Abstract
Recent advances in chromatography and mass spectrometry (MS) have made rapid and deep proteomic profiling possible. To maximize the performance of the recently produced Orbitrap hybrid mass spectrometer, we have developed a protocol that combines improved sample preparation (including optimized cellular lysis by extensive bead beating) and chromatographic conditions (specifically, 30-cm capillary columns packed with 1.7-μm bridged ethylene hybrid material) and the manufacture of a column heater (to accommodate flow rates of 350–375 nl/min) that increases the number of proteins identified across a single liquid chromatography–tandem MS (LC-MS/MS) separation, thereby reducing the need for extensive sample fractionation. This strategy allowed the identification of up to 4,002 proteins (at a 1% false discovery rate (FDR)) in yeast (Saccharomyces cerevisiae strain BY4741) over 70 min of LC-MS/MS analysis. Quintuplicate analysis of technical replicates reveals 83% overlap at the protein level, thus demonstrating the reproducibility of this procedure. This protocol, which includes cell lysis, overnight tryptic digestion, sample analysis and database searching, takes ∼24 h to complete. Aspects of this protocol, including chromatographic separation and instrument parameters, can be adapted for the optimal analysis of other organisms.
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References
Tabb, D.L. et al. Repeatability and reproducibility in proteomic identifications by liquid chromatography-tandem mass spectrometry. J. Proteome Res. 9, 761–776 (2010).
Liu, H., Sadygov, R.G. & Yates, J.R. III. A model for random sampling and estimation of relative protein abundance in shotgun proteomics. Anal. Chem. 76, 4193–4201 (2004).
Washburn, M.P., Wolters, D. & Yates, J.R. III. Large-scale analysis of the yeast proteome by multidimensional protein identification technology. Nat. Biotechnol. 19, 242–247 (2001).
Hebert, A.S. et al. The one hour yeast proteome. Mol. Cell Proteomics 13, 339–347 (2014).
Senko, M.W. et al. Novel parallelized quadrupole/linear ion trap/Orbitrap tribrid mass spectrometer improving proteome coverage and peptide identification rates. Anal. Chem. 85, 11710–11714 (2013).
Goffeau, A. et al. Life with 6000 genes. Science 274 546 563–547 (1996).
Ghaemmaghami, S. et al. Global analysis of protein expression in yeast. Nature 425, 737–741 (2003).
de Godoy, L.M. et al. Comprehensive mass-spectrometry-based proteome quantification of haploid versus diploid yeast. Nature 455, 1251–1254 (2008).
Wu, R. et al. Correct interpretation of comprehensive phosphorylation dynamics requires normalization by protein expression changes. Mol. Cell Proteomics 10, M111.009654 (2011).
Webb, K.J., Xu, T., Park, S.K. & Yates, J.R. III. Modified MuDPIT separation identified 4488 proteins in a system-wide analysis of quiescence in yeast. J. Proteome Res. 12, 2177–2184 (2013).
Nagaraj, N. et al. 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 11, M111.013722 (2012).
Kulak, N.A., Pichler, G., Paron, I., Nagaraj, N. & Mann, M. Minimal, encapsulated proteomic-sample processing applied to copy-number estimation in eukaryotic cells. Nat. Methods 11, 319–324 (2014).
Meyer, J.G. & Komives, E.A. Charge state coalescence during electrospray ionization improves peptide identification by tandem mass spectrometry. J. Am. Soc. Mass Spectrom. 23, 1390–1399 (2012).
Hahne, H. et al. DMSO enhances electrospray response, boosting sensitivity of proteomic experiments. Nat. Methods 10, 989–991 (2013).
Pirmoradian, M. et al. Rapid and deep human proteome analysis by single-dimension shotgun proteomics. Mol. Cell Proteomics 12, 3330–3338 (2013).
Huang da, W., Sherman, B.T. & Lempicki, R.A. Systematic and integrative analysis of large gene lists using DAVID bioinformatics resources. Nat. Protoc. 4, 44–57 (2009).
Richards, A., Hebert, A., Ulbrich, A., Bailey, D., Coughlin, E., Westphall, M. & Coon, J. Preparation of yeast cells for proteomic analysis by LC-MS/MS. Protocol Exchange (2015) 10.1038/protex.2015.030.
Treco, D.A. & Winston, F. Growth and manipulation of yeast. Curr. Protoc. Mol. Biol. 82, 13.2.1–13.2.12 (2008).
Elias, J.E. & Gygi, S.P. Target-decoy search strategy for increased confidence in large-scale protein identifications by mass spectrometry. Nat. Methods 4, 207–214 (2007).
Wenger, 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 11, 1064–1074 (2011).
Acknowledgements
We are grateful to A. Merrill for yeast production. We thank A. Gasch for assistance with yeast growth. This work was supported by the US National Institutes of Health (R01 GM080148) and the National Science Foundation (0701846). A.L.R. gratefully acknowledges the support from a US National Institutes of Health–funded Genomic Sciences Training Program (5T32HG002760).
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A.L.R. and A.S.H. designed experiments, performed research, analyzed data and wrote the paper; D.J.B. contributed analysis tools, analyzed data and wrote the paper; A.U. and E.E.C. contributed materials; M.S.W. analyzed data; J.J.C. designed the research and wrote the paper.
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Integrated supplementary information
Supplementary Figure 1 Distribution of the intensities of peptide precursors in the survey scan.
Red is with and black is without the addition of DMSO.
Supplementary Figure 2 Location of instrument parameters within Method Editor on the Orbitrap Fusion.
(A) MS1 resolution, MS1 detector type, and MS1 AGC target are set within the MS OT section. (B) Top speed data dependent mode and precursor priority are selected within the Decisions section. (C) MS2 detector type, MS2 isolation window, MS2 AGC target, MS2 max injection time, activation type, collision energy, detector type and scan rate are set within the MS/MS IT section.
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Supplementary Figures 1–3, Supplementary Tables 1–3 and Supplementary Data (PDF 1727 kb)
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Richards, A., Hebert, A., Ulbrich, A. et al. One-hour proteome analysis in yeast. Nat Protoc 10, 701–714 (2015). https://doi.org/10.1038/nprot.2015.040
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DOI: https://doi.org/10.1038/nprot.2015.040
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