Characterizing posttranslational modifications of amino acids in peptides that do not have a mass change using mass spectrometry

Description:
Charactering the post-translational modifications (PTMs) of peptides and proteins is a hallmark of mass spectrometry; however, these measurements become more challenging when the modification does not induce a mass shift. As one example, D-amino acid-containing peptides (DAACPs) result from an unusual PTM where an all-L peptide is isomerized at a single amino acid residue. Furthermore, as stereochemistry is not routinely evaluated in peptidomics experiments, the prevalence of this modification is not known. Here, non-targeted stereoselective enzymatic screening, liquid chromatography tandem mass spectrometry and trapped ion mobility spectrometry are being used to identify discover novel DAACPs in the central nervous system (CNS) of several animals. Endogenous peptides are extracted from the CNS and treated with the stereoselective enzyme, aminopeptidase M (APM) that degrades most all-L peptides; peptides with D-amino acids near the N-terminus resist degradation. Peptides were detected using several mass spectrometry platforms including the qTOF and trapped ion mobility qTOF instruments. DAACPs were confirmed by comparing the chromatographic, MS, and TIMS properties of stable isotope-labeled peptide standards to those of endogenous DAACP candidates and the methods have been adapted to work with single cell capillary electrophoresis MS approaches. A number of novel DAACPs have been identified, including those from previously uncharacterized proteins. The DAACPs identified indicate that the isomerase is tolerant of a range of aromatic amino acids as well as aliphatic amino acids near the N-termini of the peptide. These approaches are being adapted to a range of additional animals including mammals; our results suggest that isomerization is a more common PTM than previously thought. Current progress towards isolating and characterizing the isomerase enzymes are highlighted.

Speaker: Jonathan Sweedler - University of Illinois at Urbana-Champaign
Jonathan Sweedler is the James R. Eiszner Family Endowed Chair in Chemistry at the University of Illinois. His research interests focus on developing new approaches for assaying small volume samples, including metabolomics and peptidomics, based on mass spectrometry. He has used these tools to characterize small molecules and peptides in a range of animal models across the metazoan and in samples as small as individual cells and cellular domains. Sweedler has published more than 400 manuscripts and presented 500 invited lectures. He has received numerous awards including the ACS Award in Analytical Chemistry and the Pittsburgh Analytical Chemistry Award. He is currently the Editor-in-Chief for Analytical Chemistry.