Format

Send to

Choose Destination
Mol Cell Proteomics. 2014 Nov;13(11):3211-23. doi: 10.1074/mcp.O114.038877. Epub 2014 Aug 6.

DeMix workflow for efficient identification of cofragmented peptides in high resolution data-dependent tandem mass spectrometry.

Author information

1
From the ‡Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-17177 Stockholm, Sweden;
2
From the ‡Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-17177 Stockholm, Sweden; §Biomotif AB, Stockholm SE-182 12, Sweden.
3
From the ‡Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-17177 Stockholm, Sweden; roman.zubarev@ki.se.

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.

PMID:
25100859
PMCID:
PMC4223503
DOI:
10.1074/mcp.O114.038877
[Indexed for MEDLINE]
Free PMC Article

Supplemental Content

Full text links

Icon for HighWire Icon for PubMed Central
Loading ...
Support Center