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. 2020 Mar 16;21(6):853-859.
doi: 10.1002/cbic.201900528. Epub 2019 Oct 29.

Isotopically Labeled Clickable Glutathione to Quantify Protein S-Glutathionylation

Garrett C VanHecke  1 , Maheeshi Yapa Abeywardana  1 , Bo Huang  1 , Young-Hoon Ahn  1
Affiliations

Isotopically Labeled Clickable Glutathione to Quantify Protein S-Glutathionylation

Garrett C VanHecke et al. Chembiochem. .

Abstract

Protein S-glutathionylation is one of the important cysteine oxidation events that regulate various redox-mediated biological processes. Despite several existing methods, there are few proteomic approaches to identify and quantify specific cysteine residues susceptible to S-glutathionylation. We previously developed a clickable glutathione approach that labels intracellular glutathione with azido-Ala by using a mutant form of glutathione synthetase. In this study, we developed a quantification strategy with clickable glutathione by using isotopically labeled heavy and light derivatives of azido-Ala, which provides the relative quantification of glutathionylated peptides in mass spectrometry-based proteomic analysis. We applied isotopically labeled clickable glutathione to HL-1 cardiomyocytes, quantifying relative levels of 1398 glutathionylated peptides upon addition of hydrogen peroxide. Importantly, we highlight elevated levels of glutathionylation on sarcomere-associated muscle proteins while validating glutathionylation of two structural proteins, α-actinin and desmin. Our report provides a chemical proteomic strategy to quantify specific glutathionylated cysteines.

Keywords: S-glutathionylation; clickable glutathione; hydrogen peroxide; protein modification; proteomics; quantification.

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Conflict of interest statement

Conflict of Interest

The authors declare no conflict of interest

Figures

Figure 1.
Figure 1.. Isotopic-labeling strategy to identify and quantify glutathionylated cysteines.
(A) A scheme for quantification of glutathionylated peptides. A mutant of glutathione synthetase (GS M4) in HL-1 cells uses light or heavy azido-Ala to synthesize isotopically-labelled clickable glutathione. Upon addition of stimulus, two cohorts of lysates containing glutathionylated proteins with a light or heavy label were combined and subjected to click reaction with biotin-DADPS-alkyne, pull-down by streptavidin-beads, and tryptic digestion. Glutathionylated peptides were eluted by acidic cleavage of a DADPS linker and analyzed by LC-MS/MS, which provides a MS1-peak area ratio (RH/L) of heavy- to light-labelled peptides. (B) The structure of biotin-DADPS-alkyne with its cleavage site (dot line).
Figure 2.
Figure 2.. Evaluation of light and heavy derivatives of azido-Ala to identify glutathionylated peptides with isotopic mass difference.
(A) Light and heavy azido-Ala derivatives are used equally by GS M4 in vitro. GS M4 enzyme activity was measured with γ-Glu-Cys and ATP in the presence of two different concentrations (0.2 and 1 mM) of light azido-Ala (red) or heavy azido-Ala (blue), showing identical enzymatic rates. (B) Light and heavy azido-Ala derivatives produce the equal amount of respective azido-glutathione in cells. HEK293 expressing GS M4 (HEK293/GS M4) were incubated with light or heavy azido-Ala. Lysates were analyzed by LC-MS to detect the mass of light or heavy-labelled azido-glutathione. (C) Light and heavy azido-Ala derivatives detect the identical pattern of glutathionylation. HEK293/GS M4 cells incubated with light or heavy azido-Ala were treated with H2O2 (1 mM, 15 min). Lysates were then subjected to click reaction with Cy5-alkyne for fluorescence detection or Coomassie stains. (D) Light and heavy azido-Ala derivatives identify glutathionylated peptides with isotopic mass difference. Purified GSTO1 was glutathionylated by addition of diamide (0.2 mM, 30 min) in the presence of equal amount of light- and heavy-labelled azido-glutathione derivatives (both 1 mM). Glutathionylated GSTO1 was processed for click reaction with biotin-DDE-alkyne, tryptic digestion, pull-down, elution, and MALDI-MS analysis.[12, 20] Data are representative of 3 independent experiments.
Figure 3.
Figure 3.. Identification and quantification of glutathionylated peptides by isotopically-labelled clickable glutathione.
(A) Experimental conditions to identify glutathionylated peptides by isotopic azido-Ala, and the number of quantified glutathionylated peptides under indicated condition by LC-MS. (B) The RH/L values and the coefficient of variation (CV) of glutathionylated peptides. The box plots of RH/L values (left) and the CV percentage (right) with the median value (line), box (25–75%), and whiskers (10–90%). (C) Distribution of RH/L values of individual glutathionylated peptides. The RH/L value of 4 is indicated by a dotted line. Examples of sarcomeric proteins with cysteine sites are indicated by arrow and colors (yellow in E1 and cyan in E2). (D) Graphical display of MS1-peaks that shows relative quantification of heavy (blue)- to light (red)-labelled peptides. Selected sarcomeric or muscle-relevant proteins are shown. Retention times are indicated with MS1-peaks. (E-F) Validation of glutathionylated cysteines by Western blots. HL-1 cells expressing GS M4 were used without transfection of α-actinin or desmin. HEK293/GS M4 cells were transfected with WT or Cys mutants of α-actinin or desmin. After addition of H2O2 (15 min), glutathionylated proteins were subjected to click reaction with biotin-alkyne and pull-down with streptavidin-beads, and detected by Western blotting with individual antibodies, including α-actinin, desmin, and FLAG. Data are representative of 2 independent experiments.
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