Amino Acid Sequencing of Synthetic Peptides Using a MALDI-TOF Mass Spectrometer and Protein Sequencer

Importance of the Topic

Peptide-based therapeutics are increasingly valued for their favorable oral absorption, cell membrane permeability, and cost-efficient chemical synthesis. Accurate determination of peptide amino acid sequences is essential to verify product integrity and ensure biological function.

Objectives and Study Overview

This study presents a combined approach using a bench-top MALDI-TOF mass spectrometer (MALDI-8030) and an Edman degradation protein sequencer (PPSQ-50A) to determine complete amino acid sequences of two synthetic peptides: parathormone (34 residues, linear) and somatostatin (14 residues, cyclic).

Methodology and Instrumentation

Sample Preparation and Analytical Conditions:

• Peptide samples were applied to a MALDI plate and co-crystallized with matrices.
• CHCA matrix was used for molecular weight measurements in positive-ion mode.
• 1,5-DAN matrix enabled in-source decay (ISD) fragmentation to generate sequential fragment ions.
• Edman degradation was performed on the PPSQ-50A system using isocratic and gradient methods to identify PTH-amino acid derivatives.

Main Results and Discussion

Parathormone:

• Edman sequencing identified residues 1–33; residue 34 (Phe) was inferred from the mass difference in the MALDI spectrum.
• MALDI-ISD provided complementary fragment ions but lacked full N-terminal coverage; combining both techniques yielded the complete sequence.

Somatostatin:

• Edman degradation determined residues 1–12; Ser13 and Cys14 were missed due to side-chain losses and cyclic structure.
• MALDI-ISD generated sequential ions covering residues 9–14 and directly identified Cys, enabling full sequence assembly in combination with Edman data.

Benefits and Practical Applications

• Complete sequence determination regardless of peptide structure or database availability.
• Reduced ambiguity from side-chain modifications and enzyme-based digestion methods.
• Accurate discrimination of isobaric residues (Ile/Leu) via Edman degradation.
• Applicable to quality control and accelerated development of therapeutic peptides.

Future Trends and Applications

Advancements may include automated integration of MALDI-ISD and Edman workflows, improved fragmentation matrices, higher resolution instrumentation, and expanded de novo sequencing capabilities for longer or heavily modified peptides. Such developments could further streamline peptide analysis in drug discovery and proteomic research.

Conclusion

The combined use of the MALDI-8030 mass spectrometer and PPSQ-50A protein sequencer delivers reliable, full-length amino acid sequencing for both linear and cyclic peptides. This orthogonal approach enhances confidence in sequence verification and supports stringent quality assurance in peptide manufacturing.

Reference

• Analysis of Long-Chain Amino Acid Sequences Using a Protein Sequencer—Isocratic System, Application News No. 01-00521-EN
• Analysis of Long-Chain Amino Acid Sequences Using a Protein Sequencer—Gradient System, Application News No. 01-00549-EN
• Protein Analysis Platform Combining MALDI-TOF MS (MALDI-8020) and Edman Sequencing (PPSQ-50A Gradient System), Application News No. B105