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Rapid Commun Mass Spectrom. 2001;15(16):1416-21.

Improvement of in-gel digestion protocol for peptide mass fingerprinting by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry.

Author information

1
Laboratory of Seeds Finding Technology, Eisai Co. Ltd, Tokodai 5-1-3, Tsukuba, Ibaraki 300-2635, Japan.

Abstract

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 alpha-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.

PMID:
11507753
DOI:
10.1002/rcm.379
[Indexed for MEDLINE]

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