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J Am Chem Soc. 2010 Aug 25;132(33):11392-4. doi: 10.1021/ja103591v.

Gold nanoparticle enrichment method for identifying S-nitrosylation and S-glutathionylation sites in proteins.

Author information

1
Department of Chemistry & Biochemistry, University of Windsor, 401 Sunset Avenue, Windsor, Ontario, Canada.

Abstract

We present a simple method by which gold nanoparticles (AuNPs) are used to simultaneously isolate and enrich for free or modified thiol-containing peptides, thus facilitating the identification of protein S-modification sites. Here, protein disulfide isomerase (PDI) and dual specificity phosphatase 12 (DUSP12 or hYVH1) were S-nitrosylated or S-glutathionylated, their free thiols differentially alkylated, and subjected to proteolysis. AuNPs were added to the digests, and the AuNP-bound peptides were isolated by centrifugation and released by thiol exchange. These AuNP-bound peptides were analyzed by MALDI-TOF mass spectrometry revealing that AuNPs result in a significant enrichment of free thiol-containing as well as S-nitrosylated, S-glutathionylated, and S-alkylated peptides, leading to the unequivocal assignment of thiols susceptible to modification.

PMID:
20677743
DOI:
10.1021/ja103591v
[Indexed for MEDLINE]

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