Characterization of protein impurities and site-specific modifications using peptide mapping with liquid chromatography and data independent acquisition mass spectrometry.
Liquid chromatography (LC)-based peptide mapping is extensively used for establishing protein identity, assessing purity, and detecting post-translational modifications (PTMs) of recombinant proteins in the biopharmaceutical industry. However, current LC-UV/MS peptide mapping methods require multiple analyses and MS/MS experiments to identify protein contaminants and site-specific PTMs. This manuscript evaluated an alternative approach for protein characterization via peptide mapping employing a data independent LC-MS acquisition strategy with an alternate low and elevated collision energy scanning. The acquired peptide precursor and fragment information was utilized for effective identification of peptide sequences and site-specific modifications within a single LC run. The peptide MS signal intensities were reliably measured and used to estimate relative concentrations of PTMs and/or proteins contaminating the target protein. The method was evaluated using tryptic digests of yeast enolase and alcohol dehydrogenase. LC-eluted peptides were successfully sequenced and covered 97% target protein sequences. Protein impurities and site-specific modifications (e.g., M-oxidation and N-deamidation) were identified and quantified.
Xie
Hongwei
H
Biopharmaceutical Sciences, Waters Corporation, 34 Maple Street, Massachusetts 01757, USA. hongwei_xie@waters.com
Gilar
Martin
M
Gebler
John C
JC
D000426:Alcohol Dehydrogenase / Q000737:chemistry / Q000378:metabolism; D000595:Amino Acid Sequence; D001665:Binding Sites; D002853:Chromatography, Liquid; D013058:Mass Spectrometry; D008969:Molecular Sequence Data; D010449:Peptide Mapping / Q000379:methods*; D010455:Peptides / Q000032:analysis / Q000737:chemistry / Q000378:metabolism; D010751:Phosphopyruvate Hydratase / Q000737:chemistry / Q000378:metabolism; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism*; D013379:Substrate Specificity
D010455:Peptides; D011506:Proteins; D000426:Alcohol Dehydrogenase; D010751:Phosphopyruvate Hydratase