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Digestion and depletion of abundant proteins improves proteomic coverage

An Addendum to this article was published on 27 February 2014

Abstract

Two major challenges in proteomics are the large number of proteins and their broad dynamic range in the cell. We exploited the abundance-dependent Michaelis-Menten kinetics of trypsin digestion to selectively digest and deplete abundant proteins with a method we call DigDeAPr. We validated the depletion mechanism with known yeast protein abundances, and we observed greater than threefold improvement in low-abundance human-protein identification and quantitation metrics. This methodology should be broadly applicable to many organisms, proteases and proteomic pipelines.

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Figure 1: Schematic and description of the DigDeAPr method.
Figure 2: Analysis of proteomic metric improvements from DigDeAPr on HEK cell lysates.
Figure 3: Analysis of proteomic metrics relevant to MS- and MS/MS-based quantitation.

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Acknowledgements

This project was supported by the US National Center for Research Resources (5P41RR011823-17), National Institute of General Medical Sciences (8P41GM103533-17), National Institute of Diabetes and Digestive and Kidney Diseases (R01DK074798), National Heart, Lung, and Blood Institute (RFP-NHLBI-HV-10-5) and National Institute of Mental Health (R01MH067880). We thank J.N. Savas, C.M. Delahunty and J.K. Diedrich for comments on the manuscript.

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Contributions

B.R.F. designed experiments, performed experiments, analyzed data and wrote the manuscript. B.D.S. prepared HEK cell lysates and provided conceptual advice. K.J.W. prepared yeast lysates. T.X., J.C. and S.K.P. developed software for data analysis. J.R.Y. wrote the manuscript and provided conceptual guidance.

Corresponding author

Correspondence to John R Yates III.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Text and Figures

Supplementary Figures 1–8 and Supplementary Notes 1–6 (PDF 726 kb)

Supplementary Data

Protein and peptide raw data and comparisons from yeast and HEK-cell control and DigDeAPr analyses. (XLSX 49758 kb)

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Fonslow, B., Stein, B., Webb, K. et al. Digestion and depletion of abundant proteins improves proteomic coverage. Nat Methods 10, 54–56 (2013). https://doi.org/10.1038/nmeth.2250

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