Peptide mapping, protein characterization

Although a number of assays and technologies are available to characterize and test protein molecules, such as peptide mapping, protein sequencing, carbohydrate analysis, electrophoresis, ELISA, and mass spectroscopy, they are not as definitive as the methods used for small molecule drugs. Hence, the test for similarity is not as well defined in the case of proteins. However, as... 

C. Saint-Pierre and E. Forest, (2000). Characterization of an anti-Borrelia burgdorferi OspA conformational epitope by limited proteolysis of monoclonal antibody-bound antigen and mass spectrometric peptide mapping. Protein Sci. 9, 1002-1010. 

Once a peptide map is characterized using online MS, the chromatographic profile alone can serve as a routine analytical tool to monitor the protein s primary structure and covalent modifications, and is often used for batch release or stability testing of biopharmaceuticals. However, whenever in-depth characterization of a protein is needed, such as that required for comparability studies or reference material characterization, the peptide map should be coupled with MS to ensure a thorough examination of all peptides in the map. 

Characterization of Purified Proteins Assessing purity, 182, 555 determining size, molecular weight, and presence of subunits, 182, 566 amino acid analysis, 182, 587 limited N-terminal sequence analysis, 182, 602 peptide mapping, 182, 613 analysis for protein modifications and nonprotein cofactors, 182, 626 protein crystallization, 182, 646. 

Hancock, W. S., ed. (1996) New Methods in Peptide Mapping for the Characterization of Proteins, CRC Press, Boca Raton, Florida... 

Cobb and Novotny (7) obtained improved separations using C)8 microcolumns as a method for separating quantities on the order of 4 picomoles of tryptic peptides of phosphorylated and dephosphorylated /3-casein. Figure 4 shows two peaks with different retention times, corresponding to the phosphorylated and dephosphorylated forms of the same peptide. The rest of the peptide map is similar. Using this microcolumn, phosphorylation of a single amino acid on a protein can be detected. The method is reproducible with standard deviations smaller than 2%. Characterization of bovine /3-lg tryptic peptides by RP-HPLC on a Nucleosil Cl 8 column was also reported (123). 

The capillary LC/MS-based approach for peptide mapping performed by Arnott and colleagues features miniaturized sampleloading procedures, which are routinely amenable to small quantities of peptides. The reliable characterization of protein/peptide mixtures in conjunction with the widely used 2-DGE methods offers a powerful fingerprinting approach in the pharmaceutical industry. Low femtomole detection limits (typically <50 femtomole) with a mass accuracy of +0.5Da provide unique advantages for protein identification. Liberal parameters for mass range and unmatched masses are used for the initial protein search, whereas more conservative parameters are used to reduce the number of matches and to improve the confidence in the search. 

Quality control Protein characterization Peptide mapping Change et al., 1997... 

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. 

Peptide mapping has been widely used for characterization of protein products, and is almost always used for confirmation of identity. Generally, peptide mapping is not a very sensitive technique for detection of impurities in our experience, derivatives present at levels below a few percent are not normally detected in peptide maps, whereas HPLC of the intact protein can generally detect 0.1% of such derivatives. However, in relatively simple maps such as the V-8 protease digest of insulin, relatively good limits of detection can be achieved (5). 

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