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Submitted on October 14, 2005
Cell Biology, Harvard Medical School, Boston, MA 02115
Corresponding Author: steven_gygi{at}hms.harvard.edu
Mass spectrometers that provide high mass accuracy such as Fourier transform ion cyclotron resonance (FT-ICR) instruments are increasingly used in proteomics studies. Although the importance of accurately determined molecular masses for the identification of biomolecules is generally accepted, its role in the analysis of shotgun proteomics data has not been thoroughly studied. To gain insight into this role, we used a hybrid linear quadrupole ion trap/FT-ICR mass spectrometer (LTQ FT) for liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of a highly complex peptide mixture derived from a fraction of the yeast proteome. We applied three data-dependent MS/MS acquisition methods. The FT-ICR part of the hybrid mass spectrometer was either not exploited, used only for survey MS scans, or also used for acquiring selected ion monitoring (SIM) scans to optimize mass accuracy. MS/MS data were assigned with the SEQUEST algorithm and peptide identifications were validated by estimating the number of incorrect assignments using the composite target/decoy database search strategy. We developed a simple mass calibration strategy exploiting polydimethylcyclosiloxane background ions as calibrant ions. This strategy allowed us to substantially improve mass accuracy without reducing the number of MS/MS spectra acquired in an LC-MS/MS run. The benefits of high mass accuracy were greatest for assigning MS/MS spectra with low signal-to-noise ratio and for assigning phosphopeptides. Confident peptide identification rates from these data sets could be doubled by the use of mass accuracy information. It was also shown that improving mass accuracy at a cost to the MS/MS acquisition rate substantially lowered the sensitivity of LC-MS/MS analyses. The use of FT-ICR SIM scans to maximize mass accuracy reduced the number of protein identifications by 40%.
Revised on April 20, 2006
Accepted on April 23, 2006
Optimization and use of peptide mass measurement accuracy in shotgun proteomics
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