Analyzing protein micro-heterogeneity in chicken ovalbumin by high-resolution native mass spectrometry exposes
Yang Yang1, Arjan Barendregt, Johannis P Kamerling
1Biomolecular 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.
Insights
High-resolution native mass spectrometry revealed 59 proteoforms in chicken Ovalbumin, primarily due to phosphorylation and glycosylation. This method offers a fast, complementary approach to proteome analysis.
Area of Science:
- Biochemistry
- Analytical Chemistry
- Proteomics
Background:
- Eukaryotic proteins exhibit extensive heterogeneity.
- Characterizing diverse proteoforms is crucial for understanding protein function.
- Standard proteomic methods can be limited in capturing intact protein modifications.
Purpose of the Study:
- To qualitatively and semiquantitatively analyze proteoforms of chicken Ovalbumin.
- To demonstrate the utility of native electrospray ionization mass spectrometry (native ESI-MS) for intact protein analysis.
- To compare native ESI-MS with bottom-up shotgun proteomics.
Main Methods:
- High-resolution native electrospray ionization mass spectrometry (native ESI-MS) using a modified Exactive Orbitrap mass analyzer.
- Analysis of intact chicken Ovalbumin protein.
- Mass analysis of native protein states.
Main Results:
- Dissection of 59 distinct proteoforms in native chicken Ovalbumin.
- Identification of multiple phosphorylation sites and at least 45 different glycan structures on a single glycosylation site.
- Demonstration of rapid, low-sample-preparation mass analysis of intact proteins.
- Direct observation of modification stoichiometry.
Conclusions:
- Native ESI-MS coupled with Orbitrap analysis provides a powerful, complementary method for characterizing intact protein heterogeneity.
- This approach offers a direct view of proteoform diversity and modification stoichiometry.
- The method is fast, requires minimal sample preparation, and complements traditional bottom-up proteomics.
Abstract:
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.
