Linkers for protein interaction profiling and methods of making and using the same

Patent metadata

Field	Value
Publication number	US-11174353-B2
Application number	US-201916293191-A
Country	US
Kind code	B2
Filing date	Mar 5, 2019
Priority date	Oct 9, 2014
Publication date	Nov 16, 2021
Grant date	Nov 16, 2021

Abstract

Crosslinking compounds for effective and efficient cross-linking and identification of intermolecular and intramolecular interactions of proteins, peptides and nucleic acids.

First claim

The invention claimed is:

1. A method of cross-linking at least one of a protein, a peptide, and a nucleic acid comprising:
mixing a composition comprising the at least one of the protein, the peptide, and the nucleic acid with a crosslinker compound having a chemical structure:
Z—Y-A n -Y—Z

wherein:
n is an integer between 1 and 5;
A is an atom or a compound;
each Z is a Michael acceptor independently selected from the group consisting of a propiolate, a propiolamide, a ynone, a ynethiolate, an acrylate, and a vinylsulfone; and,
each Y is optional and is independently an organic compound optionally substituted with one or more heteroatoms selected from the group consisting of O, N, S, or P to form a crosslinked sample;

fragmenting the crosslinked sample to form crosslinked fragments; and
detecting a mass-to-charge ratio of the crosslinked fragments.

2. The method of claim 1 , wherein the composition further comprises at least one of a cell lysate, a cell culture, and a tissue sample.

3. The method of claim 1 , wherein the mixing step is conducted at a pH between pH 5 and pH 10.

4. The method of claim 1 , further comprising purifying the crosslinked sample.

5. The method of claim 1 , further comprising isolating the crosslinked fragments.

6. The method of claim 1 , wherein the fragmenting step comprises digesting the crosslinked sample with an enzymatic cleaving agent or a chemical cleaving agent.

7. The method of claim 6 , wherein the digesting comprises contacting the crosslinked sample with at least one of CNBr at pH 2, trypsin, Glu-C endoproteinase, pepsin, restriction nuclease enzymes, DNase I, RNase α-sarcin, and combinations thereof.

8. The method of claim 1 , wherein the crosslinker compound comprises at least one collision-induced dissociation group and the method further comprises dissociating at least one of the crosslinked sample and the crosslinked fragments to form a dissociated sample.

9. The method of claim 8 , wherein the dissociating comprises collisions with at least one of an inert gas, a surface, photons, thermal/black body infrared radiation, and an electron beam.

10. The method of claim 1 , further comprising ionizing at least one of the crosslinked sample and the crosslinked fragments to form an ionized fragment.

11. The method of claim 10 , wherein the ionizing comprises at least one of electrospray ionization (ESI), matrix-assisted laser desorption ionization (MALDI), and fast atom bombardment (FAB).

12. The method of claim 1 , wherein the detecting step comprises:
directing the crosslinked fragments through a first mass resolving spectrometer to select precursor ions having a first desired mass-to-charge ratio;
subjecting the precursor ions to dissociation to form product ions having a second mass-to-charge ratio; and
detecting the product ions.

13. The method of claim 1 , wherein A is selected from the group consisting of a C 1-10 alkyl, a cycloalkyl, a heteroalkyl, and a heterocycloalkyl, wherein when A is the heteroalkyl or the heterocycloalkyl, the heteroatom is selected from at least one of phosphorus (P), sulfur (S), nitrogen (N), or oxygen (O).

14. The method of claim 1 , wherein each Michael acceptor is the propiolate.

15. The method of claim 1 , wherein the crosslinker compound comprises an affinity handle (Q) selected from the group consisting of:
a) a chemical moiety that facilitates enrichment of crosslinked species from a sample;
b) a chemical moiety that facilitates precipitation or separation of the crosslinker compound from a sample;
c) at least one of biotin, a histidine residue, and PEG; and,
d) at least one of an amino acid sequence, polyhistidine, an antibody fragment, and a nucleic acid sequence.

16. The method of claim 1 , wherein the crosslinker compound comprises a molecular label (L) selected from the group consisting of: a phosphor, a radioactive atom, a atomic isotope, a fluorescent dye, a electron-dense reagent, an enzyme, biotin, digoxigenin, hapten, hydrolase, phosphatase, esterase, glycosidase, oxidotase, peroxidase, fluorescein or its derivatives, rhodamine or its derivatives, dansyl, umbel-liferone, luciferin, 2,3-dihydrophthalazinedione, a stable isotope, and a detectable proteins that incorporates a metal, radiolabel, or a phosphor.

17. The method of claim 1 , wherein each linking group (Y) independently comprises one or more cleavage sites that is capable of being cleaved by at least one of a chemical cleavage agent, an enzymatic cleavage agent, or both chemical cleavage agents and enzymatic cleavage agents.

18. The method of claim 1 , wherein each linking group (Y) comprises one or more collision-induced dissociation groups, each collision-induced dissociation group capable of forming a signature ion upon collision-induced dissociation in mass spectrometric methods.

19. The method of claim 1 , wherein the crosslinker compound has a molecular length of between about 1 angstrom and about 50 angstroms.

Assignees

• Univ Colorado Regents

Inventors

• Old William
• Chapnick Douglas A
• Mcclure-Begley Tristan D
• Gong Tao
• Worrell Brady
• Ebmeier Christopher C
• Bowman Christopher

Classifications

• G01N33/6848

  Methods of protein analysis involving mass spectrometry · CPC title

• C08K5/10

  Esters; Ether-esters · CPC title

• C08H1/00 Primary

  Macromolecular products derived from proteins (food proteins A23; glue, gelatine C09H) · CPC title