IDENTIFICATION OF DERIVATIZED PEPTIDES WITHOUT RADIOLABELS - TANDEM MASS-SPECTROMETRIC LOCALIZATION OF THE TAGGED ACTIVE-SITE NUCLEOPHILES OF 2 CELLULASES AND A BETA-GLUCOSIDASE

A new method that uses nonradioactive active site-directed enzyme inac tivators and high-performance liquid chromatography-electrospray ioniz ation tandem mass spectrometry (HPLC-ESLMS/MS) to identify labeled pep tides in a proteolytic digest is described, This method relies upon th e fragmentation of labeled peptides in a predictable and reproducible manner in the collision cell. of a tandem mass spectrometer, The exogl ycanase from Cellulomonas fimi, endoglucanase C from Clostridium therm ocellum, and the beta-glucosidase from Agrobacterium faecalis were lab eled using 2-deoxy-2-halo-beta-glycosides, digested with pepsin, and s ubjected to HPLC-ESIMS/MS analysis, scanning in the neutral loss mode, Under these conditions only peptides that lose the (known) mass of th e label are detected, Preliminary identification of candidate peptides can be achieved from the mass measured, in combination with the known sequence of the protein, Peptide identity can be confirmed through su bsequent sequencing, either via further tandem MS experiments or via t he Edman degradation, In all cafes the peptides identified in this man ner were consistent with those identified by the standard radioactive method, This mass spectrometric method represents a rapid, nonradioiso topic solution to the problem of identifying a modified peptide in a c omplex mixture, The technique is also sensitive, requiring only picomo le amounts of protein, (C) 1995 Academic Press,Inc.