2012
DOI: 10.1002/ange.201200321
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In Vivo Tagging and Characterization of S‐Glutathionylated Proteins by a Chemoenzymatic Method

Abstract: Glutathionylspermidin‐Synthetase (GspS) wurde in menschliche Zellen eingebracht, um endogenes Glutathion (GSH) mit Biotinylspermin (Biotin‐spm) für die Identifizierung von S‐thiolierten Proteinen zu markieren. Die Proteine mit Gspm‐biotin können mit einem Immunblot nachgewiesen werden (A). Eine Anreicherung mithilfe des Biotin‐Markers ermöglicht eine massenspektrometrische Proteomanalyse (B) zur ortsspezifischen Identifikation von Glutathionylierungen.

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Cited by 4 publications
(9 citation statements)
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“…Cysteine-containing peptides, namely hT28 (amino acids 200-221 of human PTP1B, ESGSLSPEHGPVVVHCSA GIGR), rT28 (amino acids 200-221 of rat PTP1B, ESGSLSPEHGPIVVHCSAGIGR), rT63 (amino acids 450-469 of rat PTPN11, QESIVDAGPVVVHCSAGIGR) and hT20-21 (amino acids 121-131 of human PTP1B, CAQYWPQKEEK), were synthesized by the Peptide Synthesis Lab at the Institute of Biological Chemistry, Academia Sinica, Taiwan. The synthesis of biotinyl-spermine (biotin-spm) and Gsp-biotin are described on previous study [19]. The pET28a-PTP1B plasmid constructed from human peripheral blood lymphocyte PTP1B cDNA by RT-PCR was kindly provided by Dr. Tzu-Ching Meng (Institute of Biological Chemistry, Academia Sinica, Taiwan).…”
Section: Methodsmentioning
confidence: 99%
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“…Cysteine-containing peptides, namely hT28 (amino acids 200-221 of human PTP1B, ESGSLSPEHGPVVVHCSA GIGR), rT28 (amino acids 200-221 of rat PTP1B, ESGSLSPEHGPIVVHCSAGIGR), rT63 (amino acids 450-469 of rat PTPN11, QESIVDAGPVVVHCSAGIGR) and hT20-21 (amino acids 121-131 of human PTP1B, CAQYWPQKEEK), were synthesized by the Peptide Synthesis Lab at the Institute of Biological Chemistry, Academia Sinica, Taiwan. The synthesis of biotinyl-spermine (biotin-spm) and Gsp-biotin are described on previous study [19]. The pET28a-PTP1B plasmid constructed from human peripheral blood lymphocyte PTP1B cDNA by RT-PCR was kindly provided by Dr. Tzu-Ching Meng (Institute of Biological Chemistry, Academia Sinica, Taiwan).…”
Section: Methodsmentioning
confidence: 99%
“…The enriched glutathionylated peptides of PTP1B were prepared using previously developed chemoenzymatic method of glutathionylspermidine tagging [19]. Briefly, the recombinant human PTP1B was incubated with 5 mM biotinylated glutathionylspermidine disulfide (Gsp-biotin disulfide) for 2 h. Fifty mM iodoacetamide was added to block free thiols, followed by acetone precipitation to remove unreacted biotinyl-spermine (biotin-spm) and Gsp-biotin, and then trypsin digestion for 16 h. The resulting Gsp-biotin S-thiolated peptides were enriched by streptavidin resin (Thermo Scientific, Waltham, MA, USA).…”
Section: Enrichment Of Glutathionylated Peptides From Purified Recombmentioning
confidence: 99%
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