Quantitative analysis of S-nitrosylated proteins.
Protein S-nitrosylation is the covalent binding of nitric oxide to specific cysteine residues in proteins. This modification influences a large number of cellular events and signaling processes. As this process is finely regulated in vivo, the level of nitrosylation changes in response to different stimuli. Since its introduction, the biotin-switch technique (BST) is the most used indirect method for the study of S-nitrosylation both in vivo and in vitro and its coupling with mass spectrometry-based proteomics lead to the identification of the S-nitroso proteome in different organisms. However, this method does not give any information about the posttranslational modification level on the same residue in different biological conditions. Quantitative proteomic methods can assess the relative change in S-nitrosylation for hundreds sites in a single experiment. Stable isotope labeling by aminoacids in cell culture (SILAC) is one of the most used and accurate quantitative techniques in MS-based proteomics. Here we present a SILAC-based method for the quantification of endogenously S-nitrosylated proteins in RAW 264.7 cells.
Mechanobiology Institute and Lipid Profiles, Centre for Life Sciences National University of Singapore, Singapore, Singapore.
Federico
F
Torta
Angela
A
Bachi
D020816:Amino Acid Motifs; D000818:Animals; D015153:Blotting, Western; D002460:Cell Line; D056148:Chromatography, Reverse-Phase; D004591:Electrophoresis, Polyacrylamide Gel; D007553:Isotope Labeling; D051379:Mice; D009569:Nitric Oxide / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000378:metabolism; D020543:Proteome / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D026403:S-Nitrosothiols / Q000737:chemistry / Q000302:isolation & purification / Q000378:metabolism*; D053719:Tandem Mass Spectrometry
D020543:Proteome; D026403:S-Nitrosothiols; D009569:Nitric Oxide; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse