Analyzing Protein Micro-Heterogeneity in Chicken Ovalbumin by High-Resolution Native Mass Spectrometry Exposes Qualitatively and Semi-Quantitatively 59 Proteoforms

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.

Hybrid mass spectrometry approaches in glycoprotein analysis and their usage in scoring biosimilarity

“…With the advent of high mass accuracy and resolving power of MS technologies, it has now become possible to detect and baseline resolve different proteoforms directly from intact proteins under pseudo-physiological native conditions, using a modified Orbitrap mass analyzer with an extended mass range19202122232425. This approach has enabled the PTM analysis of the intact ovalbumin to an unprecedented depth, where 59 different proteoforms were detected, identified and quantified from a single-shot experiment26. It is of great advantage that native MS provides a direct view on the relative abundance and overall PTM composition of different co-appearing proteoforms that are distinguishable in mass2728.…”

Evaluation of colorimetric assays for analyzing reductively methylated proteins: Biases and mechanistic insights

“…The color production in the Bradford assay occurs when the blue, anionic form of the dye is stabilized, typically through electrostatic and hydrophobic interactions. The ovalbumin glycan comprises approximately 3-4% of the total mass and is mainly composed of neutral, high-mannose- or hybrid-type oligosaccharides [50-51]. While the glycan mass alone does not explain the approximately 27% decrease in sensitivity, it is likely that the glycan competes with the dye by forming hydrogen bonds with basic amino acids, stacking with hydrophobic residues, and/or steric shielding [49].…”

“…While the glycan mass alone does not explain the approximately 27% decrease in sensitivity, it is likely that the glycan competes with the dye by forming hydrogen bonds with basic amino acids, stacking with hydrophobic residues, and/or steric shielding [49]. In addition, ovalbumin is highly phosphorylated at two sites [51], which likely further decreases the affinity for the anionic dye.…”

Coupling Capillary Zone Electrophoresis to a Q Exactive HF Mass Spectrometer for Top-down Proteomics: 580 Proteoform Identifications from Yeast

“…11–13 CZE can resolve a different set of proteoforms than RPLC because proteins with minor sequence variations or PTMs often have similar hydrophobicities but different charges. CZE-ESI-MS/MS has been employed to analyze single protein proteoforms 14–15 including pharmaceutical protein glycoform profiling, 14 and more recently, complex biological samples. 16–18 CZE can be interfaced with mass spectrometers via electrospray ionization (ESI).…”