Identification of derivatized peptides without radiolabels: tandem mass spectrometric localization of the tagged active-site nucleophiles of two cellulases and a beta-glucosidase.
A new method that uses nonradioactive active site-directed enzyme inactivators and high-performance liquid chromatography-electrospray ionization tandem mass spectrometry (HPLC-ESIMS/MS) to identify labeled peptides in a proteolytic digest is described. This method relies upon the fragmentation of labeled peptides in a predictable and reproducible manner in the collision cell of a tandem mass spectrometer. The exoglycanase from Cellulomonas fimi, endoglucanase C from Clostridium thermocellum, and the beta-glucosidase from Agrobacterium faecalis were labeled using 2-deoxy-2-halo-beta-glycosides, digested with pepsin, and subjected to HPLC-ESIMS/MS analysis, scanning in the neutral loss mode. Under these conditions only peptides that lose the (known) mass of the 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 subsequent sequencing, either via further tandem MS experiments or via the Edman degradation. In all cases the peptides identified in this manner were consistent with those identified by the standard radioactive method. This mass spectrometric method represents a rapid, nonradioisotopic solution to the problem of identifying a modified peptide in a complex mixture. The technique is also sensitive, requiring only picomole amounts of protein.
Tull
D
D
Department of Chemistry, University of British Columbia, Vancouver, Canada.
Miao
S
S
Withers
S G
SG
Aebersold
R
R
D001426:Bacterial Proteins / Q000032:analysis; D001665:Binding Sites; D002480:Cellulase / Q000032:analysis*; D002851:Chromatography, High Pressure Liquid / Q000379:methods; D003013:Clostridium / Q000201:enzymology; D004789:Enzyme Activation; D043326:Glucan 1,3-beta-Glucosidase; D006027:Glycosides / Q000494:pharmacology; D007553:Isotope Labeling; D013058:Mass Spectrometry; D010455:Peptides / Q000032:analysis*; D012231:Rhizobium / Q000201:enzymology; D001617:beta-Glucosidase / Q000032:analysis*
D001426:Bacterial Proteins; D006027:Glycosides; D010455:Peptides; D001617:beta-Glucosidase; D002480:Cellulase; D043326:Glucan 1,3-beta-Glucosidase