Analysis of Protein S‐Nitrosylation

Abstract

S-nitrosylation is the binding of an NO group to a cysteine or other thiol. Like phosphorylation, S-nitrosylation is a precisely targeted and rapidly reversible post-translational modification that serves as an on/off switch for protein function during cell signaling. However, unlike phosphorylation, S-nitrosylation of proteins occurs nonenzymatically and is mediated, at least in part, by redox-regulated chemical reactions in cells. Alterations in pH, pO(2), cellular reductants, transition metals, and UV light lead to the loss and/or gain of S-NO bonds. Due to the redox-sensitive nature of the modification, analysis of protein S-nitrosylation is technically difficult, since the S-NO bond is easily disrupted during sample preparation. In addition, the level of S-nitrosylated proteins in cells approaches the limit of detection of currently available technology. Despite these technical challenges, several useful methods have been developed recently to measure protein S-nitrosylation in biological samples, and these are described in this unit.

Quantitative site-specific reactivity profiling of S-nitrosylation in mouse skeletal muscle using cysteinyl peptide enrichment coupled with mass spectrometry

“…Twofold serial dilutions of GSNO ranging from 25 to 1.56 μM were used to generate the standard curves. A standard protocol reported previously was followed to measure the SNO signals at 540 nm [50]. Briefly, 50 μl each of the nitrosylated samples after GSNO removal was incubated with 50 μl of solution A (1% (w/v) sulfanilamide in 0.5 M HCl) or solution B (solution A containing 0.2% (w/v) HgCl 2 ) at room temperature for 5 min in separate wells of a 96-well plate.…”

Redox Regulatory Mechanism of Transnitrosylation by Thioredoxin

“…S -Nitrosothiol levels were estimated according to the procedure described by Mannick and Schonhoff (32). Briefly, 500 μg of protein from HeLa cell extract was nitrosylated with either NB, 100 μ m GSNO, GSNO plus 25 μg of oTrx1, or GSNO plus 25 μg of rTrx1 at 37 °C for 30 min.…”

Inducible Nitric Oxide Synthase Drives mTOR Pathway Activation and Proliferation of Human Melanoma by Reversible Nitrosylation of TSC2

“…Some studies have suggested that NO can activate mTOR signaling in non-cancer model systems, including murine macrophages (16), and vascular smooth muscle cells (17). However, as yet there have been no studies assessing the ability of cancer-expressed iNOS and endogenously-produced NO in physiologic concentrations to activate the mTOR pathway.…”