Disulfide Linkage

Is your protein behaving as expected?

There are several things you need to know about a protein’s structure in order to be satisfied that it will perform to expectations. The number and location of disulfide linkages – often needed to ensure correct folding and function – is one of them.

It could be that you’re about to undertake exploratory research in order to identify previously unknown linkages. Perhaps they’re not available in the literature or other prior research.

Or you might be embarking on a confirmatory study to verify and identify linkages you expect to be there. For example, you could use the information obtained to guide the digestion strategy for peptide mapping analysis. In this case, possible linkages are probably readily available in the literature.

We offer a choice of two different methods of disulfide linkage analysis, one of which will better suit your project needs: mass spectrometry alone, or a combination of liquid chromatography separation with mass spectrometry.

Our strength in this area is that we develop tailored strategies of analysis and in-house algorithms in conjunction with our clients. We view our clients as partners, and together, we develop innovative techniques to analyze disulfide linkages that don’t solely rely on traditional methods.

Note that disulfide linkage analysis is a key part of ICH Q6B.

Technical information

Disulfide linkages are an important structural feature of many proteins. Intra-chain bonds form or stabilize the tertiary structure of a protein (for example, the zinc-binding domain of zinc-finger proteins) and inter-chain disulfide-bonds covalently link protein subunits together (for example, the insulin a- and b-chain).

The process for identifying disulfide linkages includes:

• Investigating the differential appearance of the linked peptides before and after reduction of the disulfide bond, and
• Analyzing the fragment spectra of the linked peptides using mass spectrometry

Peptide mapping may be helpful to confirm the primary structure and aid in determining the most appropriate enzymatic digestion strategy for the disulfide-bridge analysis.

N- and C-terminal sequencing using MALDI-ISD can be used to determine the presence of free sulfhydryl groups or disulfide linkages within these regions.

Finally, electrophoresis, such as reducing and non-reducing SDS-PAGE to identify disulfide-linked subunits of a protein, may also be undertaken.

Your contact for Disulfide Linkages at Protagen AG

Dr Katja Aschermann Head of Business Development Protein Services. Her team will be happy to assist you with your project. Phone: +49 (0) 231 9742-6300 Email: salesproteinservices@protagen.de