Accelerated digestion for high-throughput proteomics analysis of whole bacterial proteomes

Journal of Microbiological Methods
Ali R VaezzadehDenis F Hochstrasser

Abstract

In bottom-up proteomics, rapid and efficient protein digestion is crucial for data reliability. However, sample preparation remains one of the rate-limiting steps in proteomics workflows. In this study, we compared the conventional trypsin digestion procedure with two accelerated digestion protocols based on shorter reaction times and microwave-assisted digestion for the preparation of membrane-enriched protein fractions of the human pathogenic bacterium Staphylococcus aureus. Produced peptides were analyzed by Shotgun IPG-IEF, a methodology relying on separation of peptides by IPG-IEF before the conventional LC-MS/MS steps of shotgun proteomics. Data obtained on two LC-MS/MS platforms showed that accelerated digestion protocols, especially the one relying on microwave irradiation, enhanced the cleavage specificity of trypsin and thus improved the digestion efficiency especially for hydrophobic and membrane proteins. The combination of high-throughput proteomics with accelerated and efficient sample preparation should enhance the practicability of proteomics by reducing the time from sample collection to obtaining the results.

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Citations

Feb 4, 2010·Expert Review of Proteomics·Sabine Matallana-SurgetRuddy Wattiez
Jan 25, 2011·Journal of Proteomics·Lilla TuriákKároly Vékey
Dec 16, 2011·Electrophoresis·Filip DyckaJanette Bobalova
Apr 21, 2012·Analytical Biochemistry·Hui ZhouRichard S Lee
Feb 2, 2013·Journal of Proteome Research·Linda SwitzarWilfried M A Niessen
Dec 12, 2017·Mass Spectrometry Reviews·Qiwei ZhangJianjun Li
Nov 14, 2017·Expert Review of Proteomics·Róisín O'FlahertyGordan Lauc
Apr 5, 2016·Expert Review of Proteomics·Irena Trbojević-AkmačićGordan Lauc
Jun 4, 2021·The Journal of Cell Biology·Yun QuanHuilin Zhou

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