The Impact of Different Alkylation Quenching Methods on Tryptic Activity and Protein Identification in Proteomics Sample Preparation

ABSTRACT The reduction and alkylation steps are crucial in shotgun proteomics sample preparation to ensure efficient protein digestion and prevent the reformation of artefactual disulfide bonds following proteolysis. Excessive alkylation reagents can lead to overalkylation side reactions, compromising the quality of proteomics sample detection. Previous research has predominantly focused on comparing the effects of various types or concentrations of reducing agents or alkylating reagents for proteomic sample preparation. However, there is a lack of studies systematically comparing the utilization of quenching agents for alkylation reactions and investigating their specific impact on tryptic digestion activity in proteomics sample preparation under conditions of excessive alkylation reagents. In this study, we comprehensively compared the impacts of three different alkylation quenching methods (including cysteine quenching, dithiothreitol [DTT] quenching, and no quenching) on proteomic sample preparation. The upstream sample processing included reduction with DTT or tris(2‐carboxyethyl)phosphine (TCEP), followed by alkylation with iodoacetamide (IAA) or chloroacetamide (CAA). Our study demonstrates that the choice of quenching method significantly affects the number of identified proteins and peptides, missed cleavage rates at lysine or arginine residues during trypsin digestion, and the occurrence of overalkylation side reactions. Importantly, our findings indicate that cysteine quenching effectively preserves trypsin activity, ensuring high‐quality protein sample preparation. This study provides a systematic analysis of various alkylation quenching methods in proteomic sample preparation and offers optimized experimental protocols and valuable data references for proteomics studies.