title: Functional proteomics approaches for the identification of transnitrosylase and denitrosylase targets.
abstract: Protein S-nitrosylation is a dynamic post-translational modification (PTM) of specific cysteines within a target protein. Both proteins and small molecules are known to regulate the attachment and removal of this PTM, and proteins exhibiting such a function are transnitrosylase or denitrosylase candidates. With the advent of the biotin switch technique coupled to high-throughput proteomics workflows, the identification and quantification of large numbers of S-nitrosylated proteins and peptides is now possible. Proper analysis and interpretation of high throughout and quantitative proteomics data will help identify specific transnitrosylase and denitrosylase target peptide sequences and contribute to an understanding of the function and regulation of specific S-nitrosylation events. Here we describe the application of a quantitative proteomics approach using isotope-coded affinity tags (ICAT) in the biotin switch approach for the identification of transnitrosylation and denitrosylation targets of thioredoxin 1, an enigmatic protein with both reported transnitrosylase and denitrosylase activities.
authors: Center for Advanced Proteomics Research and Department of Biochemistry and Molecular Biology, UMDNJ-New Jersey Medical School Cancer Center, Newark, NJ 07103, United States.
authors: Changgong
authors: C
authors: Wu
authors: Andrew Myles
authors: AM
authors: Parrott
authors: Tong
authors: T
authors: Liu
authors: Annie
authors: A
authors: Beuve
authors: Hong
authors: H
authors: Li
mesh_terms: D000595:Amino Acid Sequence; D019921:Biotinylation; D045744:Cell Line, Tumor; D002846:Chromatography, Affinity; D015180:Electrophoresis, Gel, Two-Dimensional; D006801:Humans; D010446:Peptide Fragments / Q000737:chemistry; D011499:Protein Processing, Post-Translational; D040901:Proteomics; D026403:S-Nitrosothiols / Q000737:chemistry* / Q000378:metabolism; D013194:Staining and Labeling; D013879:Thioredoxins / Q000737:chemistry* / Q000378:metabolism
chemical_list: D010446:Peptide Fragments; D026403:S-Nitrosothiols; D013879:Thioredoxins
keywords: 2DE; ACN; Acetonitrile; BB; BCA; BST; Bicinchoninic acid; Binding buffer; Biotin switch; Biotin-HPDP; Biotin-switch technique; CID; Collision-induced dissociation; Cys32 and Cys35 to Ser Trx1 mutant; DDA; Data-dependent analysis; ENOA; ESI; Electrospray ionization; FDR; False discovery rate; GAPDH; GSNO; Glyceraldehyde 3-phosphate dehydrogenase; Human α-enolase; ICAT; ICAT heavy reagent; ICAT light reagent; ICAT-H; ICAT-L; IEF; Isobaric tag for relative and absolute quantitation; Isoelectric focusing; Isotope-coded affinity tags; LB; Lysis buffer; MALDI; MMTS; MRM; MS/MS; Matrix-assisted laser desorption ionization; Methyl methanethiosulfonate; Multiple-reaction monitoring; N-[6-(Biotinamido)hexyl]-3′-(2′-pyridyldithio)-propionamide; NADPH; NB; Nicotinamide adenine dinucleotide phosphate; PTM; Post-translational modification; Proteomics; RB; Resuspension buffer; S-Nitrosylated cysteine; S-Nitrosylated thioredoxin 1; S-Nitrosylation buffer; S-nitrosoglutathione; S-nitrosylation; SILAC; SNO-Cys; SNO-Trx1; SNO-site identification; SNOSID; Stable isotope labeling with amino acids in cell culture; TFA; TMT; Tandem Mass Tags; Tandem mass spectrometry; Thioredoxin; Trifluoroacetic acid; Trx; Trx reductase; Trx(C32S/C35S); TrxR; Two-dimensional gel electrophoresis; iTRAQ