Effects of Heat Shock Treatment on Enzymatic Proteolysis for LC-MS/MS Quantitative Proteome Analysis

Abstract : Various efforts have been developed to improve sample preparation steps, which strongly depend on hands-on processesfor accurate and sensitive quantitative proteome analysis. In this study, we carried out heating the sample prior to trypsin di gestion usingan instrument to improve the tryptic digestion process. The heat shock generated by the system efficiently denatured proteins in thesample and increased the reproducibility in quantitative proteomics based on peptide abundance measurements. To demonstrate theeffectiveness of the protocol, three cell lines (A human lung cancer cell line (A549), a human embryonic kidney cell line (HEK293T),and a human colorectal cancer cell line (HCT-116)) were selected and the effect of heat shock was compared to that of normal trypticdigestion processes. The tryptic digests were desalted and analysed by LC-MS/MS, the results showed 57 and 36% increase in thenumber of identified unique peptides and proteins, respectively, than conventional digestion. Heat shock treated samples showed highernumbers of shorter peptides and peptides with low inter-sample variation among triplicate runs. Quantitative LC-MS/MS analysis ofheat shock treated sample yielded peptides with smaller relative error percentage for the triplicate run when the peak areas were com-pared. Exposure of heat-shock to proteomic samples prior to proteolysis in conventional digestion process can increase the digestionefficiency of trypsin resulting in production of increased number of peptides eventually leading to higher proteome coverage.Keywords : Denaturation, mass spectrometry, proteolysis, Heat shock