Peptide Mapping
The goal of peptide mapping is to obtain detailed information about a protein. This can take the form of protein identity testing, product variability and/or identification, and quantification of modifications. Often this is referred to as "fingerprinting", because the selectivity and specificity of peptide mapping when linked to mass spec is sensitive enough for characterization of single amino acid differences. Peptides, for mapping, can be generated from full length proteins by both chemical and enzymatic means. The four major steps in peptide mapping are: isolation and purification of protein, selective cleavage of peptide bonds, separation of peptides, followed by detection, and analysis.
Selection of the cleavage type, separation parameters and mass spec method is developed for each sample type and often compared to a standard processed under identical conditions. Commonly, a protein is digested in a buffered solution using a proteolytic enzyme and the peptides are separated using reversed phase HPLC under acidic conditions with detection by UV in tandem with an electrospray mass spectrometer.
Tryptic peptide map of apo-transferrin
Sequence analysis by MSMS of K233-R245 peptide
Peptide maps have been used to gain insight into protein structure, provide information about specific protein domains and their interactions as well as tracking cell culture and manufacturing processes. Post-translational modifications, glycan structure identification and disulfide connectivity are frequently determined through peptide mapping. Protein pharmaceutical companies will employ peptide mapping for monitoring pharmacokinetics and pharmacology (ADME).
The key factors in peptide mapping are robustness and reproducibility. A validated peptide map will provide sufficient specificity to establish consistency and stability of each protein's unique characteristics. Because of the unique nature of each protein and each projects goals other approaches may be employed such as in-gel digestion of proteins and MALDI-TOF analysis of the peptides.
The staff at Analytical Ventura is highly skilled in these techniques and happy to work with our clients to develop just the right approach to suit their needs.
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