to the mass of the PTM. The complete sets of assay lists used in this study can be found in the supplemental Tables S2. It should be noted that though the assay list contains between 6 and 100 transitions for the peptides, the targeted peptides were scored and identified using the six most intense transitions from that assay list (see below).
Affiliation: From the ‡Department of Biology, Institute of Molecular Systems Biology, ETH Zurich, 8093 Zurich, Switzerland;