Introduction
A disulfide bond is a covalent bond formed between the sulfur atoms of two cysteines and is a common
post-translational modification in proteins. Their presence ensures that the protein folds correctly, remains
stable, maintains normal function, etc. As a critical quality attribute of proteins, the analysis of disulfides is
critical for the development and identification of biotherapeutics.
Analytical methods to characterize the integrity of protein disulfide bond patterns have continued to evolve and
today range from classical electrophoretic methods such as SDS-PAGE and CE, absorbance spectroscopy for
thiol-reactive labeling and Edman sequencing, to state-of-the-art MS analysis. Notably, mass spectrometry is the
most prominent tool for protein disulfide bond analysis today. Most LC-MS/MS methods for protein sample analysis are
based on bottom-up approaches. Briefly, the technique requires proteolytic digestion of relevant proteins, followed
by identification based on the quality of the resulting peptides, often relying on database comparisons. Bottom-up
MS is the most widely used method for disulfide bond analysis compared to the limitations of other methods, such as
high-purity sample requirements and low throughput.
Fig. 1. Disulfide bond analysis workflow.
(Lakbub J C, et al., 2018)
Our Services
Creative Proteomics has extensive experience in protein characterization by mass spectrometry,
especially protein sequence analysis. At Creative Proteomics, our scientists have introduced a
comprehensive protein disulfide analysis solution to help you with your research, including normal, closely spaced,
and mismatched disulfide bonds analysis, etc.
According to ICH Q6B guidelines, the biotherapeutics should determine the number and location of any free sulfhydryl
and/or disulfide bonds whenever possible. Peptide mapping (under reducing and non-reducing conditions), mass
spectrometry, or other appropriate techniques may be useful for this assessment. We introduce a highly sensitive
HPLC-MS/MS platform for this purpose to analysis disulfide bonds in multiple samples from different sources.
Here, we provide one-stop disulfide bond analysis services, including sample preparation, protein purification,
enzymatic hydrolysis, LC-MS/MS identification and bioinformatics analysis.
Simple disulfide bond analysis workflow:
- Sample preparation: We have developed well-established sample handling techniques, such as alkylation of free
thiol side chains in the native protein state by adding reagents such as IAM, IAA or NEM, avoiding the formation
of artificial disulfides during subsequent proteolytic digestion bond, and maximizing the preservation of native
protein structure.
- Enzymatic digestion: Specific enzymes, usually trypsin, pepsin, and Glu-C are used to form abundant peptides.
- LC-MS/MS identification: We use LC-MS/MS to accurately detect all peptides of a protein and identify multiple
post-translational modifications of these peptides and map disulfide bonds.
- Bioinformatics analysis: Disulfide linkage and free sulfur analysis are achieved using different software such
as pLink-SS and SlinkS, and bioinformatics analysis will be performed.
- Detailed report: A report you received containing experimental procedures, MS raw data files, liquid
chromatography and mass spectrometer parameters, disulfide bond and cysteine analysis results, and
bioinformatics analysis.
What Can We Offer You?
Our laboratory technicians have extensive technical experience in elucidating disulfide bond structures. Our featured
services include but not limited to:
- Analysis of unknown disulfide linkages in proteins.
- Verify that the disulfide bonds in the protein are mismatched.
- Predict and analyze whether proteins are folded correctly.
- Identifying proteins, and providing disulfide bond mapping.
- Bioinformatics analysis.
Advantages of Our Services
Here, we provide a comprehensive service for disulfide bond analysis according to ICH Q6B guidelines, our service
features are:
- Analyze proteins at the individual protein level and at the proteome level.
- High-throughput, quantitative identification and analysis of disulfide bonds in multiple proteins.
- Maintain and analyze proteins in their native state.
Applications
- Map the disulfide bond sites of unknown proteins.
- Verify disulfide bonds and folding of protein samples.
Thanks to our powerful mass spectrometry sequencing platform, Creative Proteomics provides a
one-stop shop for complex disulfide bonds. Our experienced scientists work with you to develop tailor-made
analytical solutions. All you need to do is send us your samples, communicate your research goals with our experts,
and we'll go through the next steps and provide you with a full technical report. Please feel free to contact us with any questions regarding disulfide bond analysis.
References
- Lakbub J C, Shipman J T, Desaire H. (2018) Recent mass spectrometry-based techniques and considerations for
disulfide bond characterization in proteins. Anal Bioanal Chem. 410(10):2467-2484.
- Daniel W. (2018) Analysis of disulfide bond formation in therapeutic proteins[D]. Chemical Biology.
(4)1:81-98.
