Written by Yuning Wang, PhD

October 21, 2021

What is peptide mapping?

Peptide mapping is a widely used analytical technique to identify or verify a protein’s primary structure (amino acid sequence and chemical modifications). Briefly, peptide mapping identifies a protein by first determining peptides’ molecular weights and then matching these peptide masses to a protein’s calculated theoretical mass.

How is peptide mapping performed?

With peptide mapping, scientists analyze peptides generated from digestion of an isolated protein, or a protein mixture. The basic workflow of peptide mapping involves enzymatic digestion of the protein followed by separation of the resulting peptides via liquid chromatography prior to mass spectrometry assessment. The procedure generates a ‘fingerprint’ or set of unique peptides of the interrogated protein. The masses of the unique peptides identified are compared to theoretical masses from sequences found in a proteomics database. Unlike de novo protein sequencing, which resolves protein sequences down to the individual amino acid, peptide mapping is dependent on sequence databases to identify a protein of interest.

Why is peptide mapping needed?

As monoclonal antibody and recombinant protein drugs are becoming the fastest growing sectors of the pharmaceutical industry, careful inspection of the complete product properties (e.g., purity, biochemical properties, and stability) is critical throughout the development and manufacturing processes. Of particular importance is the heterogeneity of antibodies. The latter’s various post-translational modifications often occur over the molecule’s lifecycle. These modifications (Figure 1) play significant contributions on antigen-binding, folding, and biological function of antibodies, consequently and directly impacting drug efficacy, stability, and safety.

One of the most common PTMs for antibodies, glycosylation, plays an important role in regulating many drug side effects including antibody-dependent cellular cytotoxicity and phagocytosis. In addition to PTMs, other forms of heterogeneity can be also introduced during manufacturing and upon long-term storage, such as aggregation, degradation, and contamination. Not only can these changes result in manufacturing problems, but the administration of such proteins may also give rise to severe immunogenicity in patients.

Figure 1. Infographics showing types of antibody heterogeneity that can be analyzed by peptide mapping.

Impacts of not utilizing peptide mapping

Failing to identify a product’s heterogeneity can impact reproducibility and hamper drug development. Recent studies have shown that at least 50% of reagent antibodies cannot recognize their targets specifically, and a half or more preclinical studies cannot be reproduced. The Food and Drug Administration (FDA) and European Medicines Agency (EMA) both recommend extensive evaluation and testing of such changes including protein sequences, higher-order structures, and PTMs. Thus, having a robust workflow that includes routine monitoring and characterization of the protein is necessary to ensure product quality and reproducibility. To ensure the latter but minimize cost and time, a tool, such as peptide mapping, that secures sequence confirmation represents an attractive prospect.

Limitations of traditional peptide mapping

Traditional Peptide Mapping Suffers from Low Resolution

Since peptide mapping does not employ tandem mass spectrometry and peptide fragment data analysis (as would be employed in de novo protein sequencing), it suffers from low resolution. Foregoing assessment at the amino acid level results in the inability to distinguish between isobaric or same-mass residues.

Isobaric amino acids play a huge role in antibody binding as they are frequently found in the complementary determining regions (CDRs). In 2017, we sequenced 200 monoclonal antibodies, and found that CDRs with no isoleucine or leucine residues are very rare: almost all antibodies contain at least one isobaric amino acid in their CDRs1 (Figure 2). Without de novo protein sequencing, traditional peptide mapping is unable to tell amino acids apart.

Figure 2. Frequency of antibodies expressing isoleucine or leucine in their complementarity determining regions (CDRs). A total of 200 monoclonal antibodies were sequenced using our de novo protein sequencing platform (REmAb®).

Traditional Peptide Mapping May Be Impacted by Bias

As such, bias may be introduced when performing database searching; peptide X which shares the same mass of peptide Y might be identified as the same despite one having isoleucine, and the other having leucine, as an example. Other isobaric residues such as GlyAla and Gln with a mass difference of 0.000002 may also confound traditional peptide mapping approaches.

Traditional Peptide Mapping Is Lengthy and Costly

Because it depends on database searching, it may not be clear whether coverage is good enough to properly identify the protein simply based on the mass spec experiment. If a significant portion of the protein sequence is not covered by the tandem mass spec results, the experiment may have to be repeated.

The latter may result in delays, and limit the utility of the technique as a routine sequence check. Using tandem mass spec instrumentation that would have higher resolution, together with purpose-built bioinformatics software that would allow coverage to be assessed in real-time presents an attractive solution that would minimize time and budget spent.

MATCHmAb™: Next Generation Proteomics for Rapid Peptide Mapping

At Rapid Novor, we have developed a fast and robust peptide mapping platform, MATCHmAb™. MATCHmAb™ was developed from our established de novo protein sequencing REmAb® technology for precise sequence verification. As a result, MATCHmAb™ is capable of overcoming various conventional peptide mapping challenges. In contrast to traditional peptide mapping, MATCHmAb™ is

  • Faster
  • High-throughput (able to confirm protein sequences in hours to days)
  • Capable of resolving a sequence at the amino acid level

MATCHmAb™offers an easy check for long-lasting peace of mind. If you would like to find out more about how MATCHmAb™ can help with your project, contact us HERE.


1. McDonald, Z.L., Q; Stajduhar, A; Taylor, P; Krieger, JR; Ma, B, Large Scale Study of the W-ion Isoleucine and Leucine Determination (WILD™) Method in Antibody De Novo Protein Sequencing, in 66th American Society for Mass Spectrometry. 2018: San Diego.

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We Have Sequenced 3000+ Antibodies and We Are Eager to Help You.

Through next generation protein sequencing, Rapid Novor enables timely and reliable discovery and development of novel reagents, diagnostics, and therapeutics. Thanks to our Next Generation Protein Sequencing and antibody discovery services, researchers have furthered thousands of projects, patented antibody therapeutics, and ran the first recombinant polyclonal antibody diagnostics

Talk to our scientists. We have sequenced over 4000 antibodies and we are eager to help you.