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. 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. The ability to measure differences in protein expression has become key to understanding biological phenomena (1Walther T.C. Mann M. Mass spectrometry–based proteomics in cell biology.J. Cell Biol. 2010; 190: 491-500Crossref PubMed Scopus (307) Google Scholar, 2Mallick P. Kuster B. Proteomics: a pragmatic perspective.Nat. Biotechnol. 2010; 28: 695-709Crossref PubMed Scopus (319) Google Scholar). Owing to cost, speed, and accessibility, transcriptomic analysis is often used as a proteomic proxy (3Schena M. Shalon D. Davis R.W. Brown P.O. Quantitative monitoring of gene-expression patterns with a complementary-DNA microarray.Science. 1995; 270: 467-470Crossref PubMed Scopus (7609) Google Scholar, 4DeRisi J.L. Iyer V.R. Brown P.O. Exploring the metabolic and genetic control of gene expression on a genomic scale.Science. 1997; 278: 680-686Crossref PubMed Scopus (3695) Google Scholar). That said, mRNA is a genetic intermediary and cannot inform on the myriad of post-translational regulation processes (5Gygi S.P. Rochon Y. Franza B.R. Aebersold R. Correlation between protein and mRNA abundance in yeast.Mol. Cell. Biol. 1999; 19: 1720-1730Crossref PubMed Scopus (3185) Google Scholar, 6Grimsrud P.A. Swaney D.L. Wenger C.D. Beauchene N.A. Coon J.J. Phosphoproteomics for the Masses.Chem. Biol. 2010; 5: 105-119Google Scholar, 7Hebert Alexander S. Dittenhafer-Reed Kristin E. Yu W. Bailey Derek J. Selen Ebru S. Boersma Melissa D. Carson Joshua J. Tonelli M. Balloon Allison J. Higbee Alan J. Westphall Michael S. Pagliarini David J. Prolla Tomas A. Assadi-Porter F. Roy S. Denu John M. Coon Joshua J. Calorie restriction and sirt3 trigger global reprogramming of the mitochondrial protein acetylome.Mol. Cell. 2013; 49: 186-199Abstract Full Text Full Text PDF PubMed Scopus (477) Google Scholar). For the past decade considerable effort has been invested in maturing proteomic technology to deliver information at a rate and cost commensurate to transcriptomic technologies. Historically yeast, with its 6600 open reading frames, has been the preferred proteomic technology test-bed (8Cherry J.M. Hong E.L. Amundsen C. Balakrishnan R. Binkley G. Chan E.T. Christie K.R. Costanzo M.C. Dwight S.S. Engel S.R. Fisk D.G. Hirschman J.E. Hitz B.C. Karra K. Krieger C.J. Miyasato S.R. Nash R.S. Park J. Skrzypek M.S. Simison M. Weng S. Wong E.D. Saccharomyces Genome Database: the genomics resource of budding yeast.Nucleic Acids Res. 2012; 40: D700-D705Crossref PubMed Scopus (1144) Google Scholar). In 2003, Weissmann and colleagues measured approximate expression levels of each yeast gene using either GFP or TAP tags (9Ghaemmaghami S. Huh W.-K. Bower K. Howson R.W. Belle A. Dephoure N. O'Shea E.K. Weissman J.S. Global analysis of protein expression in yeast.Nature. 2003; 425: 737-741Crossref PubMed Scopus (2993) Google Scholar). This seminal work established that ∼4500 proteins are expressed during log-phase yeast growth. Subsequent mass spectrometry-based studies have confirmed this early estimate (9Ghaemmaghami S. Huh W.-K. Bower K. Howson R.W. Belle A. Dephoure N. O'Shea E.K. Weissman J.S. Global analysis of protein expression in yeast.Nature. 2003; 425: 737-741Crossref PubMed Scopus (2993) Google Scholar, 10de Godoy L.M.F. Olsen J.V. Cox J. Nielsen M.L. Hubner N.C. Frohlich F. Walther T.C. Mann M. Comprehensive mass-spectrometry-based proteome quantification of haploid versus diploid yeast.Nature. 2008; 455: 1251-1254Crossref PubMed Scopus (737) Google Scholar, 11Wu R. Dephoure N. Haas W. Huttlin E.L. Zhai B. Sowa M.E. Gygi S.P. Correct Interpretation of Comprehensive Phosphorylation Dynamics Requires Normalization by Protein Expression Changes.Mol. Cell. Proteomics. 