Capture compounds, collections thereof and methods for analyzing the proteome and complex compositions

• US 20100248264A1
• Filed: 02/26/2010
• Published: 09/30/2010
• Est. Priority Date: 01/16/2003
• Status: Active Grant

• Current Assignee: Caprotec Bioanalytics GmbH
• Original Assignee: Caprotec Bioanalytics GmbH
• Inventors: Siddiqi, Suhaib Mahmood, Kõster, Hubert, Yip, Ping, Marappan, Subramanian, Little, Daniel Paul, Hassman, Chester Frederick III, Grealish, Matthew Peter
• Granted Patent: US 9,034,798 B2
• Time in Patent Office: Days
• US Class Current: 435/7.400
• CPC Class Codes: C07C 2603/74 Adamantanes
• CPC Class Codes: C07C 69/76 Esters of carboxylic acids having a carboxyl group bound to a carbon atom of a six-membered aromatic ring
• CPC Class Codes: C07C 69/96 Esters of carbonic or haloformic acids
• CPC Class Codes: C07D 207/404 with only hydrogen atoms or radicals containing only hydrogen and carbon atoms directly attached to other ring carbon atoms, e.g. succinimide
• CPC Class Codes: C07D 207/46 with hetero atoms directly attached to the ring nitrogen atom
• CPC Class Codes: C07D 209/48 with oxygen atoms in positions 1 and 3, e.g. phthalimide
• CPC Class Codes: C07D 211/60 Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals
• CPC Class Codes: C07D 211/78 Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen
• CPC Class Codes: C07D 213/79 Acids; Esters
• CPC Class Codes: C07D 215/48 Carbon atoms having three bonds to hetero atoms with at the most one bond to halogen
• CPC Class Codes: C07D 239/54 as doubly bound oxygen atoms or as unsubstituted hydroxy radicals
• CPC Class Codes: C07D 239/545 with other hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
• CPC Class Codes: C07D 241/44 with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to carbon atoms of the hetero ring
• CPC Class Codes: C07D 261/12 Oxygen atoms
• CPC Class Codes: C07D 271/07 with oxygen, sulfur or nitrogen atoms, directly attached to ring carbon atoms, the nitrogen atoms not forming part of a nitro radical
• CPC Class Codes: C07D 303/14 by free hydroxyl radicals
• CPC Class Codes: C07D 303/16 by esterified hydroxyl radicals
• CPC Class Codes: C07D 317/36 Alkylene carbonates; Substituted alkylene carbonates
• CPC Class Codes: C07D 401/12 linked by a chain containing hetero atoms as chain links
• CPC Class Codes: C07D 405/10 linked by a carbon chain containing aromatic rings
• CPC Class Codes: C07D 405/12 : linked by a chain containing hetero atoms as chain links
• CPC Class Codes: C07D 417/12 : linked by a chain containing hetero atoms as chain links
• CPC Class Codes: C07D 417/14 : containing three or more hetero rings
• CPC Class Codes: C07D 495/04 : Ortho-condensed systems
• CPC Class Codes: C07D 519/00 : Heterocyclic compounds containing more than one system of two or more relevant hetero rings condensed among themselves or condensed with a common carbocyclic ring system not provided for in groups C07D453/00 or C07D455/00
• CPC Class Codes: G01N 33/6803 : General methods of protein analysis not limited to specific proteins or families of proteins
• CPC Class Codes: G01N 33/6842 : Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
• CPC Class Codes: G01N 33/6845 : Methods of identifying protein-protein interactions in protein mixtures
• CPC Class Codes: G01N 33/6848 : Methods of protein analysis involving mass spectrometry

Abstract

Capture compounds and collections thereof and methods using the compounds for the analysis of biomolecules are provided. In particular, collections, compounds and methods are provided for analyzing complex protein mixtures, such as the proteome. The compounds are multifunctional reagents that provide for the separation and isolation of complex protein mixtures. Automated systems for performing the methods also are provided.

