Enzymatic approaches for obtaining amino acid sequence: on-target ladder sequencing

Peptide sequencing by mass spectrometry (MS) is usually based on detecting mass differences associated with various amino acids in the polymer chain. Post-source decay (PSD) and MS/MS spectra may yield internal peptide sequences. However, determination of the order of the first two, and sometimes the last few, amino acids in the peptide is often problematic without additional experiments. Several enzymatic approaches have proven useful for identifying the N- and C-terminal residues. They involve the use of carboxypeptidases and aminopeptidases to produce peptide ladders for rapid analysis by matrix-assisted laser desorption/ionization mass spectrometry (MALDI-MS). This unit describes the ladder sequence method to generate amino acid sequence information from low- to subpicomole quantities of peptides. It can be performed directly on the sample stage, thus minimizing potential sample losses to vials and through sample transfer.