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

Abstract

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.

Figure 1.

HPLC-FOX instrumentation setup. The HPLC system is to the right of the frame. The fluidics module of the Fox Photolysis System is bypassed, with the second microtee output leading directly to the flash photolysis cell.

Figure 2.

Measurement of hydroxyl radical scavenging by NaCl at concentrations up to 1M. A) FOX inline adenine dosimetry measurements from triplicate samples at different concentrations of NaCl. B) LC-MS analysis for FOX oxidation of myoglobin and GluB in the presence of different concentrations of NaCl from the same triplicate samples. Error bars represent one standard deviation.

Figure 3.

HPLC and LC-MS analysis of OVA-AP reaction. A) WAX separation of OVA-AP reaction after (blue) 40 minutes, (magenta) 130 minutes or (black) 48 hours. Each peak represents a different phosphoproteoform of OVA in the dynamic system. The regions of each peak subjected to FOX are highlighted in yellow. B) LC-MS results identify the three peaks from the WAX separation as non-, mono-, and di-phosphorylated proteoforms, with the monophosphorylated form being almost solely phosphoSer68.

Figure 4.

Molecular dynamics simulation results show a significant difference between non-phosphoSer-344 and phosphoSer-344. The phosphoSer-344 showed the phosphate group interacted with Lys189 to stabilize the loop; when Ser344 is dephosphorylated, the loop is unstructured.

Figure 5.

Comparison of residue level LC-FOX data plotted on the structure of OVA. A) Change in LC-FOX footprint between non-phosphorylated and mono-phosphorylated OVA. B) Change in LC-FOX footprint between non-phosphorylated and di-phosphorylated OVA. Dominant site(s) of phosphorylation are highlighted in a yellow circle. Residue color represents fold change in oxidation, while green residues represent no statistically significant change in oxidation. Grey residues showed no measurable oxidation. C) Volcano plot of the LC-FOX data, color coded to match panels A and B.