Utilization of Liquid Chromatography–Mass Spectrometry and High-Resolution Ion Mobility–Mass Spectrometry to Characterize Therapeutically Relevant Peptides with Asparagine Deamidation and Isoaspartate
Andrew B. Dykstra, Thomas G. Lubinsky, Heidi Vitrac, Iain D. G. Campuzano, Pavel V. Bondarenko, Ashli R. Simone
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引用次数: 0
Abstract
Rapid identification of asparagine (Asn) deamidation and isoaspartate (isoAsp) in proteins remains a challenging analytical task during the development of biological therapeutics. For this study, 46 therapeutically relevant peptides corresponding to 13 peptide families (13 unmodified peptides and 33 modified peptides) were obtained; modified peptides included Asn deamidation and isoAsp. The peptide families were characterized by three methods: reversed-phase ultrahigh performance liquid chromatography–mass spectrometry (RP-UHPLC-MS); flow injection analysis high-resolution ion mobility–mass spectrometry (FIA-HRIM-MS); and shortened gradient RP-UHPLC-HRIM-MS. UHPLC-MS data acquisition was 2 h per injection, in contrast to high-throughput 1 min data acquisition of the FIA-HRIM-MS technique. A rapid 2D peptide map has been demonstrated by combining shortened gradient RP-UHPLC with HRIM, to optimize the resolution of the Asn-, Asp-, and isoAsp-containing peptides, increasing the likelihood of detecting peptides containing these quality attributes with expedited data acquisition. Additionally, this paper provides an ion mobility calibration data set for therapeutically relevant peptides (unmodified and modified) over an ion-neutral collisional cross-section range of 300–800 Å2.
期刊介绍:
Analytical Chemistry, a peer-reviewed research journal, focuses on disseminating new and original knowledge across all branches of analytical chemistry. Fundamental articles may explore general principles of chemical measurement science and need not directly address existing or potential analytical methodology. They can be entirely theoretical or report experimental results. Contributions may cover various phases of analytical operations, including sampling, bioanalysis, electrochemistry, mass spectrometry, microscale and nanoscale systems, environmental analysis, separations, spectroscopy, chemical reactions and selectivity, instrumentation, imaging, surface analysis, and data processing. Papers discussing known analytical methods should present a significant, original application of the method, a notable improvement, or results on an important analyte.