Analyzing protein micro-heterogeneity in chicken ovalbumin by high-resolution native mass spectrometry exposes qualitatively and semi-quantitatively 59 proteoforms.
Taking chicken Ovalbumin as a prototypical example of a eukaryotic protein we use high-resolution native electrospray ionization mass spectrometry on a modified Exactive Orbitrap mass analyzer to qualitatively and semiquantitatively dissect 59 proteoforms in the natural protein. This variety is largely induced by the presence of multiple phosphorylation sites and a glycosylation site that we find to be occupied by at least 45 different glycan structures. Mass analysis of the intact protein in its native state is straightforward and fast, requires very little sample preparation, and provides a direct view on the stoichiometry of all different coappearing modifications that are distinguishable in mass. As such, this proof-of-principal analysis shows that native electrospray ionization mass spectrometry in combination with an Orbitrap mass analyzer offers a means to characterize proteins in a manner highly complementary to standard bottom-up shot-gun proteome analysis.
Biomolecular Mass Spectrometry and Proteomics, Bijvoet Center for Biomolecular Research and Utrecht Institute for Pharmaceutical Sciences, University of Utrecht , Padualaan 8, 3584 CH Utrecht, The Netherlands.
Yang
Y
Yang
Arjan
A
Barendregt
Johannis P
JP
Kamerling
Albert J R
AJ
Heck
D000818:Animals; D002645:Chickens; D006031:Glycosylation; D008958:Models, Molecular; D010047:Ovalbumin / Q000737:chemistry* / Q000378:metabolism; D010766:Phosphorylation; D011487:Protein Conformation; D021241:Spectrometry, Mass, Electrospray Ionization / Q000379:methods*
D010047:Ovalbumin