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Abstract
A reliable and effective analytical method for discovering and characterizing isomerized residues in physiologically active peptides is essential for their comprehensive characterization. Complete structural detail facilitates the determination of a peptide’s biological roles and meets the increasingly stringent demands for peptide-based therapeutics. Here, a comprehensive untargeted analytical workflow predicts possible peptide isomers from peptidomics data and then localizes the isomerized residues using collision-induced dissociation-trapped ion mobility spectrometry (CID-TIMS) and protein isoaspartyl methyl transferase (PIMT) activity. The approach allows discovery and characterization of isomerized isoaspartate(isoAsp) residues and D-amino acids within the peptide. Potential isomeric peptide candidates are first identified from peptide-spectrum matches (PSMs) obtained after a database search by applying a defined retention-time window and comparing the differential ion-mobility values of precursor ions corresponding to nominally identical peptides. CID-TIMS is then utilized to locate the isomerized amino acid(s) within the predicted isomers by comparing the mobilities of fragment ions detected at those retention times. Finally, PIMT is used to label isoAsp residues for confirmation. This integrated workflow enabled localization of isoAsp residues in eight peptides from the rat hypothalamus. These peptides are from six prohormones, including proenkephalin, pro-melanin-concentrating hormone, secretogranin II, pituitary adenylate cyclase-activating polypeptide, and prosomatostatin. To show its broad utility, our workflow successfully identified a D-amino acid-containing form of small cardioactive peptide B, FMRFamide, and another D-amino acid-containing peptide from an uncharacterized protein in the sea slug A. californica. The presented discovery workflow effectively discovered novel isomerized residues in endogenous peptides directly from complex biological samples.
Supplementary materials
Title
Untargeted discovery and Localization of Isomerized Residues in Neuropeptides
Description
Additional experimental details, methods, and protocols for data analysis, additional results, including the localization of isomerized residues in the synthetic standard by CID-TIMS (Figure S1), EIC, and tandem MS spectra of a methylated sequence of prohormones of identified isoAsp-containing peptides (Figures S2-S4), and EIC of SCPB standards. Potential peptides with isomerized residues (Table S1 & S2).
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