As part of our protein mass spectrometry services, BioPharmaSpec provides a peptide mapping service that allows you to meet the structural characterization requirements of the ICH Q6B guidelines, understand the primary structure of your protein, assess post translational modifications (PTMs) and make key development decisions based on this knowledge.

Peptide Mapping Definition

The principle of peptide mapping is that controlled enzymic breakdown (or digestion) of the protein will produce peptides that are amenable to analysis by mass spectrometry. The masses of the peptides obtained are then compared to the predicted masses of peptides from a theoretical digest. Comparing the observed data with the theoretical data can be used to confirm the structure of the protein.

Any variations in the masses can be investigated to determine their cause (such as post translational modifications, see below). The use of liquid chromatography mass spectrometry (LC-MS) allows peptides to be readily separated and analyzed.

BioPharmaSpec uses state-of-the-art mass spectrometric instrumentation and techniques for peptide mapping analyses. Mass spectrometry is a powerful analytical tool that will provide you with a wealth of precise and detailed information on the primary structure of a protein.

Peptide Mapping of Biosimilars

Peptide mapping is a useful fingerprint comparability tool during batch-to-batch or biosimilar-to-innovator comparability studies.

For example, in the above image of a biosimilar to reference product comparability study, it can be seen that Biosimilar batches 2 and 3 differ from the Reference Product around the 12 minute mark. The mass spectrometric data generated from the peptide map can be used to investigate the structure of these components and, if necessary, orthogonal tests such as icIEF can then be performed to corroborate the reasons for differences in the peptide mapping data.

Peptide Mapping Analysis

Peptide mapping procedures at BioPharmaSpec follow the basic steps outlined below:

  1. Reduction and alkylation of the protein of interest
    • This step is used to reduce disulfide bridges and block the generated free thiol. Reduction of disulfide bridges opens the protein structure and allows proteolytic digestion to be more efficient. In the case of multi-chain proteins which have disulfide bridges linking the chains (e.g. monoclonal antibodies) this step will also break the bridges between the chains.
  2. Digestion of the protein using enzymes
    • This step breaks the protein down into peptides. The enzymes used are chosen based on the theoretical sequence of the protein and a knowledge of how the enzymes will theoretically digest the protein. In this way, an informed choice of enzymes(s) can be made that should result in the best peptide mapping analysis.

  1. Analysis of the digested peptides using on-line Reverse Phase-High Performance Liquid Chromatography with Ultraviolet (UV) and Electrospray-Mass Spectrometric detection (LC/ES-MS).
    • LC separates the peptides based on polarity (reverse phase-LC).
    • Mass spectrometer generates mass information for each peptide as it elutes from the LC column. Fragment ion information is also generated, which allows sequence information from the peptides to be determined. This sequence information serves to confirm peptide identity and provides primary sequence information.
    • The mass spectrometers at BioPharmaSpec are also capable of generating fragmentation information from the peptides as they enter the mass spectrometer source from the LC column.
    • This is either selective (MS/MS) or non-selective (MSe), depending on the requirements of the study and can be considered as a form of tandem mass spectrometry.

Applications of Peptide Mapping

Peptide mapping by mass spec will provide a large amount of information on the primary structure of your protein including:

Sequence confirmation

As mentioned above, a peptide mapping method will involve digesting the protein into peptides and analyzing these peptides using mass spectrometry. Using an appropriate digestion strategy, all of the protein chain can be converted to suitable peptides for peptide mapping analysis. These peptides can be assigned using mass spectrometry. The use of multiple digestion strategies results in the production of overlapping peptides which tie the sequence of the peptides together along the protein chain. The intact mass generated from each peptide along with the fragmentation data for that peptide can confirm the sequence against a theoretical amino acid sequence.

It should be noted that certain residues such as Isoleucine and Leucine have the same mass and so mass spectrometric procedures cannot differentiate between them. For studies requiring full primary amino acid sequencing, Edman chemistry is performed on collected peptides containing these residues in order to give unambiguous identification

In cases where there is no theoretical sequence or it is a regulatory requirement to confirm the sequence unambiguously, de novo primary amino acid sequencing should be performed.


