IDENTIFICATION OF PROTEINS BY MATRIX-ASSISTED LASER-DESORPTION IONIZATION MASS-SPECTROMETRY FOLLOWING IN-GEL DIGESTION IN LOW-SALT, NONVOLATILE BUFFER AND SIMPLIFIED PEPTIDE RECOVERY

Matrix-assisted laser desorption ionization-mass spectrometry is an ef ficient analytical method for large-scale identification of proteins s eparated by two-dimensional polyacrylamide gel electrophoresis. Follow ing in-gel digestion, the salt present in the peptide extracts is usua lly removed by chromatography prior to analysis. Desalting is a labor- intensive and time-consuming step, limiting the total number of sample s that can be processed daily. We improved the daily sample output by performing the in-gel protein digestion in low-salt, nonvolatile buffe r and simplifying the recovery of the generated peptides, collecting t hem in a small volume by sonication. This technique is routinely used for identification of proteins of Haemophilus influenzae and human bra in. The methodology described facilitates the analytical process and a llows the analysis of hundreds of proteins per day. Furthermore, it re presents an essential step toward process automation. (C) 1997 Academi c Press.