Peptide mapping services

  • Confirm the amino acid sequence of proteins and antibodies
  • Quantify PTMs
  • Check for amino acid substitutions and identify degradation products

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

Peptide mapping using mass spectrometry is one of the most valuable methods for verifying the amino acid sequence of proteins. Therefore, the analysis is an integral part of the protein characterization needed for biologics, as stated in ICH Q6B guidelines.

You can apply peptide mapping to confirm protein identity for lot release and to analyze product-related...

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

Peptide mapping using mass spectrometry is one of the most valuable methods for verifying the amino acid sequence of proteins. Therefore, the analysis is an integral part of the protein characterization needed for biologics, as stated in ICH Q6B guidelines.

You can apply peptide mapping to confirm protein identity for lot release and to analyze product-related impurities with stability and forced degradation studies.

Careful selection of proteases for the digest can optimize the sequence coverage. Using our peptide mapping services, you will also get detailed information about post-translational modifications – including deamidations and oxidations.

Service 1: Peptide Mapping

  • Digestion with trypsin cleaving after Arg and Lys residues.
  • Analysis of the resulting peptide mixture by single injection UV-LC-MS/MS
  • Peptide map of observed peptides against the theoretical sequence
  • Results within two weeks

Service 2: Quantitative Peptide Mapping

  • Digestion with suitable proteases based on bioinformatics analysis of the protein sequence
  • Analysis of the resulting peptide mixture by triplicate injection UV-LC-MS/MS
  • Peptide map of observed peptides against the theoretical sequence
  • Quantification of post-translational modifications
  • Results within four weeks

Common modifications we look for include Asn/Gln deamidation, N-term pyroglu, C-terminal Lys truncation, and M/W oxidation.

Service add-ons:

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

The process:

  • 1 Contact us to discuss the best possible solution and get project proposal
  • 2 Analysis phase led by Alphalyse principal investigator
  • 3 You receive a report with a clear presentation of the findings
  • 4 Follow-up by phone or email

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We offer customized solutions, contact us to discuss your project.

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Testimonials

  • "Not only do we gain access to their hands, but we also get to pick their brains for mass spectrometry knowledge."

    Protein characterization for optimization of manufacturing processes.

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  • "With the results from the extensive characterization, we identified a scale-up problem and could optimize the process to increase mAb stability."

    Combining mass spec analyses for mAb characterization and batch-to-batch comparison

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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

We cleave the protein/antibody into smaller peptides using a specific protease in our Mass Spectrometric Peptide Mapping services. The peptides are analyzed by mass spec, and then the observed peptides are correlated to the protein/antibody amino acid sequence for peptide identification.

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

Detailed characterization of a protein/antibody requires careful investigation of the protein/antibody sequence to select the best proteases by mass spec. We use UV LC-MS/MS to observe the peptides.

Peptide mapping analysis of mAb

Peptide mapping analysis of mAb

High-flow mass spec with UV

The peptide mapping using high-flow LC-MS/MS is performed on an Agilent 1200 system running at 0.2 ml/min equipped with a 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 is 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

Antibody sequence coverage map

Sample preparation

Sample requirements for peptide mapping analysis

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

We suggest you provide samples with a minimum of 80% chromatographic purity. Let us know if your samples have glycerol, detergents, or high salt content.

Provide samples in solid form or in solution. Reach out if you have gel bands you would like to analyze.

Minimum sample amount: ~ 100 microgram protein and ~ 0.5 milligram antibody.

Related services

You can combine this analysis with other analyses such as:

Meet the experts

For this type of analysis our experts include:

Do you need help?
Li Peng Lundgren

PhD in Protein Chemistry

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Do you need help?
Stine Thyssen

PhD in Health Science

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