Enrichable Protein\nFootprinting for Structural Proteomics

Protein footprinting is a useful method for studying\nprotein higher\norder structure and conformational changes induced by interactions\nwith various ligands via addition of covalent modifications onto the\nprotein. Compared to other methods that provide single amino acid-level\nstructural resolution, such as cryo-EM, X-ray diffraction, and NMR,\nmass spectrometry (MS)-based methods can be advantageous as they require\nlower protein amounts and purity. As with other MS-based proteomic\nmethods, such as post-translational modification analysis, enrichment\ntechniques have proven necessary for both optimal sensitivity and\nsequence coverage when analyzing highly complex proteomes. Currently\nused reagents for footprinting via covalent labeling, such as hydroxyl\nradicals and carbodiimide-based methods, do not yet have a suitable\nenrichment method, limiting their applicability to whole proteome\nanalysis. Here, we report a method for enrichable covalent labeling\nbuilt upon the GEE/EDC system commonly used to covalently label aspartic\nacid and glutamic acid residues. Novel labeling reagents containing\nalkynyl functionality can be “clicked” to any azido-containing\nmolecule with copper-catalyzed azide–alkyne cycloaddition (CuAAC),\nallowing for enrichment or further labeling. Multiple azide- and alkyne-containing\nGEE-like molecules were tested, and the most efficient method was\ndetermined to be the EDC-facilitated coupling of glycine propargyl\namide (GPA) to proteins. The pipeline we report includes clicking\nvia CuAAC to a commercially available biotin-azide containing a photocleavable\nlinker, followed by enrichment using a streptavidin resin and subsequent\ncleavage under ultraviolet light. The enrichment process was optimized\nthrough the screening of clickable amines, coupling reagents, and\nenrichment scaffolds and methods with pure model proteins and has\nalso been applied to complex mixtures of proteins isolated from the\nmodel plant, <i>Arabidopsis thaliana</i>, suggesting that\nour method may ultimately be used to measure protein conformation\non a proteomic scale.