S-Nitrosylation Service

What is S-Nitrosylation?

S-nitrosylation is a reversible post-translational modification of a cysteine residue, which is mediated by nitric oxide (NO). To date, more than 3000 proteins have been identified in cell as being S-nitrosylated[1]. As an important post-translational modification, S-nitrosylation modification can change the conformation, activity and interaction of proteins, and thus regulate cell signalling, redox balance, immune response, vascular function and cell cycle. In addition, it has an important role in physiological processes such as vasodilation, nerve conduction, cell growth and division, and also closely affects the occurrence, development and progression of cardiovascular diseases, neurodegenerative diseases, cancer and other diseases[2][3].

Figure 1：The occurrence and effect of S-Nitrosylation.

Mechanism of S-Nitrosylation

In mammalian cells, S-nitrosylation is a spontaneous reaction mediated by NO produced by NO synthases (NOS). The degrees of S-nitrosylation could range from mono-(single cysteine) to poly-S-nitrosylation (multiple cysteines). The initial SNO occurs only at select cysteines that are (i) proximal to NOS, (ii) within an acid/base motif, (iii) within a hydrophobic region, and (iv) in a non-sterically hindered space. These S-nitrosothiol signals can be transferred to distant proteins via transnitrosylation. During transnitrosylation, the charged amino acids in the signature motifs facilitate electrostatic protein-protein interactions. Then, the donor protein (S-nitrosylase)—with higher redox potential—passes the NO-group to the acceptor protein, while getting denitrosylated. About ten S-nitrosylases have, so far, been identified, such as S-nitrosoglutathione (GSNO), hemoglobin, thioredoxin, glyceraldehyde 3-phosphate dehydrogenase (GAPDH), caspase-3, and cyclin-dependent kinase 5 (CDK5). In contrast, denitrosylation is a process that reverts S-nitrosylation through enzymatic reactions. The balance between S-nitrosylation and denitrosylation determines the overall degree of S-nitrosylation. Two denitrosylases have, so far, been well characterized: thioredoxin/thioredoxin reductase (Trx/TrxR) and glutathione/S-nitrosoglutathione reductase (GSH/GSNOR)^[1][3].

Our S-Nitrosylation Service

S-nitrosylation can impart such diverse effects on target proteins, determining the precise modification sites and functional impacts of individual cysteine S-nitrosation events in order to gain a comprehensive understanding of the role of this modification in biological processes. Creative Proteomics has established a highly sensitive LC-MS/MS pipeline that can analyze S-nitrosylated cysteines in both eukaryotic and prokaryotic organisms. Furthermore, we have optimized our protocol, to facilitate more rapid and sensitive site mapping service for S-nitrosylated cysteines. We rely on Thermo Fisher Easy-nLC 1000 and Thermo Fisher LTQ Obitrap ETD to provide S-nitrosylated proteins analysis service packages. We will take care of all aspects of the project, including protein extraction, biotin labeling, proteolysis, peptide enrichment, mass spectrometry analysis, raw mass spectrometry data analysis, and bioinformatics analysis.

Workflow of Our S-Nitrosylation Analysis Service

Classically, the workflow of our S-nitrosylation includes protein extraction, Biotin-Switch Assay and enrichment of S-nitrosylated peptides, identification of S-nitrosylation sites, and relative quantification of changes for identified S-nitrosylation sites. Additionally, the application of MS analysis can be complemented by incorporating TMT labeling techniques to enhance the precision of relative quantitation between samples.

Figure 2: Workflow of our S-Nitrosylation analysis service.

• Sample lysis and protein extraction.
• All free sulfhydryl group are blocked.
• The S-nitrosylated cysteine are reduced by ascorbic acid.
• The free sulfhydryl group are switched by biotin.
• Protein digestion and biotinized peptide enrichment
• Biotinized peptide were detected by LC-MS/MS.