2011; 10 (M111.009654)Abstract Full Text Full Text PDF Scopus (143) Google Scholar, 12Webb K.J. Xu T. Park S.K. Yates J.R. Modified MuDPIT separation identified 4488 proteins in a system-wide analysis of quiescence in yeast.J. Proteome Res. 2013; 12: 2177-2184Crossref PubMed Scopus (48) Google Scholar). With this knowledge, we hereby define comprehensive proteome analysis as an experiment that detects ∼90% of the expressed proteome (≥ 4000 proteins for yeast). Note others have used the term “nearly complete” for this purpose; we posit that comprehensive has identical meaning (i.e. including many, most, or all things) (13Merriam-Webster's Dictionary, 2004, 11th Ed.Google Scholar). Initial MS-based proteomic analyses of yeast, each identifying up to a few hundred proteins, were conducted using a variety of separation and MS technologies (14Figeys D. Ducret A. Yates J.R. Aebersold R. Protein identification by solid phase microextraction[mdash]capillary zone electrophoresis[mdash]microelectrospray[mdash]tandem mass spectrometry.Nat Biotech. 1996; 14: 1579-1583Crossref PubMed Scopus (167) Google Scholar, 15Shevchenko A. Jensen O.N. Podtelejnikov A.V. Sagliocco F. Wilm M. Vorm O. Mortensen P. Shevchenko A. Boucherie H. Mann M. Linking genome and proteome by mass spectrometry: Large-scale identification of yeast proteins from two dimensional gels.Proc. Natl. Acad. Sci. U. S. A. 1996; 93: 14440-14445Crossref PubMed Scopus (1298) Google Scholar, 16Link A.J. Eng J. Schieltz D.M. Carmack E. Mize G.J. Morris D.R. Garvik B.M. Yates J.R. Direct analysis of protein complexes using mass spectrometry.Nat. Biotech. 1999; 17: 676-682Crossref PubMed Scopus (2071) Google Scholar). Yates and co-workers reported the first large-scale yeast proteome study in 2001 with the identification of 1483 proteins following ∼ 68 h of mass spectral analysis, i.e. 0.4 proteins were identified per minute (17Washburn M.P. Wolters D. Yates J.R. Large-scale analysis of the yeast proteome by multidimensional protein identification technology.Nat. Biotech. 2001; 19: 242-247Crossref PubMed Scopus (4077) Google Scholar). Their method—two dimensional chromatography coupled with tandem mass spectrometry—has provided a template for large-scale protein analysis for the past decade (18Beausoleil S.A. Jedrychowski M. Schwartz D. Elias J.E. Villen J. Li J.X. Cohn M.A. Cantley L.C. Gygi S.P. Large-scale characterization of HeLa cell nuclear phosphoproteins.Proc. Natl. Acad. Sci. U.S.A. 2004; 101: 12130-12135Crossref PubMed Scopus (1236) Google Scholar, 19Zanivan S. Gnad F. Wickstrom S.A. Geiger T. Macek B. Cox J. Fassler R. Mann M. Solid tumor proteome and phosphoproteome analysis by high resolution mass spectrometry.J. Proteome Res. 2008; 7: 5314-5326Crossref PubMed Scopus (114) Google Scholar, 20Ledvina A.R. Beauchene N.A. McAlister G.C. Syka J.E.P. Schwartz J.C. Griep-Raming J. Westphall M.S. Coon J.J. Activated-ion electron transfer dissociation improves the ability of electron transfer dissociation to identify peptides in a complex mixture.Anal. Chem. 2010; 82: 10068-10074Crossref PubMed Scopus (61) Google Scholar). By incorporating an offline first dimension of separation with more extensive fractionation (80 versus 15) Gygi et al. expanded on this work in 2003 (21Peng J. Elias J.E. Thoreen C.C. Licklider L.J. Gygi S.P. Evaluation of multidimensional chromatography coupled with tandem mass spectrometry (LC/LC-MS/MS) for large-scale protein analysis: the yeast proteome.J. Proteome Res. 2002; 2: 43-50Crossref Scopus (1378) Google Scholar). That said, the modest increase in identified proteins (1504) required 135 h of analysis, reducing the protein per minute count to 0.2. 