Direct and indirect detection methods for the analysis of S-nitrosylated peptides and proteins.
Covalent binding of nitric oxide to specific cysteine residues in proteins is a key event in cellular redox signal transduction. This modification influences both physiological and pathological processes, such as cardiovascular, neurological, and cancer-associated events. Even though, since its introduction, the biotin switch technique is the most used indirect method for the study of S-nitrosylation both in vivo and in vitro, during the last years modifications of this method have emerged, allowing more efficient sample enrichment and the precise identification of the modified aminoacidic sites. At the same time, to bypass the difficulties generated by the multiple chemical reaction steps required by these labeling methods, the direct identification of the SNO groups by mass spectrometry is emerging as a useful tool in this field, although, until now, it has been limited to the study of synthetic or purified recombinant proteins. Here we present two different techniques, developed in our laboratories, for detection of S-nitrosylation: the first is based on a modification of the biotin switch technique and is called His-tag switch, and the second is a direct mass spectrometry-based method used to detect in vivo generated SNO groups.
Biomolecular Mass Spectrometry Unit, Division of Genetics and Cell Biology, San Raffaele Scientific Institute, Milano, Italy.
Federico
F
Torta
Lisa
L
Elviri
Angela
A
Bachi
D000818:Animals; D019411:Clinical Laboratory Techniques; D006801:Humans; D013058:Mass Spectrometry / Q000379:methods; D009603:Nitroso Compounds / Q000032:analysis* / Q000378:metabolism; D010446:Peptide Fragments / Q000032:analysis* / Q000378:metabolism; D010449:Peptide Mapping / Q000379:methods; D011499:Protein Processing, Post-Translational; D011506:Proteins / Q000032:analysis* / Q000378:metabolism
D009603:Nitroso Compounds; D010446:Peptide Fragments; D011506:Proteins