Cracking Proteoform Complexity of Ovalbumin with Anion-Exchange Chromatography–High-Resolution Mass Spectrometry under Native Conditions

Posttranslational modifications of proteins play fundamental roles in protein function in health and disease. More than 600 different types of posttranslational modifications are known, many of them being of extremely low abundance, causing subtle changes in physicochemical properties and posing an extreme challenge to analytical methods required for their characterization. Here, we report the development of a novel pH gradient-based anion-exchange chromatography method, which can be directly interfaced to Orbitrap-based mass spectrometry for the comprehensive characterization of proteoforms at the intact protein level under native conditions. The analysis of four different proteins demonstrates outstanding chromatographic selectivity, while the mass spectra obtained are of excellent quality enabling the identification of proteoforms, including near isobaric variants, spanning 4 orders of magnitude in abundance. An in-depth analysis of ovalbumin from chicken egg white yields the identification and relative quantification of more than 150 different proteoforms, including fragmented and dimeric forms. More than 20 different ovalbumin charge variants together with their glycoform distributions are identified and quantified, many of which have not been reported previously.