Updates of the in-gel digestion method for protein analysis by mass spectrometry

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Abstract

The in‐gel digestion of proteins for analysis by liquid chromatograph mass spectrometry has been used since the early 1990s. Although several improvements have contributed to increasing the quality of the data obtained, many recent publications still use sub‐optimal approaches. We present updates of the in‐gel digestion protocol. We show that alternative reducing, alkylating agent reactions and tryptic digestion buffers increase peptide and protein identification and reduce incubation times. Our results indicate that a simultaneous and short, high temperature reduction and alkylation reaction using Tris(2‐carboxyethyl)phosphine hydrochloride (TCEP) and chloroacetamide (CAA) with a subsequent gel wash improve protein identification and sequence coverage, diminish peptide side reactions. Additionally, use of 4‐(2‐Hydroxyethyl)piperazine‐1‐ethanesulfonic acid buffer (HEPES) allows a significant reduction in the digestion time improving trypsin performance and increasing the peptide recovery. The updates of the in‐gel digestion protocol described here are efficient and offer flexibility to be incorporated in any proteomic laboratory.

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