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. Updates of the in‐gel digestion protocol has been presented in the study. It has been shown that alternative reducing, alkylating agent reactions, and tryptic digestion buffers increase peptide and protein identification and reduce incubation times. The results indicate that a simultaneous and short, high temperature reduction and alkylation reaction using Tris(2‐carboxyethyl)phosphine hydrochloride and chloroacetamide with a subsequent gel wash improve protein identification and sequence coverage, and diminish peptide side reactions. Additionally, use of 4‐(2‐hydroxyethyl)piperazine‐1‐ethanesulfonic acid buffer 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.
Highlights
The in-gel digestion of proteins for analysis by liquid chromatograph mass investigation on reducing and alkylating reagents increased the number spectrometry has been used since the early 1990s
The addition of TCEP increased the number of identified tryptic miss-cleaved peptides by 7% compared to the DTT methods and that difference was reduced to 3% by heating the TCEP/CAA reaction at 70 °C for 5 min while reducing significantly the incubation time, as previously described for DTT[17] (Figure 2C, Supporting Information)
Replacing the ammonium bicarbonate (ABC) digestion buffer with HEPES, permitted a shorter incubation time and provided the best peptide coverage (Figure 1 and Figure 2, Supporting Information)
Summary
The in-gel digestion of proteins for analysis by liquid chromatograph mass investigation on reducing and alkylating reagents increased the number spectrometry has been used since the early 1990s. Our work collates recent advances in tryptic digestion to create beneficial upbuffers increase peptide and protein identification and reduce incubation dates of the in-gel digestion protocol.[9,10]. We show superior protein identification reduction and alkylation reaction using Tris(2-carboxyethyl)phosphine hydrochloride and chloroacetamide with a subsequent gel wash improve protein identification and sequence coverage, and diminish peptide side reactions. Tryptic in-gel digestion is well established as an efficient and simple method to prepare proteins for identification and quantification by MS.[1,2] The gel delivers excellent results when mass separation is required or compounds incompatible with MS cannot be excluded from protein extraction protocols.[3,4] Previous efforts to reduce incubation times have focused on the tryptic digestion step while maintaining efficiency by adding additives or increas-
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