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One-hour proteome analysis in yeast


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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Figure 1: Structure of the in-house-manufactured column heater.
Figure 2: Column fabrication.
Figure 3: Effect of MS1 AGC target, resolution and MS2 max inject time on the number of MS/MS scans, PSMs and unique peptides.
Figure 4: Yeast peptide and protein identifications for all replicates.
Figure 5: Unique peptides and proteins identified over the LC-MS/MS gradient.
Figure 6: Effect of gradient length on peptide and protein identifications.


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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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Authors and Affiliations



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.

Corresponding author

Correspondence to Joshua J Coon.

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The authors declare no competing financial interests.

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.

Supplementary Figure 3 Orbitrap Fusion scan sequence.

Supplementary information

Supplementary Text and Figures

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).

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