Protein Characterization-ProtTech Inc.

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Disulfide Bond Analysis

Correct disulfide bond linkage is essential for the correctly folded protein 3D structure and the biological function of protein. ProtTech provides two types of service for characterization of protein disulfide bond linkage: disulfide bond linkage analysis, which is to determining the number and the sites of all disulfide bonds in protein, and free thiol assay, which uses a colorimetric assay to determine the overall ratio of free thiol in a protein sample. With the cutting edge technology we have developed for analyzing protein disulfide bonds, we can analyze complicated disulfide bonds linkage including closely spaced disulfide bonds and complicated mispaired disulfide bonds.



  1. 1. Analyzing unknown S-S bond linkage in novel proteins.

  2. 2. Analyzing disulfide bond linkage in refolded proteins for predict whether a protein is correctly folded.

  3. 3. Confirmation of correct disulfide bond linkage in protein therapeutics.


In disulfide bond linkage analysis, we have developed different procedures for analyzing different types of linkages: normal disulfide linkage, closely spaced disulfide linkage and mismatched disulfide bond linkage.

Determining normal disulfide linkages is mainly based on differential LC-MS and LC-MS/MS analysis of non-reduced and reduced samples. We routinely achieve >3X coverage for disulfide bond linkage peptides to ensure accurate and complete analysis.

For closely spaced disulfide bond linkage, we have developed the technique to carry out a stepwise cystine reduction and differential cysteine alkylation to separate two or more overlapping disulfide bonds.

For mispaired disulfide bonds in which one Cys residue may involve in several different linkages, we have developed a method to quantitatively analyze the ratio of different linkages for each Cys residue.


Our proprietary S2Map software is capable of detecting all those different types of disulfide bond linkages in a protein and can detect low level mismatched disulfide bond linkage which is often a difficult and time-consuming task even for an experienced scientist.

Protein free-sulfhydryl measurement (based on Ellman’s assay). In this assay, a protein test sample, together with a serial of standard L-Cys samples of known concentration, reacts with 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.

Service Terms

Sample Requirement: We typical need 50 ug of protein with >80% purity for a disulfide bond linkage analysis.  More samples may be needed to analyze samples with closely spaced disulfide bonds or mismatched disulfide bonds. Protein free-sulfhydryl measurement typically requires 0.5mg for each sample.

Turnaround Time: Normally 2-5 weeks depending on the difficulty of a project.

Price:  Price of a disulfide bond linkage analysis service will be project- dependent.  We’ll work out an optimal procedure for each project based on that information. Please send us your sample information and we’ll send you a formal quote within 12 hours.  Also please contact us if you need sign confidentiality agreement with us before sending out your sample information.