Proteomic and mass spectroscopic quantitation of protein S-nitrosation differentiates NO-donors.
Protein S-nitrosation has been argued to be the most important signaling pathway mediating the bioactivity of NO. This post-translational modification of protein thiols is the result of chemical nitrosation of cysteine residues. The term NO-donors covers very different chemical classes, from clinical therapeutics to probes of routine use in chemical biology; their different chemistry is predicted to result in distinctive biology regulated by protein S-nitrosation. To measure the extent of protein S-nitrosation by NO-donors, a proteomic mass spectrometry method was developed, which quantitates free thiol versus nitrosothiol for each modified cysteine residue, coined d-Switch. This method is adapted from the biotin switch (BST) method, used extensively to identify S-nitrosated proteins in complex biological mixtures; however, BST does not quantitate free thiol. Since glutathione-S-transferase P1-1 (GST-P1) has been proposed to be a biological "NO-carrier", GST-P1 was used as a reporter protein. The 5 different chemical classes of NO-donors compared by d-Switch demonstrated very different profiles of protein S-nitrosation and response to O(2) and cysteine, although all NO-donors were oxidants toward GST-P1. The low limits of detection and the ability to use established MS database searching allowed facile generalization of the d-Switch method. Therefore after incubation of neuronal cell cultures with nitrosothiol, it was possible to quantitate not only S-nitrosation of GST-P1 but also many other proteins, including novel targets such as ubiquitin carboxyl-terminal esterase L1 (UCHL1). Moreover, d-Switch also allowed identification of non-nitrosated proteins and quantitation of degree of nitrosation for individual protein thiols.
Department of Medicinal Chemistry and Pharmacognosy, College of Pharmacy, University of Illinois at Chicago, Chicago, Illinois 60612-7231, USA.
Vaishali
V
Sinha
Gihani T
GT
Wijewickrama
R Esala P
RE
Chandrasena
Hua
H
Xu
Praneeth D
PD
Edirisinghe
Isaac T
IT
Schiefer
Gregory R J
GR
Thatcher
D001710:Biotin / Q000031:analogs & derivatives / Q000378:metabolism; D002478:Cells, Cultured; D003545:Cysteine / Q000031:analogs & derivatives / Q000737:chemistry / Q000378:metabolism; D051549:Glutathione S-Transferase pi / Q000378:metabolism*; D006801:Humans; D013058:Mass Spectrometry; D009447:Neuroblastoma / Q000378:metabolism; D009569:Nitric Oxide / Q000378:metabolism*; D020030:Nitric Oxide Donors / Q000737:chemistry* / Q000378:metabolism; D015538:Nitrosation; D011506:Proteins / Q000737:chemistry* / Q000378:metabolism; D040901:Proteomics; D026422:S-Nitrosoglutathione / Q000378:metabolism; D026403:S-Nitrosothiols / Q000737:chemistry / Q000378:metabolism / Q000494:pharmacology
D020030:Nitric Oxide Donors; D011506:Proteins; D026403:S-Nitrosothiols; D009569:Nitric Oxide; D026422:S-Nitrosoglutathione; D001710:Biotin; C021315:S-nitrosocysteine; D051549:Glutathione S-Transferase pi; D003545:Cysteine