Proteomic methods for analysis of S-nitrosation

Abstract

This review discusses proteomic methods to detect and identify S-nitrosated proteins. Protein S-nitrosation, the post-translational modification of thiol residues to form S-nitrosothiols, has been suggested to be a mechanism of cellular redox signaling by which nitric oxide can alter cellular function through modification of protein thiol residues. It has become apparent that methods that will detect and identify low levels of S-nitrosated protein in complex protein mixtures are required in order to fully appreciate the range, extent and selectivity of this modification in both physiological and pathological conditions. While many advances have been made in the detection of either total cellular S-nitrosation or individual S-nitrosothiols, proteomic methods for the detection of S-nitrosation are in relative infancy. This review will discuss the major methods that have been used for the proteomic analysis of protein S-nitrosation and discuss the pros and cons of this methodology.

Fig. 1

The biotin switch assay (after Jaffrey et al. [42]). Protein thiols (-SH), S-nitrosothiols (-SNO) and disulfides (-SSR) are treated with MMTS to convert all thiols to disulfides (-SSCH₃). MMTS is removed and the proteins are incubated with ascorbate to convert –SNO to –SH. The nascent –SH is then labeled with biotin via a disulfide bond using biotin-HPDP.

Fig. 2

Reduction of protein RSNO by ascorbate and DTT. Human bronchial epithelial cells were incubated with CysNO (50 μM) for 30 minutes. Cells were lyzed in HEPES buffer containing 10 mM NEM, 100 μM DTPA and 10 μM neocuproine. Either DTT or ascorbate was added, and aliquots were taken at the indicated time points. For each aliquot, the protein was precipitated to remove excess reducing agent, and total RSNO was measured by tri-iodide-based chemiluminescence after treatment with sulfanilamide.

Fig. 3

Biotinylation of proteins after DTT reduction. Human bronchial epithelial cells were incubated without (top) or with (bottom) 10 μM CysNO and then subjected to the biotin switch assay using DTT (5 mM) in place of ascorbate. Proteins were separated in 2 dimensions and biotinylation was examined by Western blot analysis.

Fig. 4

Analysis of protein modification by DIGE. (Top) Schematic of the DIGE labeling scheme using two maleimido-cyanine-based dyes Cy3 (green) and Cy5 (red). RSNO or NO treatment will modify some fraction of the protein thiols as illustrated on the left side of the figure. Both the treated and untreated samples are sequentially incubated with NEM, reducing agent (in this case DTT) and then either Cy3 or Cy5. These samples are pooled at equal protein concentrations are run in 2 dimensions. (Bottom) Human bronchial epithelial cells were treated with or without 5 μM CysNO, and protein was treated as illustrated in the top panel. Pooled protein was run in 2 dimensions and detected by fluorescence scanning.