Future directions of structural mass spectrometry using hydroxyl radical footprinting

Abstract

Hydroxyl radical protein footprinting coupled to mass spectrometry has been developed over the last decade and has matured to a powerful method for analyzing protein structure and dynamics. It has been successfully applied in the analysis of protein structure, protein folding, protein dynamics, and protein-protein and protein-DNA interactions. Using synchrotron radiolysis, exposure of proteins to a 'white' X-ray beam for milliseconds provides sufficient oxidative modification to surface amino acid side chains, which can be easily detected and quantified by mass spectrometry. Thus, conformational changes in proteins or protein complexes can be examined using a time-resolved approach, which would be a valuable method for the study of macromolecular dynamics. In this review, we describe a new application of hydroxyl radical protein footprinting to probe the time evolution of the calcium-dependent conformational changes of gelsolin on the millisecond timescale. The data suggest a cooperative transition as multiple sites in different molecular subdomains have similar rates of conformational change. These findings demonstrate that time-resolved protein footprinting is suitable for studies of protein dynamics that occur over periods ranging from milliseconds to seconds. In this review, we also show how the structural resolution and sensitivity of the technology can be improved as well. The hydroxyl radical varies in its reactivity to different side chains by over two orders of magnitude, thus oxidation of amino acid side chains of lower reactivity are more rarely observed in such experiments. Here we demonstrate that the selected reaction monitoring (SRM)-based method can be utilized for quantification of oxidized species, improving the signal-to-noise ratio. This expansion of the set of oxidized residues of lower reactivity will improve the overall structural resolution of the technique. This approach is also suggested as a basis for developing hypothesis-driven structural mass spectrometry experiments.

Figure 1

A schematic representation of MS-based hydroxyl radical footprinting experiments. Proteins and their complex are exposed to x-ray beam for a different time intervals. Oxidized proteins are subjected to proteolysis and mass spectrometry analysis. LC-MS is employed to separate out peptides and quantitatively measure the extent of modification. Dose-response profiles are plotted for each modified peptides within proteins sequences. Modification rates derived from free proteins and their complex are calculated and compared. The specific oxidation sites are determined by MS/MS analysis.

Figure 2

Ribbon representation of the subunit structure of horse plasma gelsolin highlighting the six homologues domains such as S1 (blue), S2 (magenta), S3 (green), S4 (cyan), S5 (yellow), S6 (red), the C-terminal tail (orange) and the subdomain linkers (grey).

Figure 3

Progress curve at 10 μM (A), 100 μM (B) and 5 mM (C) Ca²⁺ for peptides comprised of residues 49–72, 722–748 and 431–454 within gelsolin protein after exposure for 50 ms.

Figure 4

Dose-response curves for the oxidation of Angiotensin II (DRVYIHPF) peptide as a function of exposure time. A, dose-response curve for the Arg oxidation derived from DDA analysis. B, dose-response curve for the His oxidation derived from DDA analysis. C, dose-response curve for the Arg oxidation derived from SRM analysis. B, dose-response curve for the His oxidation derived from SRM analysis. Fragment ion b₆ was monitored in all SRM experiments.

Figure 5

DDA vs SRM analysis of Angiotensin II peptide exposed for 40 ms. (A) DDA of doubly protonated ion with m/z= 523.6 oxidized on His; (B) DDA of doubly protonated ion with m/z= 526.2 that oxidized on His; (C) DDA of doubly protonated ion with m/z= 520.8 that oxidized on His; (D) SRM analysis of transition 523.6→784.5; (E) SRM of transition 526.2→789.4; (F) SRM of transition 520.8→778.3. Fragment ion b₆ was monitored in all SRM experiments.