title: Determination of Protein Thiol Reduction Potential by Isotope Labeling and Intact Mass Measurement.
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 top-down mass spectrometry recently 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 liquid chromatography-mass spectroscopy (LC-MS) analyses and does not necessarily require top-down fragmentation. In addition to analyzing homogeneous protein samples, we also demonstrate that our methodology can be used to determine thiol E(⊕) measurements in samples that contain mixtures of proteins. Thus, the combination of experimential methodology and data analysis regime has the potential to make such measurements in a high-throughput manner and in a manner that is more accessible to a broad community of protein scientists.
authors.affiliation: EaStCHEM School of Chemistry, University of Edinburgh , David Brewster Road, Edinburgh EH9 3FJ, United Kingdom.
authors.forename: Sophie E
authors.initials: SE
authors.lastname: Thurlow
authors.affiliation: Chemistry and Forensics, Rosalind Franklin Building, Nottingham Trent University , Clifton Campus, Clifton Lane, Nottingham, NG11 8NS, United Kingdom.
authors.forename: David P
authors.initials: DP
authors.lastname: Kilgour
authors.affiliation: EaStCHEM School of Chemistry, University of Edinburgh , David Brewster Road, Edinburgh EH9 3FJ, United Kingdom.
authors.forename: Dominic J
authors.initials: DJ
authors.lastname: Campopiano
authors.affiliation: EaStCHEM School of Chemistry, University of Edinburgh , David Brewster Road, Edinburgh EH9 3FJ, United Kingdom.
authors.forename: C Logan
authors.initials: CL
authors.lastname: Mackay
authors.affiliation: EaStCHEM School of Chemistry, University of Edinburgh , David Brewster Road, Edinburgh EH9 3FJ, United Kingdom.
authors.forename: Pat R R
authors.initials: PR
authors.lastname: Langridge-Smith
authors.affiliation: EaStCHEM School of Chemistry, University of Edinburgh , David Brewster Road, Edinburgh EH9 3FJ, United Kingdom.
authors.forename: David J
authors.initials: DJ
authors.lastname: Clarke
authors.affiliation: EaStCHEM School of Chemistry, University of Edinburgh , David Brewster Road, Edinburgh EH9 3FJ, United Kingdom.
authors.forename: Colin J
authors.initials: CJ
authors.lastname: Campbell
mesh_terms: D000478:Alkylation; D006801:Humans; D007553:Isotope Labeling; D013058:Mass Spectrometry; D010084:Oxidation-Reduction; D040901:Proteomics; D011994:Recombinant Proteins / Q000737:chemistry / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000378:metabolism*; D013879:Thioredoxins / Q000737:chemistry / Q000378:metabolism*
chemical_list: D011994:Recombinant Proteins; D013438:Sulfhydryl Compounds; D013879:Thioredoxins