Claims

1. A method for assessing interactions of a molecule Y with targets and non-targets, comprising:

(a) selecting a molecule whose interactions are to be assessed, and providing a capture compound that presents the molecule, wherein;

the capture compound has the formula;

2. The method of claim 1, wherein Y is selected from among a pharmaceutical drug or fragment, intermediate, metabolite or prodrug thereof, an enzyme substrate, inhibitor or co-factor, a receptor ligand, a transition state analog, or an ATP analog;

3. The method of claim 1, wherein Q is biotin or an oligonucleotide.

4. The method of claim 1, wherein Z is an amino acid.

5. The method of claim 1, wherein X is a photoactivatable group.

6. The method of claim 2, wherein X is a photoactivatable group.

7. The method of claim 1, wherein the activating condition for activating X is exposure to light, exposure to a change in pH or exposure to a change in temperature.

8. The method of claim 1 wherein the moiety Y is linked to the moiety Z in different orientations via different points of attachments on the Y moiety.

9. The method of claim 1, wherein:

X is selected from among an azide or a diazarine;

Z is an amino acid; and

Q is biotin or an oligonucleotide.

10. The method of claim 9, wherein Y is an enzyme substrate, inhibitor or co-factor.

11. The method of claim 1, wherein Y is an enzyme substrate, inhibitor or co-factor.

12. The method of claim 1, wherein X is

13. The method of claim 10, wherein X is

14. The method of claim 1, wherein following step (a) or (c), the capture compounds are immobilized on a solid support via Q, which binds to the surface of the support or a molecule thereon.

15. The method of claim 1, wherein Z is a moiety that is cleavable prior to or during mass spectrometric analysis of proteins bound to the capture compound.

16. The method of claim 1, wherein Z is a moiety that is not cleavable prior to or during mass spectrometric analysis of proteins bound to the capture compound.

17. The method of claim 1, wherein Q is an oligonucleotide or oligonucleotide analog.

18. The method of claim 15, wherein Z is a photocleavable, acid cleavable, alkaline cleavable, oxidatively cleavable, or reductively cleavable group.

19. The method of claim 1, wherein Z has the formula:

(S¹)_tM(R¹⁵)_a(S²)_bL, wherein;

S¹ and S² are spacer moieties;

t and b are each independently 0 or 1;

a is an integer from 0 to 4;

M is a central moiety possessing three or more points of attachment;

R¹⁵ is a monovalent group independently selected from Y²R¹⁸;

Y² is a divalent group independently having any combination of the following groups;

a direct link, arylene, heteroarylene, cycloalkylene, >

C(R¹⁷)₂, C(R¹⁷)═

C(R¹⁷), >

C═

C(R²³)(R²⁴), >

C(R²³)(R²⁴), C═

C, O, >

S(A)_u, >

P(D)_v(R¹⁷), >

P(D)_v(ER¹⁷), >

N(R¹⁷), >

N(COR¹⁷), >

N⁺(R²³)(R²⁴), >

Si(R¹⁷)₂ and >

C(E);

where u is 0, 1 or 2;

v is 0, 1, 2 or 3;

A is O or NR¹⁷;

D is S or O; and

E is S, O or NR¹⁷;

R¹⁷ and R¹⁸ are each independently selected from the group consisting of hydrogen, halo, pseudohalo, cyano, azido, nitro, SiR²⁷R²⁸R²⁵, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, aryl, aralkyl, aralkenyl, aralkynyl, heteroaryl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, heterocyclyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, hydroxy, alkoxy, aryloxy, aralkoxy, heteroaralkoxy and NR¹⁹R²⁰;

R¹⁹ and R²⁰ are each independently selected from hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heteroaryl, heteroaralkyl and heterocyclyl;

R²³ and R²⁴ are selected from (i) or (ii) as follows;

(i) R²³ and R²⁴ are independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl and heteroaryl;

or

(ii) R²³ and R²⁴ together form alkylene, alkenylene or cycloalkylene;

R²⁵, R²⁷ and R²⁸ are each independently a monovalent group selected from hydrogen, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, aryl, aralkyl, aralkenyl, aralkynyl, heteroaryl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, heterocyclyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, hydroxy, alkoxy, aryloxy, aralkoxy, heteroaralkoxy and NR¹⁹R²⁰;

