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Peptide Mapping

Gemini Bio provides a LC-MS/MS based peptide mapping service for the confirmation of protein primary structure. By using multiple proteolytic digestions and MS/MS based peptide sequencing, we guarantee results with complete sequence coverage and highly reliable sequence assignments.




  • For the peptide mapping service, each protein sample is digested with 2 or 3 proteolytic enzymes (depending on the sequence of the protein) and  the resultant peptide mixture is then analyzed by LC-MS/MS. The MS/MS data are then searched by a proprietary software to generate a peptide map. The peptide map from each digest is then assembled together to generate the complete sequence of the protein. In a comparative structural and functional analysis of biopharmaceuticals, HPLC-UV chromatograms are also generated and analyzed both qualitatively and quantitatively and correlated with MS/MS data to assign each chromatographic peak to peptides. Any minor structural differences and the ratio between a test sample and reference will be reported to the client.


  • To achieve complete coverage of an entire protein sequence, especially for  large proteins. It is often necessary to use multiple proteolytic digestions including proteases with non-specific cleavage. However, accurately assigning MS/MS data from non-specific digested protein samples is much more difficult than that from specific digestions (e.g. trypsin). To overcome that difficulty, we have developed a proprietary software platform that can analyse MS/MS data of any non-specific digestion, which enables us to offer our peptide mapping service with a 100% sequence coverage,  a highly reliable MS/MS based peptide assignment and a fast turnaround.


  • Peptide mapping is a standard method to confirm a protein's sequence. It is also used in combination with other techniques in the determination of protein N- and C-termini, in disulfide bond mapping and identification of protein post-translational modifications. It is one of the most important techniques in comparability studies of biologics.


  • We offer two types of peptide mapping services to meet the different requirements of our clients:


  1. Analytical Peptide Mapping and

  2. Micro Peptide Mapping


Analytical Peptide Mapping is suitable for development/process stage samples with high purity and amounts >0.5mg.

Micro peptide mapping is suitable for research/discovery stage samples with amounts less than 100ug and  lower purity. The details of these two procedures are described below.

Analytical Peptide Mapping

  • In analytical peptide mapping, protein samples are digested with multiple enzymes, and trypsin and chymotrypsin are often two default enzymes if the protein for the analysis has a “normal” amino acid sequence. The peptides from each digestion are injected onto a HPLC system with a 4.6mm ID reverse phase column. The eluted peptides are detected by both UV-Visible detector to generate a HPLC profile and an on-line coupled tandem mass spectrometer to generate MS and MS/MS data. MS and MS/MS data are searched by our bioinformatics software to generate peptide sequences. Each sample is analyzed at least twice to ensure a high reproducibility of the data. The HPLC profile and peptide sequence data are then correlated to generate an integrated profile with each HPLC peak assigned to a peptide sequence. Then, the integrated profiles from multiple  digestions are combined to generate a final peptide map that cover 100% of the protein sequence. 

  • In comparative studies, the peptide maps from test samples and reference samples are analyzed and compared. Any detectable difference in HPLC chromatograms is investigated with corresponding MS and MS/MS data. The quantitative measurement and structural characterization of detected differences are reported to the client.

  • The analytical peptide mapping service is conducted following ICH Q6B guidelines for the characterization of the physicochemical properties of biologic products and substances.  We’ll make our best effort to meet other specific requirements of our customers in order to comply with the regulations of other government agencies.

Micro Peptide Mapping

  • Our micro peptide mapping is often used for analyzing research/discovery stage protein samples where the sample amount is often below 100µg and its purity is low. In order to get a complete coverage of protein with low amounts of sample, we use a Nano LC-MS/MS system to achieve ultra-high sensitivity.

  • The analysis process is similar to that described in analytical peptide mapping procedure, with a major difference that a UV-Vis detection is not included. Therefore, the peptide map is generated only based on MS and MS/MS data. Similarly, a 100% sequence coverage is routinely achieved by using multiple enzyme digestions.  A peptide map with 100% sequence coverage and the peptide sequence table based on MS/MS data will be reported  to clients.

  • In the case that quantitative measurement of certain peptides are needed, for example, a protein has heterogeneous N-termini and the ratio of each N-terminal species  needs to be determined,  we will quantify the peptide of interest with EIC (Extracted Ion Chromatogram). We have carried out a parallel study to compare EIC based peptide quantitation with UV-Vis chromatogram-based peptide quantitation in which we found that EIC based quantitation, although less accurate, can provide reliable data for peptide quantitation.



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