Characterising polypeptides

1 . A method for characterising a polypeptide or a population of polypeptides, which method comprises the steps of:

(a) contacting a sample comprising one or more polypeptides with a cleavage reagent which cleaves one or more polypeptides on the C-terminal side of a lysine residue to produce peptide fragments;

(b) optionally deactivating the cleavage reagent;

(c) contacting the sample with a lysine reactive agent to cap ε-amino groups;

(d) removing those peptide fragments having capped ε-amino groups; and

(e) recovering the C-terminal peptide fragments.

2 . A method according to claim 1 , wherein peptide fragments having capped ε-amino groups are removed by capturing them on a solid phase and C-terminal peptides are recovered in solution.

3 . A method according to claim 2 , wherein the lysine reactive agent is covalently attached to a solid phase.

4 . A method according to claim 2 , wherein the peptide fragments having capped ε-amino groups are removed by affinity capture and wherein the lysine reactive agent comprises biotin and the solid phase is an avidinated solid phase.

5 . A method according to claim 1 , wherein the lysine reactive agent comprises a hindered Michael reagent.

6 . A method according to claim 1 , wherein the hindered Michael agent comprises a compound having the following structure:

wherein X is an electron withdrawing group that is capable of stabilising a negative charge; the R groups independently comprise a hydrogen, a halogen, an alkyl, an aryl, or an aromatic group with the proviso that at least one of the R groups comprises a sterically hindering group; and the group R 2 comprises a hydrogen, a halogen, a hydrocarbon group, an electron withdrawing group and/or a linker capable of attachment to an affinity capture functionality or a solid phase support.

7 . A method according to claim 6 , wherein one R comprises a methyl or phenyl group.

8 . A method according to claim 6 , wherein at least one R comprises an electron withdrawing group.

9 . A method according claim 6 , wherein at least one R comprises a cyclic or heterocylic aromatic ring or fused ring.

10 . A method according to claim 6 , wherein X comprises an —SO 2 R 1 group, wherein R 1 comprises an alkyl group or an aryl group, including aromatic groups cyclic groups, fused cyclic groups, and heterocyclic groups.

11 . A method according to claim 10 , wherein R 1 comprises an electron withdrawing group.

12 . A method according to claim 10 , wherein the ring comprises a phenyl, pyridyl, naphthyl quinolyl, pyrazine, pyrimidine or triazine ring structure.

13 . A method according to claim 6 , wherein the X group is substituted with an electron withdrawing group.

14 . A method according to claim 13 , wherein the electron withdrawing group is selected from halogens, such as fluorine chlorine, bromine or iodine, and nitro and nitrile groups.

15 . A method according to claim 6 , wherein the X group comprises a structure capable of promoting water solubility.

16 . A method according to claim 1 , wherein the cleavage agent comprises a sequence-specific cleavage agent.

17 . A method according to claim 1 , wherein the cleavage agent comprises a peptidase, or cyanogen bromide.

18 . A method according to claim 17 , wherein the peptidase comprises Lys-C.

19 . A method according to claim 1 , wherein the sample of step (a) comprises a sub-cellular fraction.

20 . A method according to claim 1 , which further comprises preparing the sample of step (a) by liquid chromatography.

21 . A method for assaying for one or more specific polypeptides in a test sample, which comprises performing a method according to claim 1 , wherein the sequence of the specific polypeptide is determined by assaying the resulting C-termini for a predetermined C-terminal sequence of amino acid residues.

22 . A method of characterising one or more mixtures of polypeptides, which method comprises the following steps:

(a) recovering one or more C-terminal peptides from the mixtures by employing one or more of the methods as defined in claim 1;

(b) detecting the peptides by mass spectrometry.

23 . A method for determining the expression profile of a sample, which method comprises characterising one or more mixtures of polypeptides according to a method as defined in claim 22 .

24 . A method according to claim 22 , which method comprises determining the identity of each of the peptides detected by mass spectrometry.

25 . A method according to claim 22 , which method comprises identifying the quantity of each of the peptides detected by mass spectrometry.

26 . A method for characterising a polypeptide or a population of polypeptides, which method comprises contacting a sample comprising one or more polypeptides with a lysine reactive agent to attach the agent to ε-amino groups, wherein the lysine reactive agent comprises a hindered Michael reagent.