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 (22de Godoy L.M.F. Olsen J.V. de Souza G.A. Li G.Q. Mortensen P. Mann M. Status of complete proteome analysis by mass spectrometry: SILAC labeled yeast as a model system.Genome Biol. 2006; 7: R50Crossref PubMed Scopus (231) Google Scholar). From these three pioneering studies we begin to see the impact of mass spectrometer acquisition rate on the depth and rate of proteome analysis. The most recent application of such technology to the yeast proteome, however, The Mann work used a hybrid linear ion trap-ion cyclotron resonance Fourier transform instrument (LTQ-FT) that delivered MS2 scans at a rate of ∼650 ms (23Syka J.E.P. Marto J.A. Bai D.L. Horning S. Senko M.W. Schwartz J.C. Ueberheide B. Garcia B. Busby S. Muratore T. Shabanowitz J. Hunt D.F. Novel linear quadrupole ion trap/FT mass spectrometer: Performance characterization and use in the comparative analysis of histone H3 post-translational modifications.J. Proteome Res. 2004; 3: 621-626Crossref PubMed Scopus (333) Google Scholar). The earlier studies, i.e. Yates and Gygi, relied on the considerably slower scanning (1–3 s/scan) three-dimensional ion trap technology. In 2008, using the novel Orbitrap hybrid mass spectrometer, Mann and colleagues reported on the first comprehensive analysis of the yeast proteome by identifying nearly 4000 proteins (10de Godoy L.M.F. Olsen J.V. Cox J. Nielsen M.L. Hubner N.C. Frohlich F. Walther T.C. Mann M. Comprehensive mass-spectrometry-based proteome quantification of haploid versus diploid yeast.Nature. 2008; 455: 1251-1254Crossref PubMed Scopus (737) Google Scholar). Extensive fractionation (24Swaney D.L. Wenger C.D. Coon J.J. Value of using multiple proteases for large-scale mass spectrometry-based proteomics.J. Proteome Res. 2010; 9: 1323-1329Crossref PubMed Scopus (330) Google Scholar) and triplicate analysis of each fraction rendered the study a considerable time investment at ∼144 analysis hours (0.5 proteins/minute). In 2010 our group achieved similar comprehensive analysis, but improved sequence coverage, using fractionation and multiple proteases (24Swaney D.L. Wenger C.D. Coon J.J. Value of using multiple proteases for large-scale mass spectrometry-based proteomics.J. Proteome Res. 2010; 9: 1323-1329Crossref PubMed Scopus (330) Google Scholar). That work, however, required even longer analysis time (0.2 proteins/min). And that was the state-of-the-art as recently as three years ago. Doubtless we, the proteomics community, had achieved one momentous goal—comprehensive coverage of the yeast proteome. Still, obtaining this depth was not routine as it mandated days of MS analysis and a considerable amount of expert labor. In 2012, with new, even faster scanning, quadrupole-Orbitrap technology (Q-OT, Q-Exactive), Mann and colleagues dispatched the concept of fractionation, improved the quality of sample preparation, and placed emphasis on higher quality online separations (25Nagaraj N. Alexander Kulak N. Cox J. Neuhauser N. Mayr K. Hoerning O. Vorm O. Mann M. System-wide perturbation analysis with nearly complete coverage of the yeast proteome by single-shot ultra HPLC runs on a bench top orbitrap.Mol. Cell. Proteomics. 2012; 11 (M111.013722)Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar). With their streamlined method they achieved detection of just over 3900 yeast proteins following four hours of MS analysis. Even more impressive this strategy translated to the identification of 16.3 proteins per minute—a 33-fold improvement over the next best comprehensive study. This success was a remarkable achievement and illustrates that comprehensive proteomic technology can indeed be executed in a time efficient manner. Time-of-flight hybrid systems, of course, can deliver very high MS2 acquisition rates, up to 100 Hz in some reports. In 2011, Muddiman and colleagues reported yeast proteome analysis using a quadrupole-TOF system (i.e. TripleTOF) operating at a much lower rate (20 Hz) MS2 scan rate (26Andrews G.L. Simons B.L. Young J.B. Hawkridge A.M. Muddiman D.C. Performance Characteristics of a New Hybrid Quadrupole Time-of-Flight Tandem Mass Spectrometer (TripleTOF 5600).Anal. Chem. 2011; 