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  • Title: Characterization of protein impurities and site-specific modifications using peptide mapping with liquid chromatography and data independent acquisition mass spectrometry.
    Author: Xie H, Gilar M, Gebler JC.
    Journal: Anal Chem; 2009 Jul 15; 81(14):5699-708. PubMed ID: 19518054.
    Abstract:
    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.
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