title: Identification of protein nitrosothiols using phosphine-mediated selective reduction.
abstract: Regulation of protein function by S-nitrosation of critical cysteines is known to be an important mechanism for nitric oxide signaling. Evidence for this comes from several different experimental approaches including the ascorbate-based biotin switch method. However technical problems with specificity and sensitivity of ascorbate reduction of S-nitrosothiols limit its usefulness and reliability. In the current study we report the use of triphenylphosphine ester derivatives to selectively reduce SNO bonds in proteins. After triphenylphosphine ester reduction, thiols were tagged with biotin or fluorescently labeled maleimide reagents. Importantly we demonstrate that these compounds are specific reductants of SNO in complex biological samples and do not reduce protein disulfides or protein thiols modified by hydrogen peroxide. Reduction proceeds efficiently in cell extracts and in whole fixed cells. Application of this approach allowed us to demonstrate S-nitrosation of specific cellular proteins, label S-nitrosoproteins in whole fixed cells (especially the nuclear compartment) and demonstrate S-nitrosoprotein formation in cells expressing inducible nitric oxide synthase.
authors.affiliation: Department of Pharmacology and Cancer Biology, Duke University Medical Center, Durham, NC 27710, USA.
authors.forename: Sheng
authors.initials: S
authors.lastname: Li
authors.forename: Hua
authors.initials: H
authors.lastname: Wang
authors.forename: Ming
authors.initials: M
authors.lastname: Xian
authors.forename: A Richard
authors.initials: AR
authors.lastname: Whorton
mesh_terms: D000818:Animals; D001710:Biotin / Q000737:chemistry; D019921:Biotinylation; D019556:COS Cells / Q000187:drug effects; D002522:Chlorocebus aethiops; D004220:Disulfides / Q000737:chemistry; D004952:Esters / Q000737:chemistry; D006861:Hydrogen Peroxide / Q000494:pharmacology; D008070:Lipopolysaccharides / Q000494:pharmacology; D008264:Macrophages / Q000187:drug effects / Q000378:metabolism; D008301:Maleimides / Q000032:analysis; D009569:Nitric Oxide / Q000378:metabolism; D015538:Nitrosation; D009943:Organophosphorus Compounds / Q000737:chemistry; D010084:Oxidation-Reduction; D010720:Phosphines / Q000737:chemistry*; D011092:Polyethylene Glycols / Q000737:chemistry; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1 / Q000378:metabolism; D011506:Proteins / Q000032:analysis* / Q000378:metabolism*; D026403:S-Nitrosothiols / Q000032:analysis* / Q000378:metabolism; D013438:Sulfhydryl Compounds / Q000737:chemistry
chemical_list: C504424:Alexa Fluor 488 C5-maleimide; D004220:Disulfides; D004952:Esters; D008070:Lipopolysaccharides; D008301:Maleimides; D009943:Organophosphorus Compounds; D010720:Phosphines; D011506:Proteins; D026403:S-Nitrosothiols; D013438:Sulfhydryl Compounds; C061896:triphenylphosphine; D009569:Nitric Oxide; D011092:Polyethylene Glycols; D001710:Biotin; D006861:Hydrogen Peroxide; D054562:Protein Tyrosine Phosphatase, Non-Receptor Type 1
references.citation: Proc Natl Acad Sci U S A. 2004 Jun 15;101(24):8945-50
references.pmid: 15184672
references.citation: Angew Chem Int Ed Engl. 2008;47(35):6598-601
references.pmid: 18642267
references.citation: Mol Pharmacol. 2005 Sep;68(3):847-54
references.pmid: 15967877
references.citation: Science. 2008 May 23;320(5879):1050-4
references.pmid: 18497292
references.citation: Nat Cell Biol. 2001 Feb;3(2):E46-9
references.pmid: 11175760
references.citation: J Am Chem Soc. 2009 Mar 25;131(11):3854-5
references.pmid: 19256495
references.citation: Am J Physiol Cell Physiol. 2007 Apr;292(4):C1263-71
references.pmid: 17092994
references.citation: Free Radic Biol Med. 2008 Apr 1;44(7):1362-72
references.pmid: 18211831
references.citation: Nat Rev Mol Cell Biol. 2005 Feb;6(2):150-66
references.pmid: 15688001
references.citation: Mol Interv. 2003 Aug;3(5):253-63
references.pmid: 14993439
references.citation: J Biol Chem. 2007 May 11;282(19):13977-83
references.pmid: 17376775
references.citation: Annu Rev Pharmacol Toxicol. 2002;42:585-600
references.pmid: 11807184
references.citation: J Mol Biol. 2005 Mar 11;346(5):1423-40
references.pmid: 15713491
references.citation: Arch Biochem Biophys. 2003 Feb 15;410(2):269-79
references.pmid: 12573287
references.citation: Org Lett. 2010 Dec 17;12(24):5674-6
references.pmid: 21080645
references.citation: J Biol Chem. 2009 Dec 25;284(52):36160-36166
references.pmid: 19847012
references.citation: Am J Physiol Lung Cell Mol Physiol. 2004 Aug;287(2):L262-8
references.pmid: 15246980
references.citation: J Am Chem Soc. 2009 Sep 23;131(37):13238-9
references.pmid: 19715315
references.citation: ACS Chem Biol. 2010 Apr 16;5(4):405-14
references.pmid: 20146502
references.citation: Biochemistry. 2000 Aug 15;39(32):9901-8
references.pmid: 10933809
references.citation: Cell. 2001 Sep 21;106(6):675-83
references.pmid: 11572774
references.citation: Biochemistry. 2001 Feb 13;40(6):1688-93
references.pmid: 11327828
references.citation: Science. 2005 Dec 23;310(5756):1966-70
references.pmid: 16373578
references.citation: Free Radic Biol Med. 2005 Apr 1;38(7):874-81
references.pmid: 15749383
references.citation: Org Lett. 2010 Sep 17;12(18):4208-11
references.pmid: 20731371
references.citation: Biochemistry. 2004 Mar 2;43(8):2314-22
references.pmid: 14979728
references.citation: Cell. 2004 Feb 20;116(4):617-28
references.pmid: 14980227
references.citation: Proc Natl Acad Sci U S A. 1996 Dec 24;93(26):15069-74
references.pmid: 8986765
references.citation: Proc Natl Acad Sci U S A. 2003 May 27;100(11):6376-81
references.pmid: 12740440
references.citation: Sci STKE. 2001 Jun 12;2001(86):pl1
references.pmid: 11752655