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Protein peptide mapping

Why map the amino acid sequence of your protein?

Peptide mapping is a useful tool for identifying/verifying the sequence of a protein and the amino acid site specific post-translational modifications. The information gleaned from peptide mapping is valuable data for the production of a variety of protein therapeutics such as monoclonal antibodies (mAbs), antibody drug conjugates (ADCs), and smaller proteins. Successful design of biosimilar proteins requires identical amino acid sequence and levels of post-translational modification. Amino acid substitutions and changes in post-translational modifications can reveal why a protein’s behavior in activity assays has changed between lots. For example, glycosylation greatly affects the half-life of a mAb and target determination within a biological system and is therefore an important characteristic to know during design and production.

The condition of the protein during storage is important to determine best storage conditions and shelf-life of the therapeutic protein. Deamidation occurs naturally during production, but additional deamidation driven by prolonged storage can lead to loss of therapeutic function. Mapping the amino acid sequence of the protein during forced degradation studies identifies deamidation “hotspots” that need to be addressed during protein production or in assigning shelf-life.

Why use the services of Novatia?

Our expert team has more than 38 years of biological mass spectrometry experience. We use electrospray ionization liquid chromatography mass spectrometry (ESI/LC/MS/MS) to characterize your digested protein. Tandem mass spectrometry in conjunction with MS1 (Novatia‚Äôs ProMass Deconvolution Software) and MS(Thermo ScientificTM PepfinderTM Software) spectra analysis is used to reliably confirm the sequence of the protease digested protein samples.  Our digestion, data acquisition and post-acquisition analysis pipeline allow us to verify the sequence, identify the sites of post-translational modifications, and rapidly return results in an easy-to-read report.  

Lysine conjugated ADC mimic Peptide mapping analysis example

ADCs are a rapidly growing therapeutic protein that combines the specificity of mAb targeting with small molecules to deliver a cytotoxic drug with reduced adverse side effects. The value used to determine the payload of the ADC is called the DAR (drug antibody ratio), and is an average of the number of drug molecules conjugated per molecule of antibody. The DAR value cannot tell you if the drug is conjugated to the correct amino acids, or what region of the heavy or light chains. Peptide mapping can identify which amino acids are drug-conjugated and the extent to which peptides are conjugated overall. In this example, the MS/MS spectra of 5 peptides from a tryptic digest of MSQC4-AF488, a lysine conjugated ADC mimic, are examined.

Figures 1 and 2 are the MS/MS spectra of peptide examples 1 (AA 156-162) and 2 (AA 163-172), sequential peptides in the light chain of MSQC4. The conjugation of AF488 to the lysine (K162) interferes with the cleavage at K162 by trypsin, and results in the longer peptide 3 (AA 156-172, K162-AF488) shown in figure 3. Figure 4 and 5 are MS/MS spectra of peptide examples 4 (AA 306-321) and 5 (AA 306-324) in the heavy chain of MSQC4. The conjugation of AF488 to the lysine (K321) interferes with the cleavage at K321 by trypsin, and results in the longer peptide 5 (AA 306-324, K321-AF488) shown in figure 5. A predicted MS/MS spectrum is shown above each of the experimental MS/MS spectrum. In addition, the y and b product ions are labeled in red for each of the experimental spectra.

Figure 1: The expected (top) and experimental (bottom) spectra of the unconjugated MSQC4 light chain tryptic peptide (AA 156-162).

Figure 2: The expected (top) and experimental (bottom) spectra of the unconjugated MSQC4 light chain tryptic peptide (AA 163-172).

Figure 3: The expected (top) and experimental (bottom) spectra of the conjugated MSQC4-AF488 light chain tryptic peptide (AA156-172).The conjugation of AF488 on K162 interferes with cleavage at K162 by trypsin, resulting in a longer peptide.

Figure 4: The expected (top) and experimental (bottom) spectra of the unconjugated MSQC4 heavy chain tryptic peptide (AA306-321).

Figure 5: The expected (top) and experimental (bottom) spectra of the conjugated MSQC4-AF488 heavy chain tryptic peptide (AA306-324). The conjugation of AF488 on K321 interferes with cleavage at K321 by trypsin, resulting in a longer peptide.

MSQC4-AF488 sequence coverage map

The peptide map of the tryptic digestion of MSQC4-AF488 is shown below.

MSQC4-AF488 modification summary

Below is summary of the modifications on the MSQC4-AF488 Lysine ADC mimic, including the lysine residues conjugated to AF488.