83: 5442-5446Crossref PubMed Scopus (223) Google Scholar). Even at this reduced rate, only 16% of the spectra were mapped to unique sequences and 1112 unique proteins identified. Because of reduced MS2 spectral quality (i.e. low signal-to-noise, S/N), even fewer unique peptide identifications were achieved at higher MS2 acquisition rates. Other studies using TOF technologies report similar results (27Cristobal A. Hennrich M.L. Giansanti P. Goerdayal S.S. Heck A.J.R. Mohammed S. In-house construction of a UHPLC system enabling the identification of over 4000 protein groups in a single analysis.Analyst. 2012; 137: 3541-3548Crossref PubMed Scopus (42) Google Scholar, 28Geromanos S. Hughes C. Ciavarini S. Vissers J.C. Langridge J. Using ion purity scores for enhancing quantitative accuracy and precision in complex proteomics samples.Anal. Bioanal. Chem. 2012; 404: 1127-1139Crossref PubMed Scopus (42) Google Scholar). For maximal proteome depth, we conclude that increased scan speed must not come at the cost of reduced spectral quality. Recently, a new Orbitrap hybrid mass spectrometer having a mass filter, a collision cell, a high-field Orbitrap analyzer, and, finally, a dual cell linear ion trap analyzer was described (Q-OT-qIT, Orbitrap Fusion) (29Senko M.W. Remes P. Song Q. Canterbury J. Blethrow J. Zabrouskov V. Lange O. Makarov A. Improving data dependent MSn Performance with a Multitasking Mass Spectrometer. American Society for Mass Spectrometry, Minneapolis, MN2013Google Scholar, 30Senko, M., Remes, P., Canterbury, J., Mathur, R., Song, Q., Eliuk, S., Mullen, C., Earley, L., Hardman, H., Blethrow, J., Bui, H., Specht, A., Lange, O., Denisov, E., Makarov, A., Horning, S., Zabrouskov, V., Novel parallelized quadrupole/linear ion trap/orbitrap tribrid mass spectrometer improves proteome coverage and peptide identification rates. Analytical Chemistry.Google Scholar). This system offers high MS2 acquisition speed of 20 Hz—double that of the Q-OT system used by Mann and colleagues. We postulated that this fresh system, with its fast scan rate, could provide comprehensive proteome analysis in record time. 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 (31Meyer J. A. Komives E. Charge state coalescence during electrospray ionization improves peptide identification by tandem mass spectrometry.J. Am. Soc. Mass Spectrom. 2012; 23: 1390-1399Crossref PubMed Scopus (45) Google Scholar, 32Hahne H. Pachl F. Ruprecht B. Maier S.K. Klaeger S. Helm D. Medard G. Wilm M. Lemeer S. Kuster B. DMSO enhances electrospray response, boosting sensitivity of proteomic experiments.Nat. Meth. 2013; 10: 989-991Crossref PubMed Scopus (175) Google Scholar). Using this novel system we report the comprehensive analysis of the yeast proteome (4002 with 1% FDR) following 1.3 h of nLC-MS2 analysis (70 min gradient). These experiments delivered an extraordinary 67 proteins per minute and demonstrate that complete analysis of the yeast proteome can be routinely performed in approximately one hour. Saccharomyces cerevisiae strain BY4741 was grown in yeast extract peptone dextrose media (1% yeast extract, 2% peptone, 2% dextrose). Four liters of media was divided between four two-liter flasks and inoculated with a starter culture (OD600 = 2.58). Cells were allowed to propagate for ∼12 generations (20 h) to an OD600 ∼2 (average of 2.18). The cells were harvested by centrifugation at 5000 rpm for 5 min, supernatant decanted, resuspended in chilled NanoPure water and all pellets were pooled together. The cells were washed two more times and centrifuged for the final pelleting at 5000 rpm for 10 min. A pellet corresponding to 5% of the total cells grown, was resuspended in lysis buffer composed of 50 mm Tris pH8, 8 m urea, 75 mm sodium chloride, 100 mm sodium butyrate, protease (Roche) and phosphatase inhibitor tablet (Roche). Yeast cells were lysed by glass bead milling (Retsch). Briefly, 2 ml of acid washed glass beads were combined with 2.5 ml of resuspended yeast cells in a stainless steel container and shaken 8 times at 30 hz for 4 min with a 1 min rest in between. Lysate protein concentration was measured by BCA (Thermo Pierce). Protein was reduced by addition of 5 mm dithiothreitol and incubated for 45 min at 55 °C. The mixture was cooled to room temperature, followed by alkylation of free thiols by addition of 15 mm iodoacetamide in the dark for 30 min. The alkylation reaction was quenched with 5 mm dithiothreitol. Urea concentration was diluted to 1.5 m with 50 mm Tris pH 8.0. Proteolytic digestion was performed by addition of Trypsin (Promega, Madison, WI), 1:50 enzyme to protein ratio, and incubated at ambient temperature overnight. An additional 1:50 bolus of trypsin was added in the morning and incubated at ambient temperature for 1 h. The digestion was quenched by addition of TFA and desalted over a tC18 Sep-Pak (Waters, Milford, MA). Reversed phase columns were prepared in-house. Briefly, a 75–360 μm inner-outer diameter bare-fused silica capillary, with a laser pulled electrospray tip, was packed with 1.7 μm diameter, 130 Å pore size, Bridged Ethylene Hybrid C18 particles (Waters) to a final length of 35 cm. The column was installed on a nanoAcquity UPLC (Waters) using a stainless steel ultra-high pressure union formatted for 360 μm outer diameter columns (IDEX) and heated to 60 °C for all runs. Mobile phase buffer A was composed of water, 0.2% formic acid, and 5% DMSO. Mobile phase B was composed of acetonitrile, 0.2% formic acid, and 5% DMSO. Samples were loaded onto the column for 12 min at 0.35 μl/min. Mobile phase B increases to 4% in the first 0.1 min then to 12% B at 32 min, 22% B at 60 min, and 30% B at 70 min, followed by a 5 min wash at 70% B and a 20 min re-equilibration at 0%B. Eluting peptide cations were converted to gas-phase ions by electrospray ionization and analyzed on a Thermo Orbitrap Fusion (Q-OT-qIT, Thermo). Survey scans of peptide precursors from 300 to 1500 m/z were performed at 60K resolution (at 200 m/z) with a 5 × 105 ion count target. Tandem MS was performed by isolation at 0.7 Th with the quadrupole, HCD fragmentation with normalized collision energy of 30, and rapid scan MS analysis in the ion trap. The MS2 ion count target was set to 104 and the max injection time was 35 ms. Only those precursors with charge state 2–6 were sampled for MS2. The dynamic exclusion duration was set to 45 s with a 10 ppm tolerance around the selected precursor and its isotopes. Monoisotopic precursor selection was turned on. The instrument was run in top speed mode with 5 s cycles, meaning the instrument would continuously perform MS2 events until the list of nonexcluded precursors diminishes to zero or 5 s, whichever is shorter. Elite runs were performed with Survey scans of peptide precursors from 300 to 1500 m/z 60K resolution (at 200 m/z) with a 1 × 106 ion count target. Tandem MS was performed by isolation at 1.8 Th with the ion-trap, CAD fragmentation with normalized collision energy of 35, and rapid scan MS analysis in the ion trap. The data dependent top 20 precursors were selected for MS2. MS2 ion count target was set to 5 × 103 and the max injection time was 125 ms. Only those precursors with charge state +2 or higher were sampled for MS2. The dynamic exclusion duration was set to 40 s with a 10 ppm tolerance around the selected precursor and its isotopes. Monoisotopic precursor selection was turned on. The raw data was processed using Proteome Discoverer (version 1.4.0.288, Thermo Fischer Scientific). MS2 spectra were searched with SEQUEST engine against a database of 6632 yeast open reading frames (ORFs) 1The abbreviations used are:ORFopen reading frameFDRfalse discovery ratePSMpeptide spectral matchesMRMmultiple reaction monitoring. (www.yeastgenome.com, February 3, 2011) (33Eng J.K. McCormack A.L. Yates Iii J.R. An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database.J. Am. Soc. Mass Spectrom. 