Hydroxyl radical mediated damage of proteins in low oxygen solution investigated using X-ray footprinting mass spectrometry.

Line G Kristensen,Sayan Gupta,Corie Y Ralston,Behzad Rad,Yan Chen,Christopher J Petzold,James M Holton

doi:10.1107/s1600577521004744

Line G Kristensen, Sayan Gupta + Show 5 more

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https://doi.org/10.1107/s1600577521004744

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Abstract

In the method of X-ray footprinting mass spectrometry (XFMS), proteins at micromolar concentration in solution are irradiated with a broadband X-ray source, and the resulting hydroxyl radical modifications are characterized using liquid chromatography mass spectrometry to determine sites of solvent accessibility. These data are used to infer structural changes in proteins upon interaction with other proteins, folding, or ligand binding. XFMS is typically performed under aerobic conditions; dissolved molecular oxygen in solution isnecessary in many, if not all, the hydroxyl radical modifications that are generally reported. In this study we investigated the result of X-ray induced modifications to three different proteins under aerobic versus low oxygen conditions, and correlated the extent of damage with dose calculations. We observed a concentration-dependent protecting effect at higher protein concentration for a given X-ray dose. For the typical doses used in XFMS experiments there was minimal X-ray induced aggregation and fragmentation, but for higher doses we observed formation of covalent higher molecular weight oligomers, as well as fragmentation, which was affected by the amount of dissolved oxygen in solution. The higher molecular weight products in the form of dimers, trimers, and tetramers were present in all sample preparations, and, upon X-ray irradiation, these oligomers became non-reducible as seen in SDS-PAGE. The results provide an important contribution to the large body of X-ray radiation damage literature in structural biology research, and will specifically help inform the future planning of XFMS, and well as X-ray crystallography and small-angle X-ray scattering experiments.

Highlights

X-ray footprinting mass spectrometry (XFMS) is a solvent accessibility-based method that is used to obtain structural information on biological macromolecules in solution
The ‘dilu- tered at third-generation synchrotron crystallography beamtion effect’ in which radiation damage is increased for lower lines, in which dose rates are on the order of MGy (Holton, protein concentrations has been previously reported in small angle X-ray scattering (SAXS) 2009)
We compared for the first time the radiation dose in an XFMS experiment with typical values calculated for MX and SAXS studies, and confirmed that for the standard XFMS experiment in which the radiation exposure is deliberately limited – typically to exposures less than a millisecond at most X-ray beamlines – no significant fragmentation or higher MW oligomer formation is observed

Summary

Introduction

X-ray footprinting mass spectrometry (XFMS) is a solvent accessibility-based method that is used to obtain structural information on biological macromolecules in solution. In the general implementation of this method, a protein or protein complex in a water-based solution is exposed to an X-ray source, and the radiolysis of the water produces hydroxyl radicals (OH). The hydroxyl radicals modify proteins and/ or cleave nucleic acid in solvent-accessible regions. Protein modifications are generally investigated using standard bottom-up liquid chromatography mass spectrometry (LCMS) analysis (Gupta et al, 2007). The XFMS method yields information on water positions within or at the surface of the macromolecule, which is used to infer structural information, such as sites of water occlusion during protein–protein interactions (Gupta et al, 2016). While the use of solvent accessibility methods to gain information on structural

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