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Updated: Jun 4, 2025
LERLIC-MS/MS for In-depth Characterization and Quantification of Glutamine and Asparagine Deamidation in Shotgun Proteomics
Published on: April 9, 2017
Utilization of Liquid Chromatography-Mass Spectrometry and High-Resolution Ion Mobility-Mass Spectrometry to
Andrew B Dykstra1, Thomas G Lubinsky2, Heidi Vitrac2
1Pre-Pivotal Attribute Sciences, Amgen, Inc, Thousand Oaks, California 91320, United States.
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.
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