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The Use of an Acid and Heat-Labile Surfactant for In-Gel Digestion

Part # PS185

Abstract

Michael Rosenblatt, Sergei Saveliev, and Marjeta Urh
Promega Corporation, 2800 Woods Hollow Road, Madison, WI 53711

SDS-PAGE is one of the most common modes of fractionation prior to MS sample preparation. High quality protein identification by mass spectrometry requires a robust in-gel digestion protocol. Incomplete peptide extraction, and extensive losses limit endusers abilities for comprehensive analysis. For example, decreases in protein coverage and the inability to detect proteins present at low nanogram levels are frequently
encountered.
We show here that with the use of the mass spectrometry-compatible surfactant ProteaseMAX™, peptide recovery from the gel is significantly improved and peptide loss is minimized leading to increased protein coverages and higher probability of protein identification for low-abundance proteins. Studies of temperature and incubation time showed that the optimal peptide recovery is achieved after 1 or 3 h depending on incubation temperature (50°C or 37°C respectively). Peptides are readily extracted from gel without the need for additional peptide extraction steps. Improvement in the in-gel protein digestion has been verified for individual proteins within a broad range of abundance for mouse membrane proteins using this enhancement.

THP08 #125

Printed in USA.