1994; 5: 976-989Crossref PubMed Scopus (5420) Google Scholar). Peptides were generated from a tryptic digestion with up to two missed cleavages, carbamidomethylation of cysteines as fixed modifications, and oxidation of methionines and protein N-terminal acetylation as variable modifications. Precursor mass tolerance was 20 ppm and product ions were searched at 0.35 Da tolerances. Peptide spectral matches (PSM) were validated using percolator based on q-values at a 1% FDR (34Brosch M. Yu L. Hubbard T. Choudhary J. Accurate and Sensitive Peptide Identification with Mascot Percolator.J. Proteome Res. 2009; 8: 3176-3181Crossref PubMed Scopus (332) Google Scholar). With proteome Discoverer, peptide identifications were grouped into proteins according to the law of parsimony and filtered to 1% FDR (35Nesvizhskii A.I. Aebersold R. Interpretation of shotgun proteomic data: the protein inference problem.Mol. Cell. Proteomics. 2005; 4: 1419-1440Abstract Full Text Full Text PDF PubMed Scopus (791) Google Scholar). For cumulative protein group identification, PSMs passing the FDR were exported to a text file and processed by a modified version of Protein Hoarder (version 2.4.1) (36Wenger C.D. Phanstiel D.H. Lee M.V. Bailey D.J. Coon J.J. COMPASS: A suite of pre- and post-search proteomics software tools for OMSSA.Proteomics. 2011; 11: 1064-1074Crossref PubMed Scopus (132) Google Scholar). The PSMs were iteratively processed in successive 1 min windows and grouped into proteins using the law of parsimony at a 1% FDR. open reading frame false discovery rate peptide spectral matches multiple reaction monitoring. Considerable gains in the depth and rate of proteomic analysis have been realized over the past decade (vide supra). These improved results stem from routine use of high mass accuracy and resolution, but also from a steady increase in MS2 acquisition rate. In the decade spanning the seminal Yates publication in 2001 and the single-shot proteome work of Mann et al. in 2012, MS2 sampling rates rose from ∼0.75 Hz to nearly 10 Hz (17Washburn M.P. Wolters D. Yates J.R. Large-scale analysis of the yeast proteome by multidimensional protein identification technology.Nat. Biotech. 2001; 19: 242-247Crossref PubMed Scopus (4077) Google Scholar, 25Nagaraj N. Alexander Kulak N. Cox J. Neuhauser N. Mayr K. Hoerning O. Vorm O. Mann M. System-wide perturbation analysis with nearly complete coverage of the yeast proteome by single-shot ultra HPLC runs on a bench top orbitrap.Mol. Cell. Proteomics. 2012; 11 (M111.013722)Abstract Full Text Full Text PDF PubMed Scopus (304) Google Scholar). Here we report on an even newer generation of mass spectrometer that comprises a mass resolving quadrupole, Orbitrap, collision cell, and linear ion trap (Q-OT-qIT, Fusion, Fig. 1) (29Senko M.W. Remes P. Song Q. Canterbury J. Blethrow J. Zabrouskov V. Lange O. Makarov A. Improving data dependent MSn Performance with a Multitasking Mass Spectrometer. American Society for Mass Spectrometry, Minneapolis, MN2013Google Scholar, 30Senko, M., Remes, P., Canterbury, J., Mathur, R., Song, Q., Eliuk, S., Mullen, C., Earley, L., Hardman, H., Blethrow, J., Bui, H., Specht, A., Lange, O., Denisov, E., Makarov, A., Horning, S., Zabrouskov, V., Novel parallelized quadrupole/linear ion trap/orbitrap tribrid mass spectrometer improves proteome coverage and peptide identification rates. Analytical Chemistry.Google Scholar). In this system, MS acquisition rate is not only boosted by the presence of a very fast scanning dual cell linear ion trap, but also by a control environment having multiple, independent processing units. 