ESNOQ, proteomic quantification of endogenous S-nitrosation

Area of Science:

• Biochemistry
• Proteomics
• Cellular Signaling

Background:

• S-nitrosation is a critical post-translational modification regulating nitric oxide (NO) signaling pathways.
• Accurate quantification of endogenous S-nitrosothiols (SNOs) is essential for understanding NO's functional roles but remains technically challenging.

Purpose of the Study:

• To develop and validate a novel method for quantifying endogenous S-nitrosation events in proteins.
• To identify and quantify S-nitrosated proteins in LPS/IFN-gamma induced RAW264.7 cells.

Main Methods:

• The Endogenous SNO Quantification (ESNOQ) method was developed, integrating stable isotope labeling by amino acids in cell culture (SILAC) with a detergent-free biotin-switch assay.
• Liquid chromatography-tandem mass spectrometry (LC-MS/MS) was employed for high-sensitivity detection and quantification of S-nitrosated peptides.

Main Results:

• The ESNOQ method demonstrated accurate quantification of endogenous S-nitrosation.
• An endogenous S-nitrosation proteome was generated, identifying 27 S-nitrosated protein targets and over 15 additional targets compared to previous studies.
• Quantitative analysis revealed site-specific S-nitrosation on cysteine residues within proteins and enrichment of targets in protein translation and glycolysis pathways.

Conclusions:

• The ESNOQ method provides a robust solution for quantifying multiple endogenous S-nitrosation events.
• This advancement enables detailed functional studies and elucidation of the complex network of S-nitrosation in cellular processes.
• Findings suggest S-nitrosation plays a significant role in regulating key cellular pathways like protein translation and glycolysis.