Utilization\nof Liquid Chromatography–Mass Spectrometry\nand High-Resolution Ion Mobility–Mass Spectrometry to Characterize\nTherapeutically Relevant Peptides with Asparagine Deamidation and\nIsoaspartate

Rapid identification of asparagine (Asn) deamidation\nand isoaspartate\n(<i>iso</i>Asp) in proteins remains a challenging analytical\ntask during the development of biological therapeutics. For this study,\n46 therapeutically relevant peptides corresponding to 13 peptide families\n(13 unmodified peptides and 33 modified peptides) were obtained; modified\npeptides included Asn deamidation and isoAsp. The peptide families\nwere characterized by three methods: reversed-phase ultrahigh performance\nliquid chromatography–mass spectrometry (RP-UHPLC-MS); flow\ninjection analysis high-resolution ion mobility–mass spectrometry\n(FIA-HRIM-MS); and shortened gradient RP-UHPLC-HRIM-MS. UHPLC-MS data\nacquisition was 2 h per injection, in contrast to high-throughput\n1 min data acquisition of the FIA-HRIM-MS technique. A rapid 2D peptide\nmap has been demonstrated by combining shortened gradient RP-UHPLC\nwith HRIM, to optimize the resolution of the Asn-, Asp-, and isoAsp-containing\npeptides, increasing the likelihood of detecting peptides containing\nthese quality attributes with expedited data acquisition. Additionally,\nthis paper provides an ion mobility calibration data set for therapeutically\nrelevant peptides (unmodified and modified) over an ion-neutral collisional\ncross-section range of 300–800 Å<sup>2</sup>.