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 lysine selective agent to cap ε-amino groups;

(b) optionally reacting the sample of polypeptides with an amine reactive reagent to block α-amino groups;

(c) digesting the sample of polypeptides with a cleavage reagent to produce peptide fragments;

(d) optionally deactivating the cleavage reagent;

(e) removing those peptide fragments having uncapped or unblocked amino groups; and

(f) recovering the N-terminal peptide fragment or fragments.

2 . A method according to claim 1 , wherein the polypeptide or polypeptides comprise one or more N-terminal amine groups that are naturally unblocked, which method comprises reacting the sample of polypeptides with an amine reactive reagent to block α-amino groups according to step (b).

3 . A method according to claim 1 , wherein the polypeptide or polypeptides comprise N-terminal amine groups that are naturally blocked, which method does not comprise reacting the sample of polypeptides with an amine reactive reagent to block α-amino groups according to step (b).

4 . A method according to claim 1 , wherein non-N-terminal peptides and/or naturally unblocked N-terminal peptides are removed by capturing them on a solid phase and N-terminal peptides are recovered in solution.

5 . 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 lysine selective agent to cap ε-amino groups;

(b) contacting the resultant capped polypeptides with an amine reactive agent which reacts with the unblocked α-amino groups at the N-termini of the polypeptides;

(c) digesting the sample of polypeptides with a cleavage agent to produce peptide fragments;

(d) optionally deactivating the cleavage reagent; and

(e) recovering N-terminal peptides that have reacted with the amine reactive agent.

6 . A method according to claim 5 , wherein N-terminal peptides are recovered by capturing them on a solid phase and non-N-terminal peptides are removed in solution.

7 . A method according to claim 6 , wherein the amine reactive agent or the lysine selective agent is attached to a solid phase.

8 . A method according to claim 6 , wherein the amine reactive agent comprises biotin and the solid phase is an avidinated solid phase.

9 . A method according to claim 6 , wherein two or more samples are reacted with differently labelled amine reactive agents, and subsequently the samples are pooled and analysed simultaneously.

10 . A method according to claim 9 , wherein at least one of the amine reactive agents is labelled with deuterium and the samples are analysed by mass spectrometry.

11 . A method according to claim 1 , wherein only one molecule of the lysine selective agent reacts with each ε-amine group available in the peptides or polypeptides.

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

13 . 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 Sub comprises a hydrogen, a halogen, a hydrocarbon group or an electron withdrawing group.

14 . A method according to claim 13 , wherein one R comprises a methyl or phenyl group.

15 . A method according to claim 13 , wherein at least one R comprises an electron withdrawing group.

16 . A method according to claim 13 , wherein at least one R comprises a cyclic or heterocylic aromatic ring or fused ring.

17 . A method according to claim 13 , 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.

18 . A method according to claim 17 , wherein R 1 comprises an electron withdrawing group.

19 . A method according to claim 17 , wherein the ring comprises a phenyl, pyridyl, naphthyl quinolyl, pyrazine, pyrimidine or triazine ring structure.

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

21 . A method according to claim 20 , wherein the electron withdrawing group is selected from halogens, such as fluorine chlorine, bromine or iodine, and nitro and nitrile groups.

22 . A method according to claim 13 , wherein the X group comprises a structure capable of promoting water solubility.

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

24 . A method according to claim 1 , wherein the cleavage agent comprises a peptidase, cyanogen bromide or BNPS-Skatole.

25 . A method according to claim 24 , wherein the peptidase comprises trypsin, Lys-C or Arg-C.

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

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

28 . 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 N-termini for a predetermined N-terminal sequence of amino acid residues.

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

(a) recovering one or more N-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.

30 . 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 26 .

31 . A method according to claim 29 , which method comprises determining the identity of each of the peptides detected by mass spectrometry.

32 . A method according to claim 28 , which method comprises identifying the quantity of each of the peptides detected by mass spectrometry.

33 . 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 selective agent to attach the agent to ε-amino groups, wherein the lysine selective agent comprises a hindered Michael reagent.

34 . A method according to claim 33 , 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 B. 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 Sub comprises a hydrogen, a halogen, a hydrocarbon group or an electron withdrawing group.

35 . 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 Sub comprises a hydrogen, a halogen, a hydrocarbon group or an electron withdrawing group.

36 . A compound according to claim 35 , wherein at least one R group comprises a methyl or phenyl group.

37 . A compound according to claim 35 , wherein at least one R group comprises an electron-withdrawing group.

38 . A compound according to claim 37 , 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.

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

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

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

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

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

40 . A kit according to claim 39 , wherein the lysine selective 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 Sub comprises a hydrogen, a halogen, a hydrocarbon group or an electron withdrawing group.

41 . A kit according to claim 40 , wherein the lysine selective 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 Sub comprises a hydrogen, a halogen, a hydrocarbon group or an electron withdrawing group.

42 . A kit according to claim 40 , wherein the a means for recovering or isolating N-terminal peptides comprises a solid phase adapted for capturing peptides comprising free α-amino groups.

43 . A method for protecting ε-amino groups in peptides and polypeptides comprising using a compound having the following structure:

wherein R 1 comprises an alkyl group or an aryl group, including aromatic groups cyclic groups, fused cyclic groups, and heterocyclic groups, 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 Sub comprises a hydrogen, a halogen, a hydrocarbon group or an electron withdrawing group.

44 . The method according to claim 43 , wherein R 1 comprises a pyridyl, quinolyl, pyrazine, pyrimidine or triazine ring structure.

45 . The method according to claim 43 , wherein at least one R group comprises a methyl or phenyl group.

46 . The method according to claim 43 , wherein at least one R group comprises an electron-withdrawing group.

47 . The method according to claim 46 , 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.

48 . The method according to claim 41 , 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.

49 . The method according to claim 48 , 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.