R¹⁵, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²³, R²⁴, R²⁵, R²⁷ and R²⁸ can be substituted with one or more substituents each independently selected from Z²;

Z² is selected from alkyl, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkenyl, hydroxy, S(O)_hR³⁵;

h is 0, 1 or 2, NR³⁵R³⁶, COOR³⁵, COR³⁵, CONR³⁵R³⁶, OC(O)NR³⁵R³⁶, N(R³⁵)C(O)R³⁶, alkoxy, aryloxy, heteroaryl, heterocyclyl, heteroaryloxy, heterocyclyloxy, aralkyl, aralkenyl, aralkynyl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, aralkoxy, heteroaralkoxy, alkoxycarbonyl, carbamoyl, thiocarbamoyl, alkoxycarbonyl, carboxyaryl, halo, pseudohalo, haloalkyl and carboxamido;

R³⁵ and R³⁶ are each independently selected from among hydrogen, halo, pseudohalo, cyano, azido, nitro, trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl, triarylsilyl, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, aryl, aralkyl, aralkenyl, aralkynyl, heteroaryl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, heterocyclyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, hydroxy, alkoxy, aryloxy, aralkoxy, heteroaralkoxy, amino, amido, alkylamino, dialkylamino, alkylarylamino, diarylamino and arylamino; and

L is a group that is cleavable prior to or during mass spectrometric analysis of the compound.

20. The method of claim 19, wherein L is a disulfide moiety, a photocleavable group, an acid cleavable group, an alkaline cleavable group, a oxidatively cleavable group, or a reductively cleavable group.

21. The method of claim 1, wherein X is selected from among a diazirine, 3-trifluoromethyldiazirine and an azide;

Z is an amino acid and Q is biotin.

22. The method of claim 21, wherein Z is selected from among serine, threonine, lysine, tyrosine and cysteine.

23. The method of claim 1, wherein the capture compound comprises a mass modifying tag linked to Z.

24. The method of claim 1, wherein the capture compound is immobilized on a solid support before step (b).

25. The method of claim 1, wherein the capture compound is immobilized on a solid support after step (b).

26. The method of claim 1, wherein the capture compounds further comprise a solubility group W that influences the solubility properties of the capture compound.

27. The method of claim 1, further comprising identifying or detecting a captured protein by mass spectrometry.

28. The method of claim 1, wherein the sample comprises a biological sample, a body tissue or fluid or a cell lysate.

29. The method of claim 1, wherein Z has the formula:

(S¹)_tM(R¹⁵)_a(S²)_b, wherein;

S¹ and S² are spacer moieties;

t and b are each independently 0 or 1;

a is an integer from 0 to 4;

M is a central moiety possessing three or more points of attachment;

R¹⁵ is a monovalent group independently selected from Y²R¹⁸;

Y² is a divalent group independently having any combination of the following groups;

a direct link, arylene, heteroarylene, cycloalkylene, >

C(R¹⁷)₂, C(R¹⁷)═

C(R¹⁷), >

C═

C(R²³)(R²⁴), >

C(R²³)(R²⁴), C≡

C, O, >

S(A)_u, >

P(D)_v(R¹⁷), >

P(D)_v(ER¹⁷), >

N(R¹⁷), >

N(COR¹⁷), >

N⁺(R²³)(R²⁴), >

Si(R¹⁷)₂ and >

C(E);

wherein;

u is 0, 1 or 2;

v is 0, 1, 2 or 3;

A is O or NR¹⁷;

D is S or O; and

E is S, O or NR¹⁷;

R¹⁷ and R¹⁸ are each independently selected from the group consisting of hydrogen, halo, pseudohalo, cyano, azido, nitro, SiR²⁷R²⁸R²⁵, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, aryl, aralkyl, aralkenyl, aralkynyl, heteroaryl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, heterocyclyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, hydroxy, alkoxy, aryloxy, aralkoxy, heteroaralkoxy and NR¹⁹R²⁰;

