C-terminal ladder sequencing via matrix-assisted laser desorption mass spectrometry coupled with carboxypeptidase Y time-dependent and concentration-dependent digestions.

The utility of matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) mass spectrometry for the analysis of C-terminal peptide ladders from carboxypeptidase Y (CPY) digestions is discussed. MALDI analysis of aliquots of an optimized time-dependent CPY digestion of ACTH 7-38 fragment allowed for the sequence of the first 19 amino acids from the C-terminus to be determined in 25 min of digestion time. A strategy for performing parallel concentration-dependent digestions on the MAL-DI plate is proven to be superior to the time-dependent approach as the method development time and practical amounts of both peptide and enzyme consumed are reduced significantly. The on-plate approach offered the same sequence information from the ACTH 7-38 fragment and was used to digest 22 peptides of various amino acid composition, size, charge, and polarity. Of the 22 peptides digested on-plate, sequence information was derived from 19 of them. A statistical analysis strategy for ladder sequencing utilizing t-statistics is offered as a method for placing confidence intervals on residue assignments.