Amino Acid Sequence Analysis of Semaglutide

Amino Acid Sequence Analysis of Semaglutide Using Edman Degradation and MALDI-TOF MS — Summary

Importance of the topic
Peptide therapeutics represent a growing middle ground between small molecules and biologics, offering high target specificity with potential for reduced side effects. Accurate verification of peptide primary structure is critical for safety, efficacy and regulatory quality control. Methods that combine sequence-specific assays with molecular-weight measurements are particularly valuable for characterizing peptides that contain non-natural amino acids or side-chain modifications that are not represented in genome databases.

Objectives and overview of the study
The study demonstrates the combined use of automated Edman degradation (PPSQ-51A/53A protein sequencer) and MALDI-TOF mass spectrometry (MALDI-8030) to:

• Determine the N-terminal sequence of semaglutide, a 31-residue GLP-1 analog containing non-natural and modified residues.
• Assess the ability to detect the non-proteinogenic residue 2‑aminoisobutyric acid (Aib) and to identify the C-terminal residue.
• Combine sequence and intact-mass data to estimate the mass contribution of a fatty-acid side-chain modification at Lys20.

Methodology and sample preparation

• Sample: Synthetic semaglutide (Merck), dissolved in 5% acetic acid to 100 pmol/µL for Edman sequencing; diluted to 10 pmol/µL in 1:1 acetonitrile/0.1% TFA for MALDI-TOF.
• Edman sequencing: 5 µL (500 pmol) applied to polybrene-treated glass fiber disks and analyzed on PPSQ-51A/53A. Two chromatographic approaches were compared: an isocratic PTH-amino acid separation (Wakopak Wakosil PTH-II column, 4.6 mm ID) and a gradient separation (Wakopak Wakosil PTH-GR column, 2.0 mm ID). Detection by UV at 269 nm using SPD-M30A high-sensitivity flow cell.
• PTH-derivatization: Standard phenylthiohydantoin (PTH) amino-acid mixture was used to establish retention times; PTH-2‑aminoisobutyric acid (PTH-Aib) standard was included to confirm identification of the non-natural residue.
• MALDI-TOF MS: CHCA matrix (α-cyano-4-hydroxycinnamic acid); measurement to obtain average molecular weight [M+H]+.

Used instrumentation

• PPSQ-51A/53A protein sequencer (Shimadzu) for automated Edman degradation and HPLC separation of PTH-amino acids.
• Columns: Wakopak Wakosil PTH-II (250 × 4.6 mm I.D.) for isocratic runs and Wakopak Wakosil PTH-GR (250 × 2.0 mm I.D.) for gradient runs.
• Detector: SPD-M30A UV detector at 269 nm (high-sensitivity flow cell).
• MALDI-8030 MALDI-TOF mass spectrometer (Shimadzu) with CHCA matrix.

Main results and discussion

• Identification of non-natural residue Aib: PTH-Aib elutes close to PTH-Arg but can be resolved using established retention-time references. In the isocratic system manual confirmation was required for cycle 2, while the gradient system provided clearer separation and higher sensitivity, allowing unambiguous detection of PTH-Aib around ~17.4 min.
• Sequence coverage and limitations: Edman degradation provided sequence assignments across much of the N-terminus. However, cycle 20 (residue Lys20) failed to produce a characteristic PTH-amino acid peak in both chromatographic modes. Possible causes include blocked or highly hydrophobic phenylthiohydantoin derivatives from the fatty-acid-modified Lys, stronger column interaction preventing elution, or degradation of the side-chain modification during chemistry.
• C-terminal identification: The gradient system detected PTH-Gly at cycle 31 and allowed confirmation of the Gly C-terminal residue. The isocratic method failed to detect the expected C-terminal PTH-products, illustrating that Edman sequencing can sometimes lose C-terminal residues due to sample loss during wash steps or sequence-dependent inefficiencies.
• MALDI-TOF mass confirmation: Intact-mass measurement returned [M+H]+ = m/z 4114.8, consistent with semaglutide. By combining the observed sequence (with the modification location known) and the intact mass, the mass attributable to the Lys20 side-chain modification was estimated at ~844.2 Da. This demonstrates how mass data complements sequence data to characterize modifications that are not identifiable by Edman chemistry alone.

Benefits and practical applications of the method

• Edman sequencing with a PTH-amino acid reference allows direct identification of non-proteinogenic residues and provides cycle-by-cycle sequence confirmation from the N-terminus.
• MALDI-TOF MS supplies rapid intact-mass data to detect mass shifts from modifications, truncations, or incorrect residues, enabling cross-validation of sequence assignments.
• The combined approach is useful in R&D and quality control for synthetic peptide drugs, especially when non-natural residues, lipidation or other side-chain modifications are present.

Limitations observed

• Edman degradation may fail to yield identifiable PTH-derivatives for residues carrying bulky hydrophobic modifications (e.g., fatty-acylated Lys), limiting direct sequencing across modification sites.
• C-terminal sequencing by Edman can be unreliable due to sample loss or cleavage inefficiencies as the reaction approaches the peptide C-terminus.
• Hydrophobic PTH-derivatives can interact strongly with reversed-phase columns, leading to poor elution unless chromatographic conditions are optimized.

Future trends and possibilities for application
Combining orthogonal analytical techniques will be increasingly necessary for characterization of complex peptide modalities such as side-chain lipidations, cyclizations and non-natural amino acids. Recommended directions include:

• Integration with tandem MS/MS (LC-MS/MS) workflows to localize modifications and confirm sequence across modification sites.
• Use of advanced fragmentation methods (ETD/ECD) or chemical derivatization strategies to improve detection of modified residues.
• Optimization of chromatographic chemistries and columns for highly hydrophobic PTH-derivatives to enhance Edman-derived identification.
• Development of standardized mass- and sequence-based QC workflows for regulatory submissions of synthetic peptide therapeutics.

Conclusion
The work illustrates that automated Edman degradation (PPSQ-51A/53A) coupled with careful chromatographic methods can identify non-natural residues such as Aib and produce reliable N-terminal sequence information for semaglutide. MALDI-TOF MS provides complementary intact-mass confirmation and enabled estimation of the mass of a Lys20 fatty-acyl modification (~844.2 Da). Limitations remain for direct identification of highly modified, hydrophobic residues and for complete C-terminal sequencing; therefore, a multi-technique analytical strategy is recommended for comprehensive characterization of modern peptide drugs.

Reference

1. Knudsen L. B., Lau J., The Discovery and Development of Liraglutide and Semaglutide. Molecular and Structural Endocrinology, 10 (155), 1–32 (2019).