Accurate S-Nitrosylation Site Mapping Service Using LC-MS/MS and TMT Labeling

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. 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.

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

Mechanism of S-Nitrosylation

S-nitrosylation is a naturally occurring modification where NO attaches to specific cysteine residues in proteins. This reaction is driven by NO generated by nitric oxide synthases (NOS) within mammalian cells. Depending on the number of cysteines involved, S-nitrosylation can occur at a single site (mono-S-nitrosylation) or at multiple sites (poly-S-nitrosylation).

Not all cysteines are equally susceptible. For a cysteine to be S-nitrosylated, it must meet several conditions:

• Be located near NOS
• Reside within a specific acidic or basic motif
• Be found in a hydrophobic region of the protein
• Be physically accessible (i.e., not buried or blocked)

From Local Events to Systemic Effects: Transnitrosylation

Once the initial S-nitrosylation occurs, the NO signal can be passed on to other proteins through transnitrosylation—a process that spreads the modification beyond the original target. This transfer relies on charged amino acid motifs that promote strong electrostatic interactions between the donor (S-nitrosylated protein) and the acceptor protein.

Key players in this cascade include known S-nitrosylases, which donate the NO group:

• S-nitrosoglutathione (GSNO)
• Haemoglobin
• Thioredoxin
• GAPDH
• Caspase-3
• CDK5

These donor proteins have a higher redox potential, allowing them to transfer the NO group and become denitrosylated in the process.

Keeping the Balance: Denitrosylation

The modification is reversible. Denitrosylation removes NO groups from proteins through enzyme-catalysed reactions, maintaining cellular redox homeostasis. Two well-characterized enzymes carry out this function:

• Thioredoxin/thioredoxin reductase (Trx/TrxR)
• Glutathione/S-nitrosoglutathione reductase (GSH/GSNOR)

Together, S-nitrosylation and denitrosylation form a regulatory cycle. Their interplay determines the overall level of protein S-nitrosylation and, consequently, affects various signalling pathways across the cell.

Our S-Nitrosylation Site Mapping Service

S-nitrosylation can dramatically alter protein behaviour, from modulating enzymatic activity to rewiring entire signalling pathways. However, to truly understand its biological role, it's critical to pinpoint exactly where these modifications occur—and what they do. That's where Creative Proteomics comes in.

We've developed a high-sensitivity LC-MS/MS-based workflow designed to identify and characterize S-nitrosylated cysteines in both eukaryotic and prokaryotic samples. Our platform enables researchers to uncover detailed, site-specific insights into this dynamic post-translational modification.

Advancing S-Nitrosylation Detection: From Biotin-Switch to TMT Labeling

Understanding NO signaling requires accurate identification of S-nitrosylated cysteine (SNO-Cys) sites. Over the years, various detection methods have been developed, with the Biotin Switch Technique (BST)—first introduced by Jaffrey in 2001—being one of the most widely used.

BST works by chemically converting SNO modifications into biotin-labeled sites, which are then detected using biotin-specific antibodies. While effective, this method presents several challenges:

• Low throughput, making it unsuitable for large-scale studies
• Limited site resolution, complicating precise localization of modified cysteines
• Risk of false positives, due to interference from endogenous biotin-like molecules during affinity enrichment

To overcome these limitations, we've adopted an advanced TMT labeling strategy. Using 6-plex Tandem Mass Tags (TMT), our approach allows for:

• Simultaneous identification and quantification of S-nitrosylation events
• Improved detection specificity and accuracy
• Scalable, multiplexed analysis across multiple biological samples

This upgraded method enhances both the depth and reliability of SNO-Cys site mapping—critical for uncovering the true complexity of NO-mediated signaling.

How the TMT Labeling Strategy Works

The TMT reagent offers a highly selective way to label cysteine-containing peptides from complex biological samples. It binds irreversibly to free thiol groups on cysteine residues, allowing researchers to confidently map redox-related modifications like S-nitrosylation.

To enrich for these labeled peptides, we use a two-step approach:

• Affinity enrichment using immobilized anti-TMT antibody resin
• Targeted elution with a dedicated TMT elution buffer

This strategy mirrors the concept behind ICAT™ (Isotope-Coded Affinity Tag) techniques, combining selective thiol tagging with high-affinity capture to streamline the workflow.

What Makes TMT Unique?

Each reagent in the iodoTMT-plex kit shares the same parent mass but differs by:

• A Cysteine-Reactive Group for thiol-specific binding
• A Mass Normalizer to ensure accurate quantification
• A Mass Reporter for multiplexed sample analysis

With the ability to label up to six different biological samples in a single run, TMT reagents significantly reduce sample complexity, expand the detectable dynamic range, and provide a robust platform for S-nitrosylation research.

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.
• Raw data interpretation and bioinformatics analysis

Technological Advantages That Set Us Apart

Expert-Led, Data-Driven

Our team of seasoned PTMs specialists ensures every project benefits from:

• Rigorous quality control protocols
• Professional data pre-processing and interpretation
• Ultra-high-resolution mass spectrometry for precise detection

Consistency You Can Trust

We guarantee high intra- and inter-assay reproducibility, providing the confidence you need in downstream data analysis and decision-making.

Multiplex, High-Throughput Capability

Our optimized workflow delivers comprehensive coverage of S-nitrosylation sites—ideal for large-scale proteomics projects or pathway-level investigations.

Cutting-Edge Instrumentation

We utilize industry-leading platforms to enhance sensitivity and resolution, including:

Exactive

Orbitrap Fusion™ Tribrid™

With these advantages, Creative Proteomics offers unmatched precision and depth for your S-nitrosylation studies.

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 & Demo

• Detailed report, including experiment procedures, parameters, etc.
• Raw data and data analysis results.

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Case Study

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

Journal: Journal of the American Heart Association
Published: 2022Main
doi: 10.1161/JAHA.121.024008

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

• Background
• Results
• Conclusions

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.

This analysis revealed a markedly increased number of S-nitrosylated residues in GSNOR⁻/⁻ placentas (459 residues corresponding to 351 proteins) compared with controls (264 residues corresponding to 198 proteins) (Figure 1A–1C).

Notably, placentas from ascorbate-treated mice exhibited a higher net number of S-nitrosylated proteins in both control and GSNOR⁻/⁻ groups—consistent with the known transnitrosylation properties of chronic ascorbate treatment—though the increase was less pronounced in the GSNOR⁻/⁻ group (Figure 1D), resulting in a reduced difference in the number of S-nitrosylated proteins between the two groups.

In line with previous findings, the majority of nitrosylated proteins were localized to the cytoplasm and nucleus across all experimental groups (Figure 1E).

To further identify the most affected signaling pathways under conditions of elevated S-nitrosylation, the researchers focused on S-nitrosylated proteins that were uniquely present in GSNOR⁻/⁻ placentas compared to B6 controls and were reversed upon ascorbate treatment. Of all the detected peptides, several S-nitrosylation residues were specific to GSNOR⁻/⁻ placentas, whereas only 16 were unique to B6 (Figures 1C and 1F). All 50 GSNOR⁻/⁻–specific S-nitrosylation residues were reversed by ascorbate. Among the corresponding proteins, 14 have previously been associated with key biological processes relevant to pregnancy, including angiogenesis, inflammation, cell migration, and apoptosis (Figure 1F), underscoring the physiological significance of these findings.

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

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.

Publication

Here are some publications in PTMs Proteomics research from our clients:

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. DOI: 10.3390/ijms22094600
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. DOI: 10.1038/s41392-023-01439-y
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. DOI: 10.1021/acschembio.3c00654