Determination of protein thiol reduction potential by isotope labeling and intact mass measurement

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Thurlow, S.E., Kilgour, D.P.A. ORCID: 0000-0002-3860-7532, Campopiano, D.J., Mackay, C.L., Langridge-Smith, P.R.R., Clarke, D.J. and Campbell, C.J., 2016. Determination of protein thiol reduction potential by isotope labeling and intact mass measurement. Analytical Chemistry, 88 (5), pp. 2727-2733. ISSN 0003-2700

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Official URL: http://doi.org/10.1021/acs.analchem.5b04195

Abstract

Oxidation/reduction of thiol residues in proteins is an important type of post-translational modification that is implicated in regulating a range of biological processes. The nature of the modification makes it possible to define a quantifiable electrochemical potential, E⊕, for oxidation/reduction that allows cysteine-containing proteins to be ranked based on their propensity to be oxidized. Measuring oxidation of cysteine residues in proteins is difficult using standard electrochemical methods but recently top-down mass-spectrometry has been shown to enable the quantification of E⊕ for thiol oxidations. In this paper we demonstrate that mass spectrometry of intact proteins can be used in combination with an isotopic labeling strategy and an automated data analysis algorithm to measure E⊕ for the thiols in both E Coli Thioredoxin 1 and Human Thioredoxin 1. Our methodology relies on accurate mass measurement of proteins using LC-MS analyses and does not necessarily require top-down fragmentation. As well as analyzing homogeneous protein samples, we also demonstrate that our methodology can be used to determine thiol E⊕ measurements in samples which contain mixtures of proteins. Thus the combination of experiential methodology and data analysis regime have the potential to make such measurements in a high-throughput manner and in a manner more accessible to a broad community of protein scientists.

Item Type:

Journal article

Publication Title:

Analytical Chemistry

Creators:

Thurlow, S.E., Kilgour, D.P.A., Campopiano, D.J., Mackay, C.L., Langridge-Smith, P.R.R., Clarke, D.J. and Campbell, C.J.

Publisher:

ACS Publications

Date:

16 February 2016

Volume:

Number: