The use of hydrolytic enzymes and multi-stage tandem mass spectrometry to analyze pyridoxal phosphate-modified peptides.

A previous approach was established that allowed direct identification of pyridoxal-5'-phosphate (PLP) bonding sites in proteins using mass spectrometry after tryptic proteolysis. The approach required peptide mass fingerprinting owing to suppressed amide backbone fragmentation in favor of side-chain elimination of diagnostic product ions from PLP-derivatized lysyl residues. While sufficient for purified proteins, unambiguous sequence determination is needed to assign PLP bonding sites in unknown proteins in complex mixtures. Here, we describe the use of hydrolytic enzymes and multi-stage tandem mass spectrometry to elucidate the amino acid sequence and PLP bonding site in PLP-modified peptides.