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 ∼2× 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. To test this hypothesis we began by performing a parametric evaluation using a complex mixture of yeast tryptic peptides eluted into the system over a 70 min gradient. We examined several settings including collisional activation mode (i.e. HCD or trap CAD), MS1 resolution, collision energy, maximum inject time, and dynamic exclusion settings. Detailed plots highlighting these results are included in Supplemental Information. Briefly, we found that MS2 analysis using HCD followed by ion trap mass analysis (low-res HCD; 80,626 MS2 events with 33,127 unique PSMs) generated more identifications compared with ion trap CAD with ion trap mass analysis (CAD; 75,973 MS2 events with 31,820 unique PSMs). This is not surprising as HCD tends to offer more random backbone fragmentation and, with the Q-OT-qIT geometry, can be accomplished slightly faster. Operation of the system with an MS1 resolving power setting of 60,000 (@ m/z 200) afforded a 20% increase in detected unique peptides over 15,000 resolving power (supplemental Fig. S1). We conclude the boosted resolving power elevates precursor signal-to-noise (S/N) ratios, allowing for improved selection of low abundance precursors, and can potentially separate otherwise unresolved precursors so that multiple MS2 events can be acquired. Of course, in this scenario such closely spaced precursors would be co-isolated (0.7 m/z isolation width); however, the selected precursor m/z annotated in the MS2 scan would be different and would facilitate identification from a chimeric MS2 scan. Increasing MS1 resolving power above 60,000 did not provide any apparent benefit for increased identifications. Thirty-five milliseconds was the optimal maximum injection time (supplemental Fig. S2). Decreasing the maximum injection time to 30 msec, or increasing it to 45 msec caused a 10% decrease in peptide identifications. We found only slight variations in peptide identifications among dynamic exclusion settings of 30, 45, and 60 s (supplemental Fig. S3). Quadrupole isolation widths from 0.5 to 1.5 m/z were examined, the best results were achieved at a value of 0.7 m/z (supplemental Fig. S4). MS1 and MS2 automatic gain control (AGC) target values of 500,000 and 7000, respectively, produced the maximum number of peptide identifications (supplemental Figs. S5 and S6). Yeast cell lysis is a critical step in achieving comprehensive proteome detection and must be executed with care. Detergents, such as SDS, or bead beating are typical approaches for yeast lysis (37Wisniewski J.R. Zougman A. Nagaraj N. Mann M. Universal sample preparation method for proteome analysis.Nat. Meth. 2009; 6: 359-362Crossref PubMed Scopus (5043) Google Scholar, 38Dephoure N. Gygi S.P. Hyperplexing: A method for higher-order multiplexed quantitative proteomics provides a map of the dynamic response to rapamycin in yeast.Sci. Signal. 2012; 5 (rs2-rs-52)Crossref PubMed Scopus (121) Google Scholar, 39Hebert A.S. Merrill A.E. Bailey D.J. Still A.J. Westphall M.S. Strieter E.R. Pagliarini D.J. Coon J.J. Neutron-encoded mass signatures for multiplexed proteome quantification.Nat. Meth. 2013; 10: 332-334Crossref PubMed Scopus (143) Google Scholar). The SDS method, used in the Mann yeast studies, has produced excellent results, but requires removal of the detergent before MS sampling. The bead beating method mixes glass beads and yeast cells in a buffer slurry, which is shaken for three, 1–4 min cycles, at 30 hz. This approach, however, can be too gentle to sufficiently lyse the yeast and, in our hands, does not efficiently extract all proteins. We aimed to avoid use of detergents and investigated a more vigorous bead beating procedure. By simply extending the number of cycles to eight (4 min each) we achieved considerably improved results (supplemental Fig. S7). Finally, we note increased identifications when lysates were not cleared of insoluble material. Zubarev a
