Selecting Reducing Agents for Native Mass Spectrometry

31 January 2024, Version 1
This content is a preprint and has not undergone peer review at the time of posting.

Abstract

In protein science, reducing agents are often added in cases where the protein, its cofactors, or its ligands are sensitive to oxidative stress. Although many native mass spectrometry (MS) workflows would benefit from maintaining reducing conditions throughout the analysis, there is a lack of consensus regarding the compatibility of reducing agents with that approach. This study systematically examines the effects of dithiothreitol (DTT), β-mercaptoethanol (βME), and tris(2-carboxyethyl)phosphine (TCEP) on the native mass spectra of protein standards. The selection and concentration of the reducing agents affected both the extent of nonspecific adduction and the charge-state distribution of the analyte. For a protein without disulfide bonds, increasing concentrations of DTT or βME resulted in shifts to higher charge states, whereas increasing concentrations of TCEP resulted in shifts to lower charge states. Based on these trends and additional properties of the reducing agents, we propose that DTT and βME are mild supercharging agents and that TCEP is a potent charge-reducing agent. The selection and concentration of the reducing agents, as well as the sample pH, also affected the extent of disulfide bond reduction, and for βME, the extent of covalent adduction of cysteine by that molecule. These results offer insights into the compatibility of reducing agents with the goal of obtaining high-quality native mass spectra. Based on our results, we present recommendations for the use of reducing agents in native MS experiments.

Supplementary materials

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Supporting Information
Description
Arrival-time distributions and discussion of ion mobility data. Additional mass spectra and plots of average charge state. Visualized structures, coordinates, and tabulated properties of selected molecules and ions.
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