US 12,146,183 B2
Compound and a method for identifying a protein using said compound
Yusheng Xie, Kowloon (HK); Rui Wang, Kowloon (HK); Liang Zhang, Kowloon (HK); and Hongyan Sun, Kowloon (HK)
Assigned to CITY UNIVERSITY OF HONG KONG, Kowloon (HK)
Filed by CITY UNIVERSITY OF HONG KONG, Kowloon (HK)
Filed on Jun. 17, 2021, as Appl. No. 17/350,934.
Claims priority of provisional application 63/040,060, filed on Jun. 17, 2020.
Prior Publication US 2021/0395795 A1, Dec. 23, 2021
Int. Cl. C12Q 1/32 (2006.01); C07D 413/12 (2006.01); C07K 7/06 (2006.01); C09K 11/06 (2006.01); C12N 9/02 (2006.01)
CPC C12Q 1/32 (2013.01) [C07D 413/12 (2013.01); C07K 7/06 (2013.01); C09K 11/06 (2013.01); C12N 9/0008 (2013.01); G01N 2333/90203 (2013.01)] 1 Claim
 
1. 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):

OG Complex Work Unit Chemistry
(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.