Technological superiority

• Professional detection and analysis capability: Experienced PTM research team, strict quality control system, together with ultra-high resolution detection system and professional data pre-processing and analysis capability, ensure reliable and accurate data.
• Reproducible: Obtain consistent and reproducible inter- and intra- assay results for data analysis.
• Multiplex, high-throughput: Deeper Coverage of S-nitrosylation Site Identification.
• High resolution and sensitivity: Q-Exactive, Q-Exactive HF, Orbitrap Fusion™ Tribrid™.

Samples Requirement for S-Nitrosylation Analysis

Note: In order to ensure the test results, please inform the buffer components if you give us proteins,

whether it contains thiourea, SDS, or strong ion salts. In addition, the sample should not contain components such as nucleic acids, lipids, and polysaccharides, which will affect the separation effect.

Results Delivery

1. Detailed report, including experiment procedures, parameters, etc.

2. Raw data and data analysis results.

How to place an order:

At Creative Proteomics, many excellent and experienced experts will optimize the experimental protocol according to your requirement and guarantee the high-quality results for protein acetylation. Creative Proteomics provides a broad range of technologies for S-Nitrosylation research that enable quantification of protein amount and S-Nitrosylation modification. Please feel free to contact us by email to discuss your specific needs. Our customer service representatives are available 24 hours a day, from Monday to Sunday.

References

1. Sharma V.; et al. S-Nitrosylation in tumor microenvironment. International Journal of Molecular Sciences. 2021; 22(9):4600.
2. Wu X.; et al. Targeting protein modifications in metabolic diseases: molecular mechanisms and targeted therapies. Signal Transduct Target Ther. 2023 May 27;8(1):220.
3. Falco JA.; et al. Identification of protein targets of S-Nitroso-Coenzyme A-Mediated S-Nitrosation using chemoproteomics. ACS Chem Biol. 2024 Jan 19;19(1):193-207.

S‐Nitrosoglutathione Reductase Deficiency Causes Aberrant Placental S‐Nitrosylation and Preeclampsia

Journal: Journal of the American Heart Association

Published: 2022

Main Technology: Botion-HDBP；TMT(Tandem Mass Tag)

Background

Preeclampsia, a leading cause of maternal and fetal mortality and morbidity, is characterized by an increase in S‐nitrosylated proteins and reactive oxygen species, suggesting a pathophysiologic role for dysregulation in nitrosylation and nitrosative stress.

The identification results of S-Nitrosation

This analysis revealed a marked increased number of S‐nitrosylated residues in GSNOR−⁄− placentas (459 corresponding to 351 proteins) compared with controls (264 S‐nitrosylated residues corresponding to 198 proteins) (Figure 1A through 1C). Importantly, placentas from ascorbate‐treated mice exhibited an increased net number of S‐nitrosylated proteins in both control and GSNOR−⁄− placentas (consistent with the transnitrosylation properties of chronic ascorbate ), but this increase was less in the GSNOR−⁄− mice (Figure 1D) decreasing the difference in number of S‐nitrosylated proteins between the 2 groups. Furthermore, we examined subcellular compartmentalization of the total proteins detected in the 4 groups. Similar to our previous study, we found majority of the nitrosylated proteins were located in the cytoplasm and the nucleus (Figure 1E). To gain insights into the most important signaling pathways affected by excess S‐nitrosylation, we examined the subset of S‐nitrosylated proteins found exclusively in GSNOR versus B6, and which exhibited denitrosylation with ascorbate. Of all the detected peptides, there were S‐nitrosylation residues unique to the GSNOR−⁄− placentas, but only 16 residues unique to the B6 placentas (Figures 1C and 1F). All 50 S‐nitrosylation residues were reversed by ascorbate. From these 50 proteins, 14 have been linked to important roles in processes essential in pregnancy, including angiogenesis, inflammation, cell migration, and apoptosis (Figure 1F), supporting the pathophysiological relevance of these proteins.

Conclusions

Therefore, deficiency of GSNOR creates dysregulation of placental S‐nitrosylation and preeclampsia in mice, which can be rescued by ascorbate. Coupled with similar findings in human placentas, these findings offer valuable insights and therapeutic implications for preeclampsia.

Figure 1:Duel‐labeling mass spectrometry revealed an increased number of SNOylated proteins in the placentas from GSNOR−⁄− animals.