Disulfide bonds, peptide mapping

Once the structural features of a reference standard of the desired protein have been well characterized, lot-to-lot confirmation of identity can be conducted using a carefully selected group of tests, wherein the lot undergoing analysis is compared to the reference standard. Tests commonly employed for this purpose are listed in Table III. Peptide mapping is perhaps the most powerful and universally used technique since it provides relatively specific confirmation of correct primary sequence and, when non-reducing conditions are employed, can be used to confirm correct disulfide bond formation. Tertiary structure is difficult to address directly on a routine (lot-to-lot) basis, and the presence of correct biological activity is often used as evidence that the correct tertiary structure is maintained. 

Deamidation, Oxidation, Disulfide bond disruption Peptide mapping... 

Comparative Peptide Mapping This procedure is used when the protein is cleaved with specific cleaving agents and the sequence of the protein is known. A simple molecular mass measurement of the peptides in a protein digest will identify the position of a disulfide bond in the protein. 

Fukuyama, Y., Iwamoto, S., and Tanaka. K. (2005) Rapid sequencing and disulfide mapping of peptides containing disulfide bonds by using... 

Peptide map analysis of a protein containing two or more cysteine residues typically employs reduction and alkylation chemistry for efficient, reliable proteolysis, reproducible chromatographic profiles, and straightforward characterization by MS. However, when protease digestion is carried out on a nonreduced protein, the disulfide bonds in the protein will maintain the covalent linkage between the peptides that are involved in the disulfide bond. In many cases, it is then possible to choose a protease that will ensure one cysteine residue in each peptide upon cleavage, such that each disulfide bond will associate with a pair of proteolytic peptides. 

Trisulfide bonds, originally elucidated in human growth hormone," " were detected recently in mAbs at low levels.The linkage between light and heavy chain is the predominant location of trisulfide bonds in mAbs. Ironically, nonreducible thioether bonds were detected previously at this same disulfide bond linkage in mAbs via peptide map analysis." " ... 

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