Free Thiol Proteomics Service
Cysteine residues in proteins contain highly nucleophilic sulfur atoms, making them among the most reactive amino acids. As important targets for protein post-translational modifications (PTMs), the thiol groups (-SH) of cysteines not only participate in enzymatic catalysis but also serve as key mediators in cellular redox signaling and regulation.
Figure 1. Summary of the oxidative modifications formed in protein thiols. (Source: Rudyk O, et al. 2014)
Under the action of reactive oxygen species (ROS), the free thiols in proteins can undergo a variety of reversible oxidative modifications, including:
- Disulfide bond formation (S-S) ——stabilizing protein structure
- S-glutathionylation (SSG) ——regulating protein function
- S-nitrosylation (SNO) ——participating in signal transduction
- S-sulfinylation (SOH) ——modulating enzyme activity
- S-acylation ——controlling protein localization
These modifications possess a unique reversibility advantage: through the action of appropriate reducing enzymes or reducing agents, oxidized cysteines can be reduced back to their free thiol state. This dynamic "switch" mechanism allows proteins to flexibly adjust their conformation, activity, and subcellular localization, thereby precisely controlling gene transcription, metabolic processes, signaling pathways, apoptosis, and protein transport—core physiological processes essential for maintaining cellular homeostasis.
Our Services
We conduct systematic proteomics research on free thiol modifications in protein samples using liquid chromatography-tandem high-resolution mass spectrometry (LC-MS/MS) technology. Through dynamic thiol labeling and enrichment techniques, we can precisely identify all thiol modification sites on proteins and generate modification heatmaps to reveal modification characteristic differences between samples. Simultaneously, we employ Label-free relative quantification, TMT/iTRAQ multi-sample labeled quantification, and other quantitative strategies, supporting quantitative comparative analysis of time-series and multi-treatment groups, with absolute quantification options available. Based on this foundation, we simultaneously detect the ratio of oxidized and reduced state cysteines, assess cellular redox homeostasis, and uncover modification changes related to disease, metabolism, and signaling pathways. Finally, through integration with multi-level data such as transcriptomics and metabolomics, we utilize GO/KEGG enrichment analysis to reveal the biological functions of modified proteins and conduct deep mechanistic analysis based on biological pathway databases.
Service Advantages
- High Specificity and Sensitivity
We employ targeted enrichment strategies that effectively capture modified peptides from complex protein mixtures, particularly those with low abundance modification sites, ensuring comprehensive and accurate detection coverage. - Comprehensive Modification Coverage
High-resolution mass spectrometry platforms combined with multi-enzymatic digestion strategies enable discovery and localization of numerous modification sites, providing you with systematic and complete modification maps for your research. - Advanced Data Analysis
We utilize industry-standard data processing software for analysis with strict quality control systems ensuring data accuracy and reliability, while providing personalized data interpretation through independently developed bioinformatics pipelines. - Professional Team and Complete Service
Our team consists of experienced proteomics specialists who strictly adhere to international quality standards throughout the entire process from sample preparation to data delivery, providing comprehensive data reports, result interpretation, and technical support for subsequent data mining and scientific paper writing.
Workflow
We first receive the sample and assess its integrity, protein concentration, and purity. Subsequently, protein quantification is performed using protein quantification reagents to ensure consistency in subsequent experiments. Finally, reduction and alkylation treatment is conducted to protect active thiols in the sample and prevent non-specific modifications during sample preparation.
Free thiols in the sample are specifically labeled using biotin or other labeling reagents. Multi-enzymatic digestion strategies are then employed to obtain peptide fragments of appropriate length, optimizing modification site coverage. Peptides are subsequently purified and desalted through affinity chromatography and solid-phase extraction to remove buffer salts and interfering substances. Finally, gradient elution is performed using liquid chromatography systems, and high-resolution mass spectrometry detection is applied to obtain parent ion and fragment ion spectra.
Multiple search engines are used for combined analysis to determine modification sites and modification types. Relative or absolute quantitative analysis is performed based on peptide abundance or reporter ion intensity. Statistical processing including multi-dimensional analysis, principal component analysis, and differential modification site screening is conducted. GO/KEGG databases are utilized for functional enrichment, pathway analysis, and function prediction. Finally, results are presented through visualization in various forms including modification heatmaps, volcano plots, and network diagrams.
Complete raw data and quality-controlled processed data are provided. Comprehensive analysis reports and figure interpretation documents are delivered. Technical support for subsequent data mining and scientific paper writing is offered.
If you have any questions about our free thiol proteomics service or need to develop a customized experimental plan based on your research requirements, please feel free to contact our technical team.
Reference
- Rudyk O, et al. Biochemical methods for monitoring protein thiol redox states in biological systems. Redox Biol. 2014 Jun 13;2:803-13.
