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Peptide Mapping by LC-MS/MS

In peptide mapping, we digest a protein with a protease to produce peptides, separate the peptides by UPLC, and then analyze those peptides by MS/MS.   The objective of a peptide map experiment is to gain a full understanding of a protein’s primary structure. 

  • Sequence coverage to confirm product identity
  • Location and extent of post-translational modifications (PTMs)

We have experience developing LC-MS/MS peptide maps for the following types of products:

  • Recombinant proteins and enzymes:  blood coagulation factors, hormones, etc.
  • Monoclonal antibodies
  • Chimeric or fusion proteins:  Fc fusions, engineered chimeric proteins
  • Recombinant protein vaccines and virus capsid proteins
  • Plasma-derived or other naturally-sourced therapeutics

Post-translational modifications

We often need to use specialized conditions to fully characterize the location and extent of post-translational modifications (PTMs) within a protein.  A one-size-fits-all approaches to peptide mapping and PTM analysis rarely works well;  a customized approach is often needed to maximize the information is often needed to provide the full amount of information about a product.  We may need to use different enzyme digests to obtain peptides that are the best size, or we may need to fine-tune MS/MS conditions if PTMs are labile or if there are multiple possible modification sites on a peptide.   Chromatographic conditions can also be important to the separation of differentially modified peptides.   We take all these factors into consideration when dealing with PTMs. 

N-glycosylation and O-glycosylation

Glycosylation can play an important role in a recombinant protein’s bioactivity and pharmacokinetics.  Glycosylation is also an important indicator of manufacturing consistency.  The glycan composition in a glycoprotein is affected by both metabolic and catabolic processes: the conditions in the bioreactor affect both synthesis and degradation of the glycans.  

A tremendous amount of information can be gleaned from glycopeptide analysis in our peptide mapping experiments, and our analysis methods can be used at all stages of product development. 

  • We have performed glycan characterization early in product development at the cell line selection stage to help clients pick a clone that produces product with the desired glycosylation profile.  
  • In cases where glycosylation is critical to potency or pharmacokinetic properties, we have analyzed process intermediate samples to ensure good column operation and that fraction pooling maximized the yield of a high-quality product.
  • For drug-substance or drug-product samples, we have used glycopeptide analysis to confirm comparability between lots. 
    • pilot scale → engineering runs → GMP
    • Process changes, new sites of production, etc.  

Glycopeptide analysis is challenging.  We use MS source ionization conditions that preserve the glycans and minimize in-source fragmentation.  This, in combination with our LC conditions that provide good chromatographic resolution of glycopeptides, ensures that we obtain accurate representation of the glycoforms at each site.  We have worked with some of the more complex glycoproteins found in nature and characterized products from cell culture as well as natural sources such as plasma and milk.  

Our glycopeptide map data will provide results that correlate well with released N-glycan profiling experiments (which we also do).   We can therefore provide important glycosylation information early in product development from samples that are not as pure as drug substance samples. If you are producing a monoclonal antibody, and are concerned about the relative amounts of fucosylated vs. a-fucosylated glycans at each site, we can get you that information.  Or maybe you are concerned about the amounts of high mannose glycans, or the level of sialylation, or other N-glycan structural features at each glycosylation site…  we can do that as well.   Perhaps you are concerned about the level of Neu5Gc-sialylated glycans – that too can be determined by our experiments.  Or maybe your protein has a site that is partially glycosylated – we can give you a relative quantitation of site occupancy.   Also note - our services are not limited to N-glycosylation analysis; we have also characterized O-glycans in both plasma-derived and recombinant proteins. 

Disulfide Mapping

Disulfide mapping provides information on the product’s higher-order-structure (HOS).   We have performed disulfide mapping of monoclonal antibodies as well as recombinant proteins purified from E. coli inclusion bodies.   In our analyses, we use digest conditions that lower the chance of scrambling during sample processing.  The data are searched for the expected disulfides, tri-sulfides, free thiols, and all possibilities of scrambled disulfides.  

Deamidation

Deamidation is a chemical modification that can occur at any time in the production/purification of a protein.  Asparagine is more readily deamidated than glutamine, and the local environment within the protein molecule can play a significant role in the susceptibility to deamidation.  For example, Asn in an Asn-Gly sequon usually has the most rapid deamidation kinetics.  Many do not realize that deamidation can also be an artifact of the peptide map experiment.  Standard digest conditions that are often used for peptide maps can cause significant amounts of in-digest deamidation, and thus the experiment overestimates the deamidation of the product.   This information may be useful for future forced-degradation and stability studies, but it does not accurately represent the inherent deamidation state of the product.  We use digest conditions that eliminate the in-digest deamidation artifact and provide an accurate quantitation of deamidation in a sample.  We also use chromatographic conditions that usually enable us to separate and resolve the Asp and iso-Asp products of deamidation, so that both deamidated forms can be included in the quantitation.  

Oxidation

We routinely monitor oxidation of Met, Tyr, Trp, Phe, and Pro residues in samples and report the % oxidized at each site.

Glycation

Glycation is the chemical modification of Lys with glucose (Glc, Hexose, or Hex).   We commonly find higher glycation levels in samples from very early in the development process where bioreactor control has not been optimized.  Hopefully, as bioreactor conditions are optimized and good process control is implemented, glycation will be minimal. 

Other Modifications

We have experience working with products that have other special modifications that need to be monitored.  If your modification is not mentioned here, contact us. 

  • N-terminal pyroGlu (monoclonal antibodies)
  • Asp beta-hydroxylation (vitamin K-dependent coagulation factors)
  • Glu gamma-carboxylation (vitamin K-dependent coagulation factors)
  • Sulfation
  • Phosphorylation
  • Acetylation
  • Methylation
  • C-terminal Lys removal and amidation of monoclonal antibody heavy chains

For more information, please contact us.