Metabolic Synthesis of Clickable Glutathione for Chemoselective Detection of Glutathionylation

Samarasinghe, Kusal T. G.; Godage, Dhanushka N. P. Munkanatta; VanHecke, Garrett C.; Ahn, Young Hoon

doi:10.1021/ja503946q

Cited by 49 publications

(58 citation statements)

References 22 publications

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“…4B) (61). To this end, glutathione synthetase, a key enzyme that catalyzes coupling of ␥Glu-Cys to Gly to form glutathione, was genetically mutated to produce an enzyme that catalyzed addition of azido-Ala in place of Gly.…”

Section: The Expanding Landscape Of the Thiol Redox Proteomementioning

confidence: 99%

The Expanding Landscape of the Thiol Redox Proteome

Yang

Carroll

Liebler

2016

Molecular & Cellular Proteomics

184

111

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Cysteine occupies a unique place in protein chemistry. The nucleophilic thiol group allows cysteine to undergo a broad range of redox modifications beyond classical thiol-disulfide redox equilibria, including S-sulfenylation (-SOH), S-sulfinylation (-SO 2 H), S-sulfonylation (-SO 3 H), S-nitrosylation (-SNO), S-sulfhydration (-SSH), S-glutathionylation (-SSG), and others. Emerging evidence suggests that these post-translational modifications (PTM) are important in cellular redox regulation and protection against oxidative damage. Identification of protein targets of thiol redox modifications is crucial to understanding their roles in biology and disease. However, analysis of these highly labile and dynamic modifications poses challenges. Recent advances in the design of probes for thiol redox forms, together with innovative mass spectrometry based chemoproteomics methods make it possible to perform global, site-specific, and quantitative analyses of thiol redox modifications in complex proteomes.

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Section: The Expanding Landscape Of the Thiol Redox Proteomementioning

confidence: 99%

The Expanding Landscape of the Thiol Redox Proteome

Yang

Carroll

Liebler

2016

Molecular & Cellular Proteomics

184

111

View full text Add to dashboard Cite

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“…1). 24 In response to ROS, azido-glutathione is glutathionylated on proteins, and the subsequent click reaction allows for identifying glutathionylated proteins (Fig. 1).…”

Section: Introductionmentioning

confidence: 99%

A clickable glutathione approach for identification of protein glutathionylation in response to glucose metabolism

et al. 2016

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Glucose metabolism and mitochondrial function are closely interconnected with cellular redox-homeostasis. Although glucose starvation, which mimics ischemic condition or insufficient vascularization, is known to perturb redox-homeostasis, global and individual protein glutathionylation in response to glucose metabolism or mitochondrial activity remains largely unknown. In this report, we use our clickable glutathione approach, which form clickable glutathione (azido-glutathione) by using a mutant of glutathione synthetase (GS M4), for detection and identification of protein glutathionylation in response to glucose starvation. We found that protein glutathionylation is readily induced in HEK293 cells in response to low glucose concentrations when mitochondrial reactive oxygen species (ROS) are elevated in cells, and glucose is the major determinant for inducing reversible glutathionylation. Proteomic and biochemical analysis identified over 1,300 proteins, including SMYD2, PP2Cα, and catalase. We further showed that PP2Cα is glutathionylated at C314 in a C-terminal domain, and PP2Cα C314 glutathionylation disrupts the interaction with mGluR3, an important glutamate receptor associated with synaptic plasticity

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“…New developments via the use of selective metabolic tagging method to identify glutathionylation [19,20] have been instrumental in elucidating the role of this particular modification. A method recently introduced by us utilizes a glutathionespecific enzyme to chemoenzymatically label and enrich Sglutathionylated proteins in human cells [19].…”

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confidence: 99%

Characteristic Tandem Mass Spectral Features Under Various Collision Chemistries for Site-Specific Identification of Protein S-Glutathionylation

Chou

Chiang

Lin

et al. 2014

J. Am. Soc. Mass Spectrom.

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Protein S-glutathionylation is a reversible post-translational modification widely implicated in redox regulated biological functions. Conventional biochemical methods, however, often do not allow such a mixed disulfide modification to be reliably identified on specific cysteine residues or be distinguished from other related oxidized forms. To develop more efficient mass spectrometry (MS)-based analytical strategies for this purpose, we first investigated the MS/MS fragmentation pattern of S-glutathionylated peptides under various dissociation modes, including collision-induced dissociation (CID), higher-energy C-trap dissociation (HCD), and electron transfer dissociation (ETD), using synthetic peptides derived from protein tyrosine phosphatase as models. Our results indicate that a MALDI-based high energy CID MS/MS on a TOF/TOF affords the most distinctive spectral features that would facilitate rapid and unambiguous identification of site-specific S-glutathionylation. For more complex proteomic samples best tackled by LC-MS/MS approach, we demonstrate that HCD performed on an LTQ-Orbitrap hybrid instrument fairs better than trap-based CID and ETD in allowing more protein site-specific S-glutathionylation to be confidently identified by direct database searching of the generated MS/MS dataset using Mascot. Overall, HCD afforded more peptide sequence-informative fragment ions retaining the glutathionyl modification with less neutral losses of side chains to compromise scoring. In conjunction with our recently developed chemo-enzymatic tagging strategy, our nanoLC-HCD-MS/MS approach is sufficiently sensitive to identify endogenous S-glutathionylated peptides prepared from non-stressed cells. It is anticipated that future applications to global scale analysis of protein S-glutathionylation will benefit further from current advances in both speed and mass accuracy afforded by HCD MS/MS mode on the Orbitrap series.

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Metabolic Synthesis of Clickable Glutathione for Chemoselective Detection of Glutathionylation

Cited by 49 publications

References 22 publications

The Expanding Landscape of the Thiol Redox Proteome

The Expanding Landscape of the Thiol Redox Proteome

A clickable glutathione approach for identification of protein glutathionylation in response to glucose metabolism

Characteristic Tandem Mass Spectral Features Under Various Collision Chemistries for Site-Specific Identification of Protein S-Glutathionylation

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