Evaluation of endogenous peptide stereochemistry using liquid chromatography-mass spectrometry-based spiking...

Functional d-amino acid-containing peptides are endogenously found throughout nature, generated through non-ribosomal peptide synthesis or through post-translational modification of ribosome-derived peptides. Despite the high functional importance of peptide stereochemistry in biomolecular recognition, identification of amino acid residue isomerization can be challenging using most common peptide characterization workflows. Here, we describe a relatively simple approach to test hypotheses regarding the stereochemistry of endogenous peptides via liquid chromatography-mass spectrometry (LC-MS) spiking experiments with synthetic isotope-labeled peptide standards. Our protocol details the synthesis of ¹³C-labeled synthetic peptide diastereomers via solid-phase peptide synthesis, their purification and characterization, and LC-MS experiments to evaluate putative stereochemical assignments. This approach does not require demanding purification of the endogenous peptide and is compatible with small quantities of endogenous extracts. Overall, this procedure complements current endogenous peptide characterization methods, granting the ability to relatively quickly verify the sequence and stereochemistry in endogenous peptide diastereomers directly in complex biological extracts.