Peptide Mapping Using Multienzyme Digestion Strategies Integrated with LC-HRMS Workflow: A Case Study.

Area of Science:

• Pharmaceutical Analysis
• Biochemistry
• Analytical Chemistry

Background:

• Peptide therapeutics offer significant therapeutic potential but present development challenges due to structural complexity.
• Advanced analytical techniques, including peptide mapping, are crucial for identifying peptide primary structure and demonstrating structural similarity.
• Regulatory agencies like the FDA and EMA recommend peptide mapping for structural verification.

Purpose of the Study:

• To describe a novel multienzyme peptide mapping workflow designed to improve sequence coverage and structural verification of peptide therapeutics.
• To demonstrate the workflow's effectiveness using exenatide and H-GLP-1 as model systems.

Main Methods:

• Integration of multienzymatic digestion with complementary specificities to generate diverse peptide maps.
• Separation using a Waters XBridge Peptide BEH C18 column and gradient elution.
• High-resolution Liquid Chromatography-Mass Spectrometry (LC-HRMS) with Orbitrap detection for precise peptide fragment identification (<5 ppm mass accuracy).

Main Results:

• Full sequence coverage was achieved for exenatide and H-GLP-1 by combining peptide maps generated with trypsin, Glu-C, and chymotrypsin.
• The workflow applied to a GLP-1 analog yielded over 95% sequence coverage and confirmed intact mass.
• Precise peptide fragment identification was obtained using high-resolution MS/MS data.

Conclusions:

• The described multienzyme peptide mapping workflow provides a reliable and stepwise approach for structural verification of peptide therapeutics.
• This method enables confident assessment of the primary structure of peptides like exenatide and H-GLP-1.
• The workflow enhances sequence coverage and structural confirmation, addressing key challenges in peptide drug development.