R¹⁹ and R²⁰ are each independently selected from hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heteroaryl, heteroaralkyl and heterocyclyl;

R²³ and R²⁴ are selected from (i) or (ii) as follows;

(i) R²³ and R²⁴ are independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl and heteroaryl;

or

(ii) R²³ and R²⁴ together form alkylene, alkenylene or cycloalkylene;

R²⁵, R²⁷ and R²⁸ are each independently a monovalent group selected from hydrogen, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, aryl, aralkyl, aralkenyl, aralkynyl, heteroaryl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, heterocyclyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, hydroxy, alkoxy, aryloxy, aralkoxy, heteroaralkoxy and NR¹⁹R²⁰;

R¹⁵, R¹⁷, R¹⁸, R¹⁹, R²⁰, R²³, R²⁴, R²⁵, R²⁷ and R²⁸ can be substituted with one or more substituents each independently selected from Z²;

Z² is selected from alkyl, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkenyl, hydroxy, S(O)_hR³⁵;

h is 0, 1 or 2, NR³⁵R³⁶, COOR³⁵, COR³⁵, CONR³⁵R³⁶, OC(O)NR³⁵R³⁶, N(R³⁵)C(O)R³⁶, alkoxy, aryloxy, heteroaryl, heterocyclyl, heteroaryloxy, heterocyclyloxy, aralkyl, aralkenyl, aralkynyl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, aralkoxy, heteroaralkoxy, alkoxycarbonyl, carbamoyl, thiocarbamoyl, alkoxycarbonyl, carboxyaryl, halo, pseudohalo, haloalkyl and carboxamido; and

R³⁵ and R³⁶ are each independently selected from among hydrogen, halo, pseudohalo, cyano, azido, nitro, trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl, triarylsilyl, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, aryl, aralkyl, aralkenyl, aralkynyl, heteroaryl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, heterocyclyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, hydroxy, alkoxy, aryloxy, aralkoxy, heteroaralkoxy, amino, amido, alkylamino, dialkylamino, alkylarylamino, diarylamino and arylamino.

30. The method of claim 1, further comprising

re-designing the moiety Y to eliminate or alter its binding interactions with a captured protein.

31. The method of claim 1, further comprising identifying a function of a captured protein.

32. The method of claim 30, wherein the alteration in binding is an increase in binding.

33. The method of claim 30, wherein the alteration in binding is a decrease in binding.

34. The method of claim 30, wherein the protein for which binding is altered is a non-target protein.

35. The method of claim 1, wherein the sample is contacted with a plurality of different capture compounds.

36. The method of claim 30, wherein the method is repeated with the re-designed moiety Y linked to a capture compound to identify non-targets that bind thereto.

37. The method of claim 1, wherein a concentration of capture compound is varied in a plurality of different reactions.

38. The method of claim 27, wherein the mass spectrometry is selected from among matrix assisted laser desorption ionization (MALDI), continuous or pulsed electrospray (ES) ionization, ionspray, thermospray and massive cluster impact mass spectrometry.

39. The method of claim 38, wherein the mass spectrometry analysis is linear time-of-flight (TOF), reflectron time-of-flight, single quadruple, multiple quadruple, single magnetic sector, multiple magnetic sector, Fourier transform, ion cyclotron resonance (ICR) or ion trap.

40. The method of claim 28, wherein the function of the protein is determined by sequence alignment, pharmacophores, homology models and protein motif correlation, liver microsomes metabolic pathways, cDNA-expressed enzymes, signal pathways and back-mapping to yeast pathways, simulations and protein/protein interaction of pull-out proteins, native polymorphisms, knock-out/knock-in, flow cytometry, therapeutic activity of the drug, or prospective genotyping and prospective phenotyping.

41. The method of claim 30, wherein:

the moiety Y is a pharmaceutical drug or fragment, intermediate, metabolite or prodrug of the pharmaceutical drug; and

redesigning the drug results in a second drug with fewer side-effects or an increased therapeutic index compared to the first drug.