Bioorthogonal Peptide Enrichment from Complex Samples Using a Rink-Amide-Based Catch-and-Release Strategy

Area of Science:

• Immunology
• Proteomics
• Biochemistry

Background:

• Antigen presentation by antigen-presenting cells (APCs) is crucial for adaptive immunity.
• Identifying low-abundance exogenous antigens in complex cell extracts is challenging.
• Mass spectrometry-based proteomics requires efficient methods for high-sensitivity antigen retrieval.

Purpose of the Study:

• To develop a selective and sensitive method for enriching antigenic peptides from APCs.
• To enable the identification of rare antigens using bioorthogonal labeling and click chemistry.
• To overcome limitations in current proteomic analysis of immune cell samples.

Main Methods:

• Utilized click-antigens: proteins engineered with azidohomoalanine (Aha) instead of methionine.
• Employed alkynyl-functionalized PEG-based Rink amide resin for covalent capture via copper-catalyzed azide-alkyne cycloaddition (CuAAC).
• Applied stringent washing steps to remove non-specific background, followed by acid-mediated peptide release.

Main Results:

• Successfully enriched and identified antigenic peptides from APC proteomes.
• Demonstrated sensitivity down to femtomole amounts of Aha-labeled antigen.
• Achieved clean and selective enrichment of bioorthogonally modified peptides from complex mixtures.

Conclusions:

• The developed method offers a promising approach for sensitive and selective enrichment of rare antigenic peptides.
• This technique enhances the capability of mass spectrometry-based proteomics in studying immune responses.
• Facilitates the identification of low-abundance exogenous antigens crucial for understanding adaptive immunity initiation.