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Review
. 2015 Mar:80:191-211.
doi: 10.1016/j.freeradbiomed.2014.09.016. Epub 2014 Sep 28.

Mass spectrometry in studies of protein thiol chemistry and signaling: opportunities and caveats

Nelmi O Devarie Baez  1 , Julie A Reisz  1 , Cristina M Furdui  2
Affiliations
Review

Mass spectrometry in studies of protein thiol chemistry and signaling: opportunities and caveats

Nelmi O Devarie Baez et al. Free Radic Biol Med. 2015 Mar.

Abstract

Mass spectrometry (MS) has become a powerful and widely utilized tool in the investigation of protein thiol chemistry, biochemistry, and biology. Very early biochemical studies of metabolic enzymes have brought to light the broad spectrum of reactivity profiles that distinguish cysteine thiols with functions in catalysis and protein stability from other cysteine residues in proteins. The development of MS methods for the analysis of proteins using electrospray ionization (ESI) or matrix-assisted laser desorption/ionization (MALDI) coupled with the emergence of high-resolution mass analyzers has been instrumental in advancing studies of thiol modifications, both in single proteins and within the cellular context. This article reviews MS instrumentation and methods of analysis employed in investigations of thiols and their reactivity toward a range of small biomolecules. A selected number of studies are detailed to highlight the advantages brought about by the MS technologies along with the caveats associated with these analyses.

Keywords: Cysteine; Kinetics; Mass spectrometry; Modifications; Peptides; Proteins; Thiol.

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Figures

Figure 1
Figure 1
Peptide bonds cleaved by MS2 fragmentation. A: Collison-induced dissociations fragment the amide bond, producing b- and y-ions. Also targeted are many PTMs (not shown). B: Electron-transfer dissociation targets the amino-α-carbon bond, producing c- and z-ions, and leaving PTMs intact.
Figure 2
Figure 2
Representative ESI-TOF mass spectra for wild type and mutant AhpC from S. typhimurium. A: Wild type AhpC is observed as the monomer (20,616 amu) and the homodimer (41,230 amu). B: The Cys165Ser AhpC mutant, devoid of a resolving Cys, allows for the facile preparation of numerous oxoforms. R-SN refers to the cyclic sulfenamide Cys-SN formed from the intramolecular amine-mediated dehydration of Cys-SOH in the gas phase and/or in solution. Unpublished Data.
Figure 3
Figure 3
Discontinuous and continuous methods for kinetic investigations using intact proteins. A: Discontinuous approaches involve quenching reaction aliquots using conditions of low pH or a size exclusion resin followed by MS analysis of each time point. B: Continuous time-resolved kinetic analyses require the direct infusion using dual syringes with reaction occurring between the mixing tee and the ion source (called the aging loop). Step-wise alterations in flow velocity allow for the observation of multiple reaction time points in a single run. The methods allow for simultaneous monitoring of all covalent and in some cases non-covalent species present at each reaction time point.
Figure 4
Figure 4
Mutant AhpC-SOH readily captures electrophiles iodoacetamide (IAM), N-ethylmaleimide (NEM), and methyl methanethiosulfonate (MMTS) widely used in thiol blocking to yield covalent Cys-SOR adducts (exact connectivity unknown). Subsequent treatment with reductant (thiols, phosphines, ascorbate) converts Cys-SOR to Cys-SH at varying degrees. [DHA: dehydroalanine.] Adapted from Reisz et al. [39]
Figure 5
Figure 5
Hydration-dehydration equilibrium relating sulfenic acid and sulfenamide. 18O-Labeling of the sulfenamide leads to the formation of the 18O-labeled Cys-SOR species. SOH/SN equilibrium in the solution phase will decrease 18O incorporation in favor of the more abundant 16O.
Figure 6
Figure 6
Protein deacylation strategies: A. alkaline methanolysis; B. aminolysis; C. hydrogenation [FA = formic acid]
Figure 7
Figure 7
Catalytic cycle of 2-Cys peroxiredoxins and ESI-TOF mass spectra revealing the hyperoxidation sensitivity differences between the human Prx2 and Prx3 enzymes in the presence of H2O2. [Prx: peroxiredoxin; Trx: thioredoxin; TrxR: thioredoxin reductase] Adapted from Haynes et al. [69].
Figure 8
Figure 8
Overview of strategies for investigating protein conformational changes by mass spectrometry. A: General scheme for the hydrogen-deuterium exchange (HDX) technique used to uncover flexible and/or solvent-exposed regions of protein tertiary and quaternary structure. B: Chemical cross-linking as a valuable means for probing protein conformation in native and perturbed states. In this example, nearby cysteine residues are cross-linked by a homobifunctional maleimide (Mal-Ph-Mal), forming a covalent cross-link stable toward proteolysis and MS ionization and fragmentation. Both intramolecular and intermolecular crosslinking events may occur depending on the unique structure of the protein or protein complex investigated. C: IMS-MS enables separation of protein conformers based on the protein tertiary structure.
Figure 9
Figure 9
Structures of selected thiol-targeted electrophiles
Figure 10
Figure 10
Tag-switch method for protein sulfhydration
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