Enrichable Protein Footprinting for Structural Proteomics.

Area of Science:

• Biochemistry
• Proteomics
• Structural Biology

Background:

• Protein footprinting analyzes protein higher-order structure and conformational changes using covalent modifications.
• Mass spectrometry (MS)-based methods offer advantages over cryo-EM, X-ray diffraction, and NMR, requiring less protein and higher purity.
• Enrichment techniques are crucial for sensitivity and coverage in MS-based proteomic analyses, but current footprinting reagents lack suitable enrichment methods.

Purpose of the Study:

• To develop an enrichable covalent labeling method for protein footprinting applicable to complex proteomes.
• To adapt the GEE/EDC system for labeling aspartic and glutamic acid residues with alkynyl functionality for click chemistry.
• To establish a pipeline for enrichment and analysis of protein conformation on a proteomic scale.

Main Methods:

• Utilized glycine propargyl amide (GPA) for EDC-facilitated coupling to proteins, introducing alkynyl functionality.
• Employed copper-catalyzed azide-alkyne cycloaddition (CuAAC) to 'click' azide-containing molecules to the labeled proteins.
• Developed an enrichment pipeline involving biotin-azide with a photocleavable linker, streptavidin resin, and UV-light cleavage.

Main Results:

• Identified EDC-facilitated coupling of GPA as the most efficient labeling method.
• Successfully demonstrated enrichment of labeled proteins from complex mixtures, including those from Arabidopsis thaliana.
• Optimized the enrichment process by screening clickable amines, coupling reagents, and enrichment scaffolds.

Conclusions:

• The developed method enables enrichable covalent labeling for protein footprinting, overcoming limitations of current techniques.
• This approach enhances MS-based analysis of protein structure and conformation, particularly in complex biological samples.
• The method holds potential for proteomic-scale measurement of protein conformation.