LAUSR

Posttranslational modifications of proteins play fundamental roles in protein function in health and disease. More than 200 different types of posttranslational modifications are known, many of them being extremely low abundant, causing subtle changes in physicochemical properties and posing an extreme challenge to analytical methods required for their characterisation. Here, we report the development of a novel pH gradient based anion exchange chromatography method which can be directly hyphenated to Orbitrap-based mass spectrometry for the comprehensive characterisation of proteoforms on 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 four orders of magnitude in dynamic range. An in-depth analysis of ovalbumin from chicken egg white yielded 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 were identified and quantified, many of which have not been previously reported.