Quantitation of protein S-glutathionylation by liquid chromatography–tandem mass spectrometry: Correction for contaminating glutathione and glutathione disulfide

Bukowski, Michael R.; Bucklin, Christopher; Picklo, Matthew J.

doi:10.1016/j.ab.2014.10.002

Cited by 8 publications

(6 citation statements)

References 49 publications

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“…Levels of GSSP in solid tissues from rats at different ages are reported in Table 4 . These values well agree with those reported by other research groups (Bukowski et al 2015 ; Arambasic et al 2013 ) that found 57 ± 42 nmol/mg protein GSSP in rat liver and 1.75 µM GSSP in rat kidneys, respectively. GSSP were found to be significantly increased in some organs (lung, heart, spleen, brain) with aging (Giustarini et al 2011 ), thus supporting a role of free radicals in this physio-pathological process (Viña et al 2007 ).…”

Section: Biomedical Applicationssupporting

confidence: 93%

“…Human platelets were exposed to 0.1, 0.3, and 1 mM disulfiram. Platelet aggregation was initiated by the addition of ADP (10 µM, final concentration) and was monitored for 10 min by continuous recording of light transmission in a platelet aggregometer (Rossi et al 2006a) (Bukowski et al 2015;Arambasic et al 2013) that found 57 ± 42 nmol/mg protein GSSP in rat liver and 1.75 µM GSSP in rat kidneys, respectively. GSSP were found to be significantly increased in some organs (lung, heart, spleen, brain) with aging (Giustarini et al 2011), thus supporting a role of free radicals in this physio-pathological process (Viña et al 2007).…”

Section: Rssp In Cultured Cells and Solid Tissuesmentioning

confidence: 99%

“…The levels we measured are in agreement with those measured by Hansen et al (Hansen et al 2009 ), who found very low levels of GSSP also in HEK and HeLa cells, with values of a factor of 1000 below the value of total protein disulfides (i.e., PSSP + GSSP). Analogously, very low levels of GSSP (i.e., 57 ± 42 nmol/mg protein) were measured in rat liver by means of LC–MS/MS (Bukowski et al 2015 ). Levels of GSSP in solid tissues from rats at different ages are reported in Table 4 .…”

Section: Biomedical Applicationsmentioning

confidence: 99%

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Measurement of S-glutathionylated proteins by HPLC

et al. 2021

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S-glutathionylated proteins (GSSP), i.e., protein-mixed disulfides with glutathione (GSH), are considered a suitable biomarker of oxidative stress. In fact, they occur within cells at low level and their concentration increases markedly under pro-oxidant conditions. Plasma is something different, since it is physiologically rich in S-thiolated proteins (RSSP), i.e., protein-mixed disulfides with various types of low molecular mass thiols (LMM-SH). However, albumin, which is largely the most abundant plasma protein, possesses a cysteine residue at position 34 that is mostly reduced (about 60%) under physiological conditions, but easily involved in the formation of additional RSSP in the presence of oxidants. The quantification of GSSP requires special attention to sample handling, since their level can be overestimated as a result of artefactual oxidation of GSH. We have developed the present protocol to avoid this methodological problem. Samples should be treated as soon as possible after their collection with the alkylating agent N-ethylmaleimide that masks –SH groups and prevents their oxidation. The GSH released from mixed disulfides by reduction with dithiothreitol is then labeled with the fluorescent probe monobromobimane and quantified by HPLC. The method can be applied to many different biological samples, comprising blood components, red blood cell plasma membrane, cultured cells, and solid organs from animal models.

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Section: Biomedical Applicationssupporting

confidence: 93%

Section: Rssp In Cultured Cells and Solid Tissuesmentioning

confidence: 99%

Section: Biomedical Applicationsmentioning

confidence: 99%

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Measurement of S-glutathionylated proteins by HPLC

et al. 2021

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“…This observation is in agreement with results obtained by Hansen et al [77], who found very low levels of PSSG also in HEK and HeLa cells, with values of a factor of 1,000 below the value of total protein disulphides (i.e., PSSP + PSSG). Analogously, very low levels of PSSG (i.e., 57 ± 42 nmol/mg protein) were measured in rat liver by means of LC-MS/MS [83], whereas we observed a significant increase in PSSG levels in the organs from aged animals [67]. The reference levels for PSSG in human plasma are widely accepted [84].…”

Section: Levels Of Pssg In Some Biological Fluidsmentioning

confidence: 50%

Assessment of glutathione/glutathione disulphide ratio and S-glutathionylated proteins in human blood, solid tissues, and cultured cells

Giustarini

Colombo

Garavaglia

et al. 2017

Free Radical Biology and Medicine

124

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“…Confidence scores are generated based on previously published experimental data as well as high-throughput arrays, text mining, genomic annotations, and computational predictions. residue(s) (6,8). A second more general limitation to this study is the THP-1 monocytic cell line used to validate the labeling technique.…”

Section: Discussionmentioning

confidence: 99%

ProteinS-Glutathionylation Mediates Macrophage Responses to Metabolic Cues from the Extracellular Environment

Ullevig

Kim

Short

et al. 2016

Antioxidants & Redox Signaling

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Aims: Protein S-glutathionylation, the formation of a mixed disulfide between glutathione and protein thiols, is an oxidative modification that has emerged as a new signaling paradigm, potentially linking oxidative stress to chronic inflammation associated with heart disease, diabetes, cancer, lung disease, and aging. Using a novel, highly sensitive, and selective proteomic approach to identify S-glutathionylated proteins, we tested the hypothesis that monocytes and macrophages sense changes in their microenvironment and respond to metabolic stress by altering their protein thiol S-glutathionylation status. Results: We identified over 130 S-glutathionylated proteins, which were associated with a variety of cellular functions, including metabolism, transcription and translation, protein folding, free radical scavenging, cell motility, and cell death. Over 90% of S-glutathionylated proteins identified in metabolically stressed THP-1 monocytes were also found in hydrogen peroxide (H 2 O 2 )-treated cells, suggesting that H 2 O 2 mediates metabolic stress-induced protein S-glutathionylation in monocytes and macrophages. We validated our findings in mouse peritoneal macrophages isolated from both healthy and dyslipidemic atherosclerotic mice and found that 52% of the S-glutathionylated proteins found in THP-1 monocytes were also identified in vivo. Changes in macrophage protein S-glutathionylation induced by dyslipidemia were sexually dimorphic. Innovation: We provide a novel mechanistic link between metabolic (and thiol oxidative) stress, macrophage dysfunction, and chronic inflammatory diseases associated with metabolic disorders. Conclusion: Our data support the concept that changes in the extracellular metabolic microenvironment induce S-glutathionylation of proteins central to macrophage metabolism and a wide array of cellular signaling pathways and functions, which in turn initiate and promote functional and phenotypic changes in macrophages. Antioxid. Redox Signal. 25, 836-851.

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Quantitation of protein S-glutathionylation by liquid chromatography–tandem mass spectrometry: Correction for contaminating glutathione and glutathione disulfide

Cited by 8 publications

References 49 publications

Measurement of S-glutathionylated proteins by HPLC

Measurement of S-glutathionylated proteins by HPLC

Assessment of glutathione/glutathione disulphide ratio and S-glutathionylated proteins in human blood, solid tissues, and cultured cells

ProteinS-Glutathionylation Mediates Macrophage Responses to Metabolic Cues from the Extracellular Environment

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