A novel strategy for the targeted analysis of protein and peptide metabolites

Abstract In many biological applications such as epitope discovery or drug metabolism studies, the detection of naturally processed exogenous proteins (e.g. vaccines or peptide therapeutics) and their metabolites is frequently complicated by the presence of a complex endogenous mixture of closely related or even identical compounds. We describe a method that incorporates stable isotope labelling of the protein of interest, allowing the selective screening of the intact molecule and all metabolites using a modified precursor ion scan. This method involves monitoring the low‐molecular‐weight fragment ions produced during MS/MS that distinguish isotopically labelled peptides from related endogenous compounds. All isotopically labelled peptides can be selected using this method. The technique makes no assumptions about the processed or post‐translational state of the peptide, and hence can selectively screen out modified peptides that would otherwise be missed by single reaction monitoring approaches. This method does not replace single reaction monitoring or regular precursor scanning techniques; instead, it is a method that can be used when the assumptions required for the former two techniques cannot be predicted. The potential for this technique to be used in metabolism and pharmacokinetic experiments is discussed with specific examples looking at the metabolism of α‐synuclein in serum and the brain.