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Improvement of in‐gel digestion protocol for peptide mass fingerprinting by matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry
Author(s) -
Katayama Hiroyuki,
Nagasu Takeshi,
Oda Yoshiya
Publication year - 2001
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.379
Subject(s) - chemistry , chromatography , mass spectrometry , bottom up proteomics , sample preparation in mass spectrometry , matrix assisted laser desorption/ionization , protein mass spectrometry , time of flight mass spectrometry , peptide mass fingerprinting , two dimensional gel electrophoresis , capillary electrophoresis–mass spectrometry , gel electrophoresis , matrix (chemical analysis) , mass spectrum , proteomics , desorption , electrospray ionization , ionization , biochemistry , ion , adsorption , gene , organic chemistry
High‐sensitivity, high‐throughput analysis of proteins for proteomics studies is usually performed by polyacrylamide gel electrophoresis in combination with mass spectrometry. However, the quality of the data obtained depends on the in‐gel digestion procedure employed. This work describes an improvement in the in‐gel digestion efficiency for matrix‐assisted laser desorption/ionization time‐of‐flight mass spectrometry (MALDI‐TOFMS) analysis. A dramatic improvement in the coverage of tryptic peptides was observed when n‐octyl glucoside was added to the buffer. Whole cell extracted proteins from S. cerevisiae were separated by two‐dimensional gel electrophoresis and stained with silver. Protein spots were identified using our improved in‐gel digestion method and MALDI‐TOFMS. In addition, the mass spectra obtained by using the matrix α‐cyano‐4‐hydroxycinnamic acid (CHCA) were compared with those obtained using 2,5‐dihydroxybenzoic acid (DHB). The DHB matrix usually gave more peaks, which led to higher sequence coverage and, consequently, to higher confidence in protein identification. This improved in‐gel digestion protocol is simple and useful for protein identification by MALDI‐TOFMS. Copyright © 2001 John Wiley & Sons, Ltd.