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Anal Chem. 2018 Dec 4;90(23):13876-13881. doi: 10.1021/acs.analchem.8b02796. Epub 2018 Nov 16.

Affinity Purification of Methyllysine Proteome by Site-Specific Covalent Conjugation.

Author information

1
Department of Biochemistry and Siebens-Drake Medical Research Institute, Schulich School of Medicine and Dentistry , Western University , London , Ontario N6A 5C1 , Canada.
2
Department of Biomedical Sciences , City University of Hong Kong , Kowloon , Hong Kong Special Administrative Region , China.
3
Department of Chemistry , The Chinese University of Hong Kong , Shatin , Hong Kong Special Administrative Region , China.

Abstract

A basic but critical step in targeted proteomics by mass spectrometry is the separation of the targeted proteins from the complex mixture of the whole proteome by affinity purification. The bait protein is usually immobilized on the surface of a solid support to enable affinity-based purification of the targeted proteome. Here, we developed a site-specific covalent immobilization of the bait protein through affinity-guided covalent coupling (AGCC) of a single cysteine residue of an SH2 domain (utilized as an affinity tag for the protein target) with an engineered ligand peptide. Site-specific covalent immobilization of a methyllysine-binding protein HP1β chromodomain on the agarose resin was used to purify the methyllysine proteome from the whole-protein mixture. This new bait immobilization led to a notably low background in the affinity purification step, markedly outperforming the conventional (His)6 tag-nickel nitrilotriacetic acid (Ni-NTA) immobilization method. Subsequent analysis of the purified proteome identified 275 lysine methylated sites and 184 methylated proteins from 332 HP1β CD-binding proteins, including 30 novel methylated proteins. This work demonstrates that a robust site-specific covalent protein immobilization method is well-suited for proteomic analysis of low-abundance proteins. This method also enables the identification of new methylated proteins and methylation sites in the methyllysine proteome.

PMID:
30395435
DOI:
10.1021/acs.analchem.8b02796
[Indexed for MEDLINE]

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