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Abstract

Proteins are selectively extracted from complex biological mixtures for subsequent analysis by methods such as mass spectrometry. Protein-binding (e.g., PDZ or SH2) or cellular compartmentalizing (e.g., membrane-targeting) domains are provided to target specific proteins, and targeted proteins are enzymatically modified by attachment of modification molecules. For example, targeted proteins can be biotinylated by the enzyme biotin protein ligase. The protein-targeting domain and protein-modifying enzyme can be immobilized on a solid surface (macroscopic planar surface or particle surface), provided as a fusion protein, or chemically coupled. The modified proteins are then exposed to complementary molecules (e.g., avidin or streptavidin) that bind strongly to the modification molecule, resulting in affinity capture of the selected proteins. Because the protein-targeting domain brings the protein in proximity to the active site of the enzyme, the method effectively broadens the substrate specificity of the enzyme, allowing affinity capture of any desired protein subset.

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First Claim