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A qualitative and quantitative evaluation of the peptide characteristics of microwave‐ and ultrasound‐assisted digestion in discovery and targeted proteomic analyses
Author(s) -
Guo Zhengguang,
Cheng Jie,
Sun Haidan,
Sun Wei
Publication year - 2017
Publication title -
rapid communications in mass spectrometry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.528
H-Index - 136
eISSN - 1097-0231
pISSN - 0951-4198
DOI - 10.1002/rcm.7913
Subject(s) - digestion (alchemy) , chemistry , trypsin , proteomics , proteome , quantitative proteomics , peptide , chromatography , sample preparation , biochemistry , enzyme , gene
Rationale Fast digestion methods can dramatically accelerate enzyme digestion and increase the throughput of proteomic analysis. However, the peptide characteristics of fast digestion methods and their performance in discovery and targeted proteomic analysis must be systematically evaluated. Methods Three digestion methods, including overnight digestion, microwave‐assisted protein enzymatic digestion (MAPED), and high‐intensity focused ultrasonic‐assisted enzymatic digestion (HIFUSAED), in trypsin or in trypsin/Lys‐C were comprehensively compared in both discovery and targeted proteomics analysis using the HeLa cell proteome. In discovery proteomic analysis, the highest numbers of peptides and proteins were identified when the sample was digested via the MAPED method with trypsin/Lys‐C. Results The fast digestion methods showed a higher mis‐cleavage rate and a lower semi‐tryptic rate than the overnight digestion method. In both label‐free quantitative analysis and targeted proteomic analysis, both fully cleaved peptides (FCPs) and mis‐cleaved peptides (MCPs) from the fast digestion methods and the overnight digestion method showed good reproducibility if they showed good abundance. Conclusions When both the FCPs and MCPs were included in the analysis, the MAPED with trypsin/Lys‐C method showed the best results for both discovery proteomic analysis and relative quantitative targeted proteomic analysis. These results will be beneficial for the application of fast digestion methods to proteomics.