Profiling thiol metabolites and quantification of cellular glutathione using FT-ICR-MS spectrometry

Sadakatali S. Gori; Pawel Lorkiewicz; Daniel S. Ehringer; Alex C. Belshoff; Richard M. Higashi; Teresa W.M. Fan; Michael H. Nantz

doi:10.1007/s00216-014-7810-z

Profiling thiol metabolites and quantification of cellular glutathione using FT-ICR-MS spectrometry

Sadakatali S. Gori
, Pawel Lorkiewicz
, Daniel S. Ehringer
, Alex C. Belshoff
, Richard M. Higashi
, Teresa W.M. Fan
, Michael H. Nantz

Producción científica: Article › revisión exhaustiva

24 Citas (Scopus)

Resumen

We describe preparation and use of the quaternary ammonium-based α-iodoacetamide QDE and its isotopologue*QDE as reagents for chemoselective derivatization of cellular thiols. Direct addition of the reagents to live cells followed by adduct extraction into n-butanol and analysis by FT-ICR-MS provided a registry of matched isotope peaks from which molecular formulae of thiol metabolites were derived. Acidification to pH 4 during cell lysis and adduct formation further improves the chemoselectivity for thiol derivatization. Examination of A549 human lung adenocarcinoma cells using this approach revealed cysteine, cysteinylglycine, glutathione, and homocysteine as principal thiol metabolites as well as the sulfinic acid hypotaurine. The method is also readily applied to quantify the thiol metabolites, as demonstrated here by the quantification of both glutathione and glutathione disulfide in A549 cells at concentrations of 34.4±11.5 and 10.1±4.0 nmol/mg protein, respectively.

Idioma original	English
Páginas (desde-hasta)	4371-4379
Número de páginas	9
Publicación	Analytical and Bioanalytical Chemistry
Volumen	406
N.º	18
DOI	https://doi.org/10.1007/s00216-014-7810-z
Estado	Published - jul 2014

Nota bibliográfica

Funding Information:
Acknowledgments This work was supported by grants from NIH (1 R01 ES022191-01, 3R01CA118434-02S1, and 1R01CA118434-01A2). The FT-ICR-MS instrumentation at the Center for Regulatory and Environmental Analytical Metabolomics mass spectrometry facility was funded by an NSF/EPSCoR grant (EPS-0447479). We also acknowledge the insightful contributions on data analysis from Mr. James Carrier and Dr. Hunter Moseley (Department of Molecular and Cellular Biochemistry, University of Kentucky), and we thank Ms. Stephanie Mattingly for her advice on how to best operate the FT-ICR-MS for isotopologue pairs of quaternary ammonium derivatives. We also thank Ms. Ramya Balasubramaniam for providing cultured cells.

Financiación

Acknowledgments This work was supported by grants from NIH (1 R01 ES022191-01, 3R01CA118434-02S1, and 1R01CA118434-01A2). The FT-ICR-MS instrumentation at the Center for Regulatory and Environmental Analytical Metabolomics mass spectrometry facility was funded by an NSF/EPSCoR grant (EPS-0447479). We also acknowledge the insightful contributions on data analysis from Mr. James Carrier and Dr. Hunter Moseley (Department of Molecular and Cellular Biochemistry, University of Kentucky), and we thank Ms. Stephanie Mattingly for her advice on how to best operate the FT-ICR-MS for isotopologue pairs of quaternary ammonium derivatives. We also thank Ms. Ramya Balasubramaniam for providing cultured cells.

Financiadores	Número del financiador
NSF-Kentucky EPSCoR	EPS-0447479
National Institutes of Health (NIH)	3R01CA118434-02S1
National Institute of Environmental Health Sciences (NIEHS)	R01ES022191

ASJC Scopus subject areas

Analytical Chemistry
Biochemistry

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@article{3d9b30eb92244d54a4b6c023b8c2bfda,

title = "Profiling thiol metabolites and quantification of cellular glutathione using FT-ICR-MS spectrometry",

abstract = "We describe preparation and use of the quaternary ammonium-based α-iodoacetamide QDE and its isotopologue*QDE as reagents for chemoselective derivatization of cellular thiols. Direct addition of the reagents to live cells followed by adduct extraction into n-butanol and analysis by FT-ICR-MS provided a registry of matched isotope peaks from which molecular formulae of thiol metabolites were derived. Acidification to pH 4 during cell lysis and adduct formation further improves the chemoselectivity for thiol derivatization. Examination of A549 human lung adenocarcinoma cells using this approach revealed cysteine, cysteinylglycine, glutathione, and homocysteine as principal thiol metabolites as well as the sulfinic acid hypotaurine. The method is also readily applied to quantify the thiol metabolites, as demonstrated here by the quantification of both glutathione and glutathione disulfide in A549 cells at concentrations of 34.4±11.5 and 10.1±4.0 nmol/mg protein, respectively.",

