Utilization of Liquid Chromatography–Mass Spectrometry and High-Resolution Ion Mobility–Mass Spectrometry to Characterize Therapeutically Relevant Peptides with Asparagine Deamidation and Isoaspartate

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.