Compound and a method for identifying a protein using said compound
AssigneeCity University of Hong Kong
InventorsYusheng Xie, Rui Wang, Liang Zhang, Hongyan Sun
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Abstract
The present invention relates to a compound for use in detecting “reader” and “eraser” proteins of lysine lipoylation. The present invention provides an affinity-based probe, referred to herein as “KPlip”, capable of interrogating the lipoylated peptide/protein interactions under native cellular environments. The chemical probe allows for the identification of potential regulators of lysine lipoylation, thus uncovering new biology related to lipoylation. There is also provided a method of using the compound to identifying proteins that interact with lipoylated proteins.
Claims1
A method for identifying a protein that binds to a lipoylated peptide comprising the steps of:
- (a) forming a first mixture by adding a portion of a first compound and a second compound into a portion of a sample containing the protein, and forming a second mixture by adding a remaining portion of the first compound and a third compound into a remaining portion of the sample containing the protein, wherein,
- the first compound has a structure of Formula (II), the second compound has a structure of Formula (III) and the third compound has a structure of Formula (IV):
- (b) subjecting the first mixture of step (a) to conditions where photo-crosslinking occurs between the protein and the first compound or the second compound of the first mixture;
- (c) adding biotin azide into the product of step (b);
- (d) subjecting the product of step (c) to conditions where click reaction occurs between biotin azide and the first compound or the second compound;
- (e) subjecting the product of step (d) to affinity purification, SDS-PAGE and liquid chromatography mass spectrum (LC-MS) analysis to remove non-specific protein binding to the second compound, while producing a first LC-MS intensity of the protein that binds to the first compound;
- (f) subjecting the second mixture of step (a) to conditions where photo-crosslinking occurs between the protein and the first compound of the second mixture;
- (g) adding biotin azide into the product of step (f);
- (h) subjecting the product of step (g) to conditions where click reaction occurs between biotin azide and the first compound;
- (i) subjecting the product of step (h) to affinity purification, SDS-PAGE and LC-MS analysis to produce a second LC-MS intensity of the protein that binds to the first compound;
- and
- (j) determining whether the first LC-MS intensity of step (e) is at least 3-fold higher than the second LC-MS intensity of step (i) in order to identify whether the protein binds to a lipoylated peptide.
Dependencies are inferred from the claim text.