keywords = "Chemoselective, Cysteine, Hypotaurine, Iodoacetamide, Metabolomics, Oxidative stress",

author = "Gori, \{Sadakatali S.\} and Pawel Lorkiewicz and Ehringer, \{Daniel S.\} and Belshoff, \{Alex C.\} and Higashi, \{Richard M.\} and Fan, \{Teresa W.M.\} and Nantz, \{Michael H.\}",

note = "Funding Information: Acknowledgments This work was supported by grants from NIH (1 R01 ES022191-01, 3R01CA118434-02S1, and 1R01CA118434-01A2). The FT-ICR-MS instrumentation at the Center for Regulatory and Environmental Analytical Metabolomics mass spectrometry facility was funded by an NSF/EPSCoR grant (EPS-0447479). We also acknowledge the insightful contributions on data analysis from Mr. James Carrier and Dr. Hunter Moseley (Department of Molecular and Cellular Biochemistry, University of Kentucky), and we thank Ms. Stephanie Mattingly for her advice on how to best operate the FT-ICR-MS for isotopologue pairs of quaternary ammonium derivatives. We also thank Ms. Ramya Balasubramaniam for providing cultured cells.",

year = "2014",

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doi = "10.1007/s00216-014-7810-z",

language = "English",

volume = "406",

pages = "4371--4379",

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AU - Gori, Sadakatali S.

AU - Lorkiewicz, Pawel

AU - Ehringer, Daniel S.

AU - Belshoff, Alex C.

AU - Higashi, Richard M.

AU - Fan, Teresa W.M.

AU - Nantz, Michael H.

N1 - Funding Information: Acknowledgments This work was supported by grants from NIH (1 R01 ES022191-01, 3R01CA118434-02S1, and 1R01CA118434-01A2). The FT-ICR-MS instrumentation at the Center for Regulatory and Environmental Analytical Metabolomics mass spectrometry facility was funded by an NSF/EPSCoR grant (EPS-0447479). We also acknowledge the insightful contributions on data analysis from Mr. James Carrier and Dr. Hunter Moseley (Department of Molecular and Cellular Biochemistry, University of Kentucky), and we thank Ms. Stephanie Mattingly for her advice on how to best operate the FT-ICR-MS for isotopologue pairs of quaternary ammonium derivatives. We also thank Ms. Ramya Balasubramaniam for providing cultured cells.

PY - 2014/7

Y1 - 2014/7

N2 - We describe preparation and use of the quaternary ammonium-based α-iodoacetamide QDE and its isotopologue*QDE as reagents for chemoselective derivatization of cellular thiols. Direct addition of the reagents to live cells followed by adduct extraction into n-butanol and analysis by FT-ICR-MS provided a registry of matched isotope peaks from which molecular formulae of thiol metabolites were derived. Acidification to pH 4 during cell lysis and adduct formation further improves the chemoselectivity for thiol derivatization. Examination of A549 human lung adenocarcinoma cells using this approach revealed cysteine, cysteinylglycine, glutathione, and homocysteine as principal thiol metabolites as well as the sulfinic acid hypotaurine. The method is also readily applied to quantify the thiol metabolites, as demonstrated here by the quantification of both glutathione and glutathione disulfide in A549 cells at concentrations of 34.4±11.5 and 10.1±4.0 nmol/mg protein, respectively.

AB - We describe preparation and use of the quaternary ammonium-based α-iodoacetamide QDE and its isotopologue*QDE as reagents for chemoselective derivatization of cellular thiols. Direct addition of the reagents to live cells followed by adduct extraction into n-butanol and analysis by FT-ICR-MS provided a registry of matched isotope peaks from which molecular formulae of thiol metabolites were derived. Acidification to pH 4 during cell lysis and adduct formation further improves the chemoselectivity for thiol derivatization. Examination of A549 human lung adenocarcinoma cells using this approach revealed cysteine, cysteinylglycine, glutathione, and homocysteine as principal thiol metabolites as well as the sulfinic acid hypotaurine. The method is also readily applied to quantify the thiol metabolites, as demonstrated here by the quantification of both glutathione and glutathione disulfide in A549 cells at concentrations of 34.4±11.5 and 10.1±4.0 nmol/mg protein, respectively.

KW - Chemoselective

KW - Cysteine

KW - Hypotaurine

KW - Iodoacetamide

KW - Metabolomics

KW - Oxidative stress

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M3 - Article

C2 - 24858467

AN - SCOPUS:84903784852

SN - 1618-2642

VL - 406

SP - 4371

EP - 4379

JO - Analytical and Bioanalytical Chemistry

JF - Analytical and Bioanalytical Chemistry

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ER -

Profiling thiol metabolites and quantification of cellular glutathione using FT-ICR-MS spectrometry

Resumen

Nota bibliográfica

Financiación

ASJC Scopus subject areas

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