SNO spectral counting (SNOSC), a label-free proteomic method for quantification of changes in levels of protein S-nitrosation

Abstract

S-Nitrosation plays an important role in regulation of protein function and signal transduction. Discovering S-nitrosated targets is a prerequisite for further functional study. However, current proteomic methods used to quantify S-nitrosation are limited in their applicability to certain types of samples, or by the need for special reagents and complex procedures to obtain the results. Here we devised a label-free proteomic method for quantification of changes in the level of protein S-nitrosation on the basis of a spectral counting strategy, called S-nitrosothiol (SNO) spectral counting (SNOSC). With this method, samples can be from any source (cells, tissues); there is no need for labelling reagents or procedures, and the results yield quantitative information. Moreover, as it is based on the irreversible biotinylation procedure (IBP) for S-nitrosation protein enrichment, false positive targets caused by the interference of intermolecular disulphide bonds are ruled out. Using SNOSC we studied S-nitrosation in the cell line RAW264.7 induced exogenously with S-nitrosoglutathione (GSNO), or induced endogenously by lipopolysaccharides/interferon-gamma (LPS/IFN-γ). We detected a significant increase in S-nitrosation of 50 proteins after exogenous induction and 17 proteins after endogenous induction. We thus demonstrate that SNOSC is a widely applicable proteomic method for fast screening of SNO proteins.

Keywords::

• S-nitrosation
• S-nitrosothiol (SNO)
• spectral counting
• irreversible biotinylation procedures (IBP)
• quantification

Acknowledgment

We are very grateful to Dr. Sarah Perrett for English editing of this manuscript. We also thank Peng Xue, Zhensheng Xie, Peng Wu and Fuquan Yang in our institutefor their technical assistance of mass spectrometry.

Declarations of interest: The authors report no conflicts of interest. The authors alone are responsible for the content and writing of the paper. This work was supported by the ‘973 Program’ (2011CB910900, 2011CB503900, 2012CB911000) and the National Natural Sciences Foundation of China (31030023).