Chromatographic properties of deamidated peptides with Asn-Gly sequences in proteomic bottom-up experiments

Studies surrounding deamidation have relied on the chromatographic and mass spectrometric differentiation of Asn containing peptides and their isomeric Asp and isoAsp products. The development of mass spectrometry analytical techniques and characterization of isomer specific fragmentation patterns has permitted the investigation of some deamidation species but has struggled to remain effective when applied and on complex samples or in high throughput scenarios. On the other hand, chromatographic separations can provide additional information to facilitate detection of deamidation. In this work the retention characteristics of deamidation products have been reported in reversed-phase separations using formic acid as an ion-pairing modifier. We found three major elution patterns depending on primary and secondary structure of Asn-Gly containing tryptic peptides. Random coil, helical conformations, and N-terminal positioning of Asn usually result in Asn < isoAsp < Asp, isoAsp < Asn < Asp, and Asn < Asp < isoAsp elution order, respectively. These trends, found from the analyses of proteomic samples, were subsequently confirmed via analytical scale UV-HPLC. Additionally, we determined the retention shifts following deamidation for twenty various separation settings used as a first-dimension fractionation for high-throughput proteomic 2D LC-MS/MS analyses.