Structural Analysis of Phosphorylation Proteoforms in a Dynamic Heterogeneous System Using Flash Oxidation Coupled In-Line with Ion Exchange Chromatography.

Protein posttranslational modifications (PTMs) are key modulators of protein structure and function that often change in a dynamic fashion in response to cellular stimuli. Dynamic PTMs are very challenging to structurally characterize using modern techniques, including covalent labeling methods, due to the presence of multiple proteoforms and conformers together in solution. We have coupled an ion exchange high-performance liquid chromatography separation with a flash oxidation system [ion exchange chromatography liquid chromatography-flash oxidation (IEX LC-FOX)] to successfully elucidate structural changes among three phosphoproteoforms of ovalbumin (OVA) during dephosphorylation with alkaline phosphatase. Real-time dosimetry indicates no difference in the effective radical dose between peaks or across the peak, demonstrating both the lack of scavenging of the NaCl gradient and the lack of a concentration effect on radical dose between peaks of different intensities. The use of IEX LC-FOX allows us to structurally probe into each phosphoproteoform as it elutes from the column, capturing structural data before the dynamics of the system to reintroduce heterogeneity. We found significant differences in the residue-level oxidation between the hydroxyl radical footprint of nonphosphorylated, monophosphorylated, and diphosphorylated OVA. Not only were our data consistent with the previously reported stabilization of OVA structure by phosphorylation, but local structural changes were also consistent with the measured order of dephosphorylation of Ser344 being removed first. These results demonstrate the utility of IEX LC-FOX for measuring the structural effects of PTMs, even in dynamic systems.