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 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.
Katayama
H
H
Laboratory of Seeds Finding Technology, Eisai Co. Ltd, Tokodai 5-1-3, Tsukuba, Ibaraki 300-2635, Japan.
Nagasu
T
T
Oda
Y
Y
D015180:Electrophoresis, Gel, Two-Dimensional / Q000379:methods; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods; D005656:Fungal Proteins / Q000737:chemistry* / Q000302:isolation & purification; D005782:Gels; D005960:Glucosides; D007202:Indicators and Reagents; D010446:Peptide Fragments / Q000737:chemistry* / Q000302:isolation & purification; D010455:Peptides / Q000737:chemistry*; D012441:Saccharomyces cerevisiae / Q000737:chemistry; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods; D014357:Trypsin
D005656:Fungal Proteins; D005782:Gels; D005960:Glucosides; D007202:Indicators and Reagents; D010446:Peptide Fragments; D010455:Peptides; C018619:octyl-beta-D-glucoside; D014357:Trypsin