27 . A method according to claim 26 , wherein the hindered Michael agent is a compound having the following structure:

wherein X is an electron withdrawing group that is capable of stabilising a negative charge: the R groups independently comprise a hydrogen, a halogen, an alkyl, an aryl, or an aromatic group with the proviso that at least one of the R groups comprises a sterically hindering group; and the group R 2 comprises a hydrogen, a halogen, a hydrocarbon group, an electron withdrawing group and/or a linker capable of attachment to an affinity capture functionality or a solid phase support.

28 . A compound having the following structure:

wherein R 1 comprises a pyridyl, quinolyl, pyrazine, pyrimidine or triazine ring structure and the R groups independently comprise a hydrogen, a halogen, or an alkyl or aryl group with the proviso that at least one of the R groups comprises a sterically hindering group; and the group R 2 comprises a hydrogen, a halogen, a hydrocarbon group, an electron withdrawing group and/or a linker capable of attachment to an affinity capture functionality or a solid phase support.

29 . A compound according to claim 28 , wherein at least one R group comprises a methyl, or phenyl group.

30 . A compound according to claim 28 , wherein at least one R group comprises an electron-withdrawing group.

31 . A compound according to claim 30 , wherein at least one R group comprises a halogen atom or a halogenated alkyl group, or a phenyl ring with one or more electron withdrawing substituents.

32 . A kit for characterising a polypeptide or a population of polypeptides, which kit comprises:

(a) a lysine reactive agent for capping ε-amino groups;

(b) a means for recovering or isolating C-terminal peptides;

(c) optionally an amine reactive reagent for labelling α-amino groups;

(d) optionally a cleavage reagent for producing peptide fragments.

33 . A kit according to claim 32 , wherein the lysine reactive agent comprises a compound having the following structure:

wherein X is an electron withdrawing group that is capable of stabilising a negative charge; the R groups independently comprise a hydrogen, a halogen, an alkyl, an aryl, or an aromatic group with the proviso that at least one of the R groups comprises a sterically hindering group; and the group R 2 comprises a hydrogen, a halogen, a hydrocarbon group, an electron withdrawing group and/or a linker capable of attachment to an affinity capture functionality or a solid phase support.

34 . A kit according to claim 33 , wherein the lysine reactive agent comprises a compound having the following structure:

wherein R 1 comprises a pyridyl, quinolyl, pyrazine, pyrimidine or triazine ring structure and the R groups independently comprise a hydrogen, a halogen, or an alkyl or aryl group with the proviso that at least one of the R groups comprises a sterically hindering group; and the group R 2 comprises a hydrogen, a halogen, a hydrocarbon group, an electron withdrawing group and/or a linker capable of attachment to an affinity capture functionality or a solid phase support.

35 . A kit according to claim 34 , wherein the means for recovering or isolating C-terminal peptides comprises an affinity capture agent attached to the lysine reactive agent, or a solid phase covalently bound to the lysine reactive agent.

36 . A method for protecting ε-amino groups in peptides and polypeptides comprising using the compound:

wherein R 1 comprises a pyridyl, quinolyl, pyrazine, pyrimidine or triazine ring structure and the R groups independently comprise a hydrogen, a halogen, or an alkyl or aryl group with the proviso that at least one of the R groups comprises a sterically hindering group; and the group R 2 comprises a hydrogen, a halogen, a hydrocarbon group, an electron withdrawing group and/or a linker capable of attachment to an affinity capture functionality or a solid phase support.

37 . The method according to claim 36 , wherein R 1 comprises a pyridyl, quinolyl, pyrazine, pyrimidine or triazine ring structure.

38 . The method according to claim 36 , wherein at least one R group comprises a methyl or phenyl group.

39 . The method according to claim 36 , wherein at least one R group comprises an electron-withdrawing group.

40 . The method according to claim 39 , wherein at least one R group comprises a halogen atom or a halogenated alkyl group, or a phenyl ring with one or more electron withdrawing substituents.

41 . The method according to claim 36 , wherein the protection is against further reaction of the ε-amino groups with Edman agents, capture agents and agents which are capable of reacting with α-amino groups.

42 . The method according to claim 41 , wherein the Edman agent comprises an isothiocyanate or an isocyanate, the capture agent comprises N-hydroxysuccinimidyl biotin and the agent which is capable of reacting with α-amino groups comprises acetic acid N-hydroxysuccinimide ester.