Peptide mapping analysis

  • Confirm the amino acid sequence of proteins
  • Comply with ICH Q6B Guidelines
  • Search for specific post-translational modifications and amino acid substitutions

Do you want to verify the sequence of your protein or antibody?

Peptide mapping using mass spec is one of the most valuable methods for verifying the amino acid sequence of proteins. The analysis is an important part of the characterization needed for biologics.

For most proteins you can obtain 100% sequence coverage by careful selection of proteases for the digest. The analysis can also provide detailed information about po...

Do you want to verify the sequence of your protein or antibody?

Peptide mapping using mass spec is one of the most valuable methods for verifying the amino acid sequence of proteins. The analysis is an important part of the characterization needed for biologics.

For most proteins you can obtain 100% sequence coverage by careful selection of proteases for the digest. The analysis can also provide detailed information about post-translational modifications including deamidation and oxidation.

A standard analysis includes

  • Digestion with trypsin cleaving after Arg and Lys residues
  • Analysis of the resulting peptide mixture by nano LC-MS/MS
  • Peptide map of observed peptides to confirm the expected amino acid sequence
  • NB: Only in-gel samples can be submitted for online order

Please send us a request if your samples are not in-gel or if you are interested in the optional services below:

Optional services

  • Multiple protease digestions e.g. Lys-C, Glu-C, chymotrypsin, Asp-N, Arg-C
  • Custom choice of both LC gradient and column
  • Search for specific post-translational modifications and amino acid substitutions
MoreLess details

The process:

  • 1 Order the standard analysis or contact us for a custom project proposal
  • 2 Analysis phase lead by Alphalyse principal investigator
  • 3 You receive a report with a clear presentation of the findings
  • 4 Follow-up by phone or email

More information:

See an example of how results are presented

Get sample report Peptide mapping analysis of mAb

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Knowledge center

More information

We would like to help you as much as possible with your project and therefore provide several kinds of customer support:

Technical details

Introduction to peptide mapping analysis

In Mass Spectrometric Peptide Mapping analysis, we cleave the protein/antibody into smaller peptides using a specific protease. The peptides are analyzed by mass spec and then the observed peptides correlated to the protein/antibody amino acid sequence.

The identified peptides in the peptide map thus confirms the specific amino acid sequences covered by the peptide map, as well as the identity of the protein/antibody. Peptide mapping using a single protease typically gives a sequence coverage of 30-90%. By using two or more proteases this coverage increases to approx. 95-100%.

Detailed characterization of a protein/antibody requires careful investigation of the protein/antibody sequence to select the best proteases, and choice of nano-flow LC-MS/MS or standard flow UV LC MS/MS to observe the peptides.

The difference between the two techniques relates to the amount of material needed for the analysis. Nano-flow can thus be conducted on low microgram protein/antibody whereas the high-flow analysis requires at least 20 microgram per analysis. The high-flow analysis also include a UV-trace of the chromatogram.

Peptide mapping analysis of mAb

Peptide mapping analysis of mAb

Nano-flow LC-MS/MS

We perform the peptide mapping using nanoLC-MS/MS on a Dionex 3000 mass spec system running at 300 nl/min equipped with C18 capillary column connected to a high-resolution Q-TOF instrument (Bruker Impact HD). The instrument runs either with a short or long gradient, and the Q-TOF set to data dependent analysis mode (DDA), switching between MS and MS/MS mode.

The analysis provides a Base Peak Chromatogram, and collects all MS/MS data for peptides with mass/charge from 200 to 1600 Da.

High-flow LC-MS/MS with UV

The peptide mapping using high-flow LC-MS/MS is performed on a Agilent 1200 system running at 0.2 ml/min equipped with C18 column connected to a high-resolution Q-TOF instrument (Bruker Impact HD). The instrument runs either with a short or long gradient, and the Q-TOF set to data dependent analysis mode (DDA), switching between MS and MS/MS mode.

The analysis provides both a UV- and a Base peak chromatogram, and all MS/MS data is collected for peptides with m/z from 200 to 1600 Da.

 

Peptide mapping analysis of mAb

Protein sequence coverage map

Sample preparation

Sample requirements for peptide mapping

Protein/Antibody peptide mapping analysis by mass spectrometry requires a pure protein/antibody in sufficient amounts to obtain good data. It is thus important to prepare samples in a clean laboratory to avoid contamination with human keratin.

NB: Only in-gel samples can be submitted for online order. Please send us a request if you would like to submit in-solution samples instead.

Minimum sample amount: ~ 20-50 microgram protein or ~ 0.5 milligram antibody.

Use Eppendorf Safe Lock tubes, or similar high-quality tubes from your lab.

Related services

Meet the experts

For this type of analysis our experts include:

Do you need help?
Jakob Bunkenborg

PhD in Analytical Chemistry

Read more
Do you need help?
Fen Yang Reske-Nielsen

PhD in Protein Chemistry

Read more
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