Post-translational Modification (PTM) Analysis by Peptide Mapping (CAT#: STEM-B-0014-ZJF)

Peptide mapping is a method for the identification of proteins, especially those obtained by rDNA technology. It involves chemical or enzymatic processing of proteins to form peptide fragments, which are then isolated and identified in a repeatable manner. This is a powerful test that is able to identify changes in almost any single amino acid that are caused by events such as a misread of a complementary DNA (cDNA) sequence or a point mutation. Peptide mapping is a comparison procedure because the information obtained confirms the primary structure of the protein, enables detection of whether the structure has changed, and demonstrates the consistency and genetic stability of the process when compared to reference substances of similar treatment. Each protein has unique characteristics that must be fully understood so that scientific and analytical methods allow the development of validated peptide maps that provide sufficient specificity.

Proteins, Biopharmaceutics

1. Isolation and purification: This procedure is necessary for analysis of bulk drugs or dosage forms containing interfering excipients and carrier proteins.
2. Selective cleavage of peptide bonds: The method chosen for the cleavage of peptide bond depends on the protein being tested. This selection process involves determination of the type of cleavage to be employed, enzymatic or chemical, and the type of cleavage agent within the chosen category.
3. Chromatographic separation: Many techniques are used to separate peptides for mapping. The selection of a technique depends on the protein being mapped. Techniques used for the separation of
peptides: Reversed-phase high performance liquid chromatography (HPLC), Ion-exchange chromatography (IEC), Hydrophobic interaction chromatography (HIC), Polyacrylamide gel electrophoresis (PAGE), Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE), Capillary electrophoresis (CE), Paper chromatography-high voltage (PCHV), and High voltage-paper electrophoresis (HVPE).
4. Analysis and identification of peptides: The use of peptide maps as a qualitative tool does not require complete characterization of individual peptide peaks. Validation of peptide mapping in support of regulatory applications requires rigorous characterisation of each of the individual peaks in the peptide map.

• Chromatographic apparatus
• Mass spectrometry apparatus
• Sample material