Abstract
We describe the comprehensive analysis of the yeast proteome in just over one hour of optimized analysis. We achieve this expedited proteome characterization with improved sample preparation, chromatographic separations, and by using a new Orbitrap hybrid mass spectrometer equipped with a mass filter, a collision cell, a high-field Orbitrap analyzer, and, finally, a dual cell linear ion trap analyzer (Q-OT-qIT, Orbitrap Fusion). This system offers high MS2 acquisition speed of 20 Hz and detects up to 19 peptide sequences within a single second of operation. Over a 1.3 h chromatographic method, the Q-OT-qIT hybrid collected an average of 13,447 MS1 and 80,460 MS2 scans (per run) to produce 43,400 (x̄) peptide spectral matches and 34,255 (x̄) peptides with unique amino acid sequences (1% false discovery rate (FDR)). On average, each one hour analysis achieved detection of 3,977 proteins (1% FDR). We conclude that further improvements in mass spectrometer scan rate could render comprehensive analysis of the human proteome within a few hours.
Highlights
Yeast, with its 6600 open reading frames, has been the preferred proteomic technology test-bed [8]
To maximize performance we developed an optimized cellular lysis approach, employed trypsin digestion, and used dimethyl sulfoxide (DMSO, 5%) as an LC additive to increase abundance of acidic peptides and unify charge state [31, 32]. Using this novel system we report the comprehensive analysis of the yeast proteome (4002 with 1% FDR) following 1.3 h of nLCMS2 analysis (70 min gradient)
The new system is equipped with a sophisticated control system that parallelizes the processes of ion injection, precursor isolation, fragmentation, and mass analysis to achieve a ϳ2ϫ boost in acquisition rates. We reasoned that this Q-OT-qIT configuration, with its 20 Hz MS2 acquisition rate, could afford a considerable gain for rapid, whole proteome analysis
Summary
Yeast, with its 6600 open reading frames, has been the preferred proteomic technology test-bed [8]. Yates and co-workers reported the first large-scale yeast proteome study in 2001 with the identification of 1483 proteins following ϳ 68 h of mass spectral analysis, i.e. 0.4 proteins were identified per minute [17]. Armed with a faster hybrid mass spectrometer capable of accurate mass measurement, Mann and colleagues achieved detection of 2003 yeast proteins in an impressive 48 h (0.7 proteins/minute) in 2006 [22]. From these three pioneering studies we begin to see the impact of mass spectrometer acquisition rate on the depth and rate of proteome analysis. In 2010 our group achieved similar comprehensive analysis, but improved sequence coverage, using fractionation and multiple proteases
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