title: DeMix workflow for efficient identification of cofragmented peptides in high resolution data-dependent tandem mass spectrometry.
abstract: Based on conventional data-dependent acquisition strategy of shotgun proteomics, we present a new workflow DeMix, which significantly increases the efficiency of peptide identification for in-depth shotgun analysis of complex proteomes. Capitalizing on the high resolution and mass accuracy of Orbitrap-based tandem mass spectrometry, we developed a simple deconvolution method of "cloning" chimeric tandem spectra for cofragmented peptides. Additional to a database search, a simple rescoring scheme utilizes mass accuracy and converts the unwanted cofragmenting events into a surprising advantage of multiplexing. With the combination of cloning and rescoring, we obtained on average nine peptide-spectrum matches per second on a Q-Exactive workbench, whereas the actual MS/MS acquisition rate was close to seven spectra per second. This efficiency boost to 1.24 identified peptides per MS/MS spectrum enabled analysis of over 5000 human proteins in single-dimensional LC-MS/MS shotgun experiments with an only two-hour gradient. These findings suggest a change in the dominant "one MS/MS spectrum - one peptide" paradigm for data acquisition and analysis in shotgun data-dependent proteomics. DeMix also demonstrated higher robustness than conventional approaches in terms of lower variation among the results of consecutive LC-MS/MS runs.
authors.affiliation: From the ‡Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-17177 Stockholm, Sweden;
authors.forename: Bo
authors.initials: B
authors.lastname: Zhang
authors.affiliation: From the ‡Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-17177 Stockholm, Sweden; §Biomotif AB, Stockholm SE-182 12, Sweden.
authors.forename: Mohammad
authors.initials: M
authors.lastname: Pirmoradian
authors.affiliation: From the ‡Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-17177 Stockholm, Sweden;
authors.forename: Alexey
authors.initials: A
authors.lastname: Chernobrovkin
authors.affiliation: From the ‡Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-17177 Stockholm, Sweden; roman.zubarev@ki.se.
authors.forename: Roman A
authors.initials: RA
authors.lastname: Zubarev
mesh_terms: D000595:Amino Acid Sequence; D045744:Cell Line, Tumor; D020869:Gene Expression Profiling; D006367:HeLa Cells; D006801:Humans; D010446:Peptide Fragments / Q000032:analysis* / Q000737:chemistry*; D020543:Proteome / Q000032:analysis; D040901:Proteomics / Q000379:methods*; D020539:Sequence Analysis, Protein / Q000379:methods; D053719:Tandem Mass Spectrometry; D057188:Workflow
chemical_list: D010446:Peptide Fragments; D020543:Proteome
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