Advancing stability proteomics for functional characterization of intact proteoforms

Introduction

High-throughput top-down proteomics has significantly advanced the characterization of intact proteoforms in complex biological systems. However, linking proteoform structure with function remains challenging. Stability proteomics methods assess protein functionality by analyzing stability under a denaturation gradient, but bottom-up proteomics techniques limit their ability to characterize intact proteoforms with modifications. To address this, we developed top-down stability proteomics methods, including Top-Down Thermal Proteome Profiling (TD-TPP) and Methionine Oxidative Footprinting in Intact Proteins (MOFIP), using thermal and chemical denaturant gradients to probe protein stability. These methods were employed to study the effect of protein modification on proteoform stability in complex samples, aiming to bridge the gap between structure and function.

Methods

We developed TD-TPP and MOFIP to probe proteoform stability in E. coli, mouse brain, and HeLa lysates. For TD-TPP, aliquots were heated (37-56 °C) and centrifuged to remove aggregates. For MOFIP, lysates were treated with H2O2 and quenched with L-methionine. They were then incubated with increasing concentrations of guanidinium hydrochloride (GdmCl) followed by H2O2 incubation and quenching. Label-free quantitative TD proteomics was performed using our one-dimensional ultra-high-pressure liquid chromatography top-down mass spectrometry (1D UPLC-TDMS) platform for deep characterization of intact proteoforms.

Results

Our top-down platform enabled the characterization and profiling of hundreds of proteoforms in complex biological samples. Using TD-TPP, we analyzed the standard proteins β lactoglobulin A&B. βLG B was slightly destabilized as a result of an amino acid substitution (D80G) to a more nonpolar residue on an exposed turn as well as the exchange to a more polar residue (V134A) internal beta-sheet. TD-TPP was further applied to HeLa lysate and determined the average melting point of the intact proteoforms was 59.3 °C for cytosolic proteoforms, consistent with previous reports.

The MOFIP was applied to evaluate solvent-accessible Met residues based on their secondary structure. Interestingly, 26 proteoforms were partially oxidized while natively folded indicating multiple populations may co-exist in cell lysate. Furthermore, proteoform stability was examined and one protein, the Ribose import binding protein (RbsB), contains 1 solvent-accessible methionine residue on domain 1 and 3 solvent-inaccessible methionine residues on domain 2. We found that the three residues on domain 2 were simultaneously oxidized upon denaturation demonstrating the potential of MOFIP to obtain domain-specific information.

Conclusions

Our novel top-down stability proteomics platforms have shown great potential for deep proteoform characterization and stability evaluation at the proteoform level.