Identification of nearest-neighbor peptides in protease digests by mass spectrometry for construction of...

Continuous-flow fast atom bombardment mass spectrometry was used for the identification of the intermediates and end products of the tryptic digest of polypeptides throughout the time-course of the reactions. Precursor/product relationships for these peptides were determined with the aid of a simple personal computer program. The C-terminal tryptic peptides were identified by performing a tryptic digest in 50% 18O-enriched buffer which resulted in labeling of all non-C-terminal peptides with 18O. This information, along with the precursor/product correlations, was used to create a sequence-ordered tryptic map of the original polypeptide. Ion intensities of intermediate hydrolysis products are compared for enzyme to substrate ratios of 1:100 and 1:1000 (w/w) over the course of the reaction. Intermediates were found to have significantly longer lifetimes when lower levels of trypsin were present.