Glycosylation is one of the most important post-translational modifications to consider during protein characterization and it is a regulatory requirement to characterize the glycans on your biopharmaceutical.

Peptide mapping data will provide you information on:

  • N-glycan population profiles
  • O-glycan population profiles
  • Location of sites of glycosylation in the protein chain
  • Degree of site occupancy

Peptide mapping will provide initial information on the glycans present.  It cannot be used as a substitution for a full investigation into glycan structure, which requires a specific set of investigations into glycosylation.

N-terminal pyroglutamination

During protein production, modifications to the protein backbone can occur. These are known as Post Translational Modifications (PTMs). There are many different types of PTMs and these can be identified through the use of peptide mapping. An important PTM for antibodies is the cyclization of the N-terminal Glutamine residue to pyroglutamate. This is a common antibody PTM and can be generated on the light and/or heavy chains. This modification reduces the overall mass of the protein and so would be detected during peptide mapping. This PTM can cause specific challenges during N-terminal sequencing using Edman chemistry.

Disulfide bridges

How many disulfide bridges your protein has and the position of these bridges will play a key role in the overall secondary and higher order structure of the molecule. The ICH guidelines (Q6B) state that the disulfide bridge pattern must be characterized. This is because disulfide bridges can be mismatched or scrambled, leading to alterations in protein folding and potentially subsequent effects on functionality, aggregation or immunogenicity.

Care must be taken when digesting proteins containing free thiols as the free thiol can initiate scrambling within the digest and the result obtained may not be consistent with the true disulfide bridge/free thiol pattern within the product.

Using a range of techniques from LC-MS, LC-MS/MS through to Nanospray, BioPharmaSpec can determine the disulfide bridge pattern in your protein to the fullest extent possible.

Heavy chain C-terminal Lysine

As well as the possible presence of pyroglutamic acid at the N-terminus of monoclonal antibodies, the C-terminus of the heavy chain can also exhibit variation in the extent to which the C-terminal Lysine residue is present. This post translational modification can be detected in intact mass analysis and confirmed by peptide mapping through an examination of the C-terminal heavy chain peptide for the plus and minus Lysine containing forms of the peptide. Orthogonal procedures such as electrophoretic profiling (icIEF) can be used to examine the effect on this post translational modification on the charge profile of the antibody.


During deamidation, the side chain amide group of asparagine or glutamine residues is converted to aspartate/isoaspartate and glutamate, respectively. This Post Translational Modification (PTM) can occur as a result of manufacturing or purification processes. It may also take place naturally over time as a result of interactions of the protein with the sample formulation components. Changes in the manufacturing, purification or formulation can lead to a change in the degree of deamidation and subsequently in the functionality of your protein.

Deamidation results in a very small change in mass (1Da). The accurate instrumentation used during peptide mapping can detect this change. Different asparagine and glutamine residues in the sequence will have different susceptibilities for deamidation. Peptide mapping can show which residues are more or less amenable to this modification. Orthogonal techniques can also be used to further confirm deamidation as the root cause of the mass change (e.g. icIEF, due to a change in isoelectric point).


Oxidation occurs most commonly on the side chain of methionine or tryptophan residues. As with deamidation, oxidation can occur as a result of process changes or simply take place naturally over time. Changes in the manufacturing or purification processes or formulation can lead to a change in the degree of oxidation and subsequently in the functionality of your protein. Oxidation is another PTM that will be detected during peptide mapping.

Product-related impurities, including degradation products.

During the production of a biologic, variants may be produced that have some changes compared to the intended structure. This could be truncated species, partially cleaved forms, species with scrambled disulfide bridges or other forms of post translational modifications.

Whilst any purification process will aim to remove process related impurities that could impact the quality of the product, an assessment of product related impurities in a sample can be used as a means to develop and adjust the manufacturing or purification process to eliminate these as far as possible from the final product. It is a requirement of ICH Q6B that product related impurities are investigated.

BioPharmaSpec offers a comprehensive protein mass spectrometry service, with our peptide mapping services at the core of our expertise. Contact our scientists now to find out how our peptide mapping analysis can be applied to your protein.