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Peptide mapping

Peptide mapping is an analytical method in which a protein or peptide is selectively fragmented into discrete peptides with enzymes or chemicals and the resulting fragments are analyzed, for example by HPLC. ICH Q6B states that peptide mapping of a drug substance or drug product using an appropriately validated procedure is frequently used to confirm desired product structure for lot release purposes.[1]

What are the steps in a peptide map?

A review by Kašička groups the steps of peptide mapping as preparation before cleavage, including purification, preconcentration, denaturation, reduction and alkylation; generation of peptide fragments by enzymatic or chemical cleavage; preparation of the resulting mixture; and separation of that mixture, in the methods the review covers by capillary electromigration coupled with mass spectrometry detection.[2]

In an early example, Hunt and colleagues produced peptides from a protein with cyanogen bromide and trypsin, fractionated them by HPLC and analyzed the fractions by tandem mass spectrometry.[3]

How are the fragments identified?

ICH Q6B states that the peptide fragments should be identified to the extent possible using techniques such as amino acid compositional analysis, N-terminal sequencing or mass spectrometry.[1]

In the peptide map of the NISTmAb reference material described by Mouchahoir and Schiel, peptide identifications from tandem mass spectra were mapped to the peaks of a reference total ion chromatogram. The authors classify enzymatic cleavage C-terminal to a residue other than lysine or arginine as non-specific cleavage for trypsin.[4]

What does a peptide map show?

Mouchahoir and Schiel describe peptide mapping as a component of the analytical toolbox used to aid identity confirmation and to monitor degradative events such as oxidation or deamidation, with the advantage of site-specific information about modifications that may arise during production, processing or storage.[4]

They add that, when a map is coupled with mass spectrometry, a change in the map can be pinpointed to a particular attribute, such as increased oxidation of a certain methionine residue or the appearance of a new sequence variant.[4]

ICH Q6B notes that truncated forms, which arise when peptide bonds are cleaved, may be detected by HPLC or SDS-PAGE, and that peptide mapping may be useful depending on the property of the variant.[1]

What is sequence coverage?

Sequence coverage is the proportion of the expected sequence accounted for by identified peptides. Mouchahoir and Schiel state that a peptide mapping method must provide a high level of sequence coverage so that no critical regions of the molecule go undetected.[4]

In their study, coverage was calculated at the peptide level: an amino acid was counted as covered if the peptide containing it was identified. They report coverage of 96.89% for the heavy chain and 100% for the light chain of the NISTmAb.[4]

What can distort a peptide map?

Mouchahoir and Schiel state that sample preparation can itself induce modifications, which may confound the evaluation of true modification levels, and that the conditions favoring effective reduction, alkylation and digestion are often the same conditions that promote unwanted modifications.[4]

They also state that the enzyme must reproducibly cleave the same locations, because missed cleavages, semi-tryptic cleavages or autolysis can add peaks to, or remove peaks from, the chromatographic profile.[4]

In the same study, the varied levels of methionine oxidation indicated that some modification could be attributed to the digestion method itself.[4]

Limitations

ICH Q6B was written for biotechnological and biological products, and the NISTmAb study concerns a monoclonal antibody reference material. The figures quoted here describe that material and that method, not other materials.[1],[4]

A peptide map examines only the regions covered by identified peptides, and ICH Q6B states that more than one test may be necessary to establish identity.[4],[1]

This page describes what the cited documents say. It is not a statement that any PepGenex material has been manufactured, tested or released under any of them.

Compound profiles

Compound profiles whose identity section records the sequence and molecular formula that an identity result is compared against.

References

  1. ICH Q6B Specifications: Test Procedures and Acceptance Criteria for Biotechnological/Biological Products. U.S. FDA guidance for industry, August 1999. FDA docket FDA-1998-D-0003
  2. Kašička V. Peptide mapping of proteins by capillary electromigration methods. J Sep Sci. 2022;45(23):4245-4279. PMID 36200755 · DOI 10.1002/jssc.202200664
  3. Hunt DF, Yates JR, Shabanowitz J, Winston S, Hauer CR. Protein sequencing by tandem mass spectrometry. Proc Natl Acad Sci U S A. 1986;83(17):6233-6237. PMID 3462691 · DOI 10.1073/pnas.83.17.6233 · PMC386476
  4. Mouchahoir T, Schiel JE. Development of an LC-MS/MS peptide mapping protocol for the NISTmAb. Anal Bioanal Chem. 2018;410(8):2111-2126. PMID 29411091 · DOI 10.1007/s00216-018-0848-6 · PMC5830484