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

High-sensitivity, high-throughput analysis of proteins for proteomics studi es is usually performed by polyacrylamide gel electrophoresis in combinatio n with mass spectrometry. However, the quality of the data obtained depends on the in-gel digestion procedure employed. This work describes an improve ment in the in-gel digestion efficiency for matrix-assisted laser desorptio n/ionization time-of-flight mass spectrometry (MALDI-TOFMS) analysis. A dra matic improvement in the coverage of tryptic peptides was observed when n-o ctyl glucoside was added to the buffer. Whole cell extracted proteins from S. cerevisiae were separated by two-dimensional gel electrophoresis and sta ined with silver. Protein spots were identified using our improved in-gel d igestion method and MALDI-TOFMS. In addition, the mass spectra obtained by using the matrix alpha -cyano-4-hydroxycinnamic acid (CHCA) were compared w ith those obtained using 2,5-dihydroxybenzoic acid (DHB). The DHB matrix us ually gave more peaks, which led to higher sequence coverage and, consequen tly, to higher confidence in protein identification. This improved in-gel d igestion protocol is simple and useful for protein identification by MALDI- TOFMS. Copyright (C) 2001 John Wiley & Sons, Ltd.