Open AccessEnhancing Accuracy in Molecular Weight Determination of Highly Heterogeneously Glycosylated Proteins by Native Tandem Mass Spectrometry
Author(s) -
Guanbo Wang,
Rob N. de Jong,
Ewald T.J. van den Bremer,
Paul W.H.I. Parren,
Albert J. R. Heck
Publication year - 2017
Publication title -
analytical chemistry
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 2.117
H-Index - 332
eISSN - 1520-6882
pISSN - 0003-2700
DOI - 10.1021/acs.analchem.6b05129
Subject(s) - chemistry , electrospray ionization , chromatography , mass spectrometry , tandem mass spectrometry , glycosylation , sodium dodecyl sulfate , glycoprotein , molecular mass , analytical chemistry (journal) , biochemistry , enzyme
The determination of molecular weights (MWs) of heavily glycosylated proteins is seriously hampered by the physicochemical characteristics and heterogeneity of the attached carbohydrates. Glycosylation impacts protein migration during sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE) and size-exclusion chromatography (SEC) analysis. Standard electrospray ionization (ESI)-mass spectrometry does not provide a direct solution as this approach is hindered by extensive interference of ion signals caused by closely spaced charge states of broadly distributed glycoforms. Here, we introduce a native tandem MS-based approach, enabling charge-state resolution and charge assignment of protein ions including those that escape mass analysis under standard MS conditions. Using this method, we determined the MW of two model glycoproteins, the extra-cellular domains of the highly and heterogeneously glycosylated proteins CD38 and epidermal growth factor receptor (EGFR), as well as the overall MW and binding stoichiometries of these proteins in complex with a specific antibody.