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Disulfide Bond Analysis

Correct disulfide bond linkage is essential for the appropriately folded structure and the biological function of a protein. Gemini Bio provides two types of services for characterization of protein disulfide bond linkages i.e. disulfide bond mapping, which is to determine the number and the sites of disulfide bonds in a protein and free thiol assay, which uses a colormetric assay to determine the overall ratio of free thiol in a protein sample.


Based on the availability and amount of a protein sample as well the client's requirements, we offer two different types of disulfide bond mapping service: i.e. analytical disulfide mapping and micro disulfide mapping.

The analytical disulfide mapping service is often used for the characterization of biopharmaceutical products and substances, as well as for comparability studies of biologics. It requires > 0.5 mg protein for each sample, and the analysis is carried by a LC-MS system with both a UV-Visible detector and an MS detector. The quantitation of free or disulfide linked peptides is based on HPLC chromatography and assignments of those peptides are based on MS and MS/MS spectra.

Our micro peptide mapping  service requires < 100 ug protein per sample. The analysis is carried out on a Nano LC-ESI-MS/MS system without a UV-visible detector.


a. Analytical Disulfide Bond Mapping

Analytical disulfide bond mapping is used to determine the number, the position and ratio of disulfide bond linkages in a protein. In this analysis, a protein sample is split into two fractions, one treated with a cysteine alkylation agent, the other is incubated with DTT to reduce all disulfide bonds completely before reacting with a Cys alkylation agent. Both fractions are digested with 2-3 proteolytic enzymes. The digestion products are then analyzed with an analytical HPLC system with a reverse-phase C18 column. Disulfide bond-linked peptides are determined by our proprietary software based on the specific MS/MS features of the peptide as well as the difference in the chromatogram between non-reduced and reduced sample. The ratio of S-S linked peptides as well as free Cys residues are determined based on the areas of corresponding peaks in the chromatogram.

We report the ratio and each detected linkage of each system residues in the protein in a project report.

Due to the use of analytical HPLC in this analysis, we require > 0.5mg protein with > 90% purity for S-S mapping. The service is used in the characterization of biologics samples at the development stage and the comparability study.

b. Micro Disulfide Bond Mapping

Micro disulfide bond mapping is often used for research stage protein samples that the disulfide linkage is unknown. The amount of a sample is often limited in a 10ug-100ug range, and the purity of the sample might be low. Therefore, it becomes necessary to use NanoLC-MS/MS system that is of more than thousands fold higher sensitivity in detection of disulfide bond linked peptides.

In micro disulfide mapping, a sample is split into 2 fractions, one with Cysteine alkylation only, and one with DTT reduction and alkylation. (Optionally, SDS-PAGE is used to further purify protein of interest.) Each fraction is then digested with 2-3 different proteolytic enzymes and digested samples were analyzed by NanoLC-ESI-MS/MS system. Our proprietary software is used to analysis MS and MS/MS data to specifically identify S-S bonds linked peptides and the determine the amino acid sequences of the linked peptides. The ratio of a peptide that does not form a disulfide bond can be determined semi-quantitatively based on EIC( extracted ion current)  of the peptides.

c. Protein-Free Sulfhydryl Measurement

Protein-free sulfhydryl measurement is based on Ellman’s assay. In this assay, a protein test sample, together with a series of standard L-Cys samples of known concentration,  is reacted with the chemical compound DTNB. The absorption at 412nm is measured for each sample by a UV-Vis spectrometer to generate a standard curve. The level of free thiol in the sample is calculated based on its 412nm reading in reference to the standard curve.

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