Gold nanoparticle enrichment method for identifying S-nitrosylation and S-glutathionylation sites in proteins.
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.
Faccenda
Adam
A
Department of Chemistry & Biochemistry, University of Windsor, 401 Sunset Avenue, Windsor, Ontario, Canada.
Bonham
Christopher A
CA
Vacratsis
Panayiotis O
PO
Zhang
Xueji
X
Mutus
Bulent
B
D000478:Alkylation; D001665:Binding Sites; D054638:Dual Specificity Phosphatase 1 / Q000737:chemistry / Q000378:metabolism*; D005978:Glutathione / Q000737:chemistry / Q000378:metabolism*; D006046:Gold / Q000737:chemistry*; D006801:Humans; D053768:Metal Nanoparticles / Q000737:chemistry*; D019704:Protein Disulfide-Isomerases / Q000737:chemistry / Q000378:metabolism*; D026403:S-Nitrosothiols / Q000737:chemistry / Q000378:metabolism*
D026403:S-Nitrosothiols; D006046:Gold; D054638:Dual Specificity Phosphatase 1; D019704:Protein Disulfide-Isomerases; D005978:Glutathione