WO2009003952A2

WO2009003952A2 - Column and method for preparing a biological sample for protein profiling

Info

Publication number: WO2009003952A2
Application number: PCT/EP2008/058300
Authority: WO
Inventors: Katleen Verleysen; Koen Sandra; Robin Tuytten; Patrick Sandra
Original assignee: Pronota NV
Current assignee: Pronota NV
Priority date: 2007-06-29
Filing date: 2008-06-27
Publication date: 2009-01-08
Anticipated expiration: 2009-12-29
Also published as: WO2009003952A3

Abstract

The present invention relates to a liquid chromatography column having a solid support comprising at least one immobilised crown ether. It also relates to a use of a crown ether for preparing a biological sample for protein profiling. The invention also relates to a method of preparing a biological sample for protein profiling, comprising the steps of: treating (2) the sample (1) with a cleavage reagent to generate peptides (7) comprising N-terminal primary amines (3, 4, 5), and sorting (9, 10) the peptides (7) by non-covalent interactions using a solid support (11, 13), wherein the non-covalent interactions are H-bridges or pi-pi (π-π) interactions. It also relates to a kit therefor.

Description

COLUMN AND METHOD FOR PREPARING A BIOLOGICAL SAMPLE FOR PROTEIN

PROFILING

FIELD OF THE INVENTION The present invention is in the field of protein profiling. It is also in the field of biomarkers. Specifically, it is in the field of techniques and columns for protein profiling.

BACKGROUND OF THE INVENTION

Biomarkers are biological indicators that signal a changed physiological state due to a disease or a therapeutic intervention. According to the Food and Drug Administration

(FDA), an ideal biomarker should be specific, sensitive, predictive, robust, able to bridge pre-clinical and clinical trials, as well as being easily accessible, using a non-invasive procedure. From this description, proteins in easily accessible body fluids (preferentially plasma or serum) are the targets of choice to identify a set of biomarkers or molecular signatures that are indicators of certain disease states or of a response to therapeutic regimens.

Biomarker discovery has historically been dominated by targeted approaches, in which candidates derived from biological knowledge are evaluated for their correlations with biological conditions. More recently, the generation of protein profiles of the proteome by mass spectrometry, to monitor differences between disease states, has gained popularity. In this global non-directed approach, sera from different patients, diagnosed with a particular disease, are profiled, and the generated protein or peptide patterns are compared with protein or peptide patterns obtained from the corresponding controls. A major challenge encountered, when using serum as a proteome source, is the high dynamic range of proteins, known to exceed 10¹¹. Furthermore, 99% of the serum protein mass can be attributed to 22 proteins (Tirumalai, R. S., et al., MoI. Cell. Proteomics 2003, 10, 1096-1 103). The presence of these proteins will 'blind' the mass spectrometric analysis. If high abundance proteins are removed, proteins of interest can be identified over a relatively high dynamic range. However, the number of proteins and, after tryptic digestion, of peptides from serum or plasma can be tremendously high. When considering only 30 000 plasma proteins, with an average of 40 tryptic peptides per protein, one can expect 1 200 000 peptides, not taking any processing or modifications into account. Separation and subsequent analysis of all peptides present in such a complex mixture is a huge challenge using existing separation technologies. Nevertheless, many advances have been made throughout recent years. Surface-Enhanced Laser Desorption Ionization Time of Flight Mass Spectrometry (SELDI- TOF MS) (Veenstra, HJ. , et al., Biochem. Biophys. Res. Commun. 2002, 292, 587-592) is a technologies based on protein pattern recognition of generated MS spectra, but has the inherent limitation of not being able to provide potential biomarker identifications because of lack of accuracy in mass determination. The well established technique of two- dimensional polyacrylamide gel-electrophoresis (2D-PAGE) (Shevchenko, A., et al. Proc. Natl. Acad. Sci. U.S.A. 1996, 93, 14440-14445; Gorg, A. et al., Proteomics 2004, 4, 3665-3685), a gel-based approach, has been and still is the method of choice. In a first dimension, proteins are separated according to their isoelectric point (pi), followed by deconvolution according to size in the second dimension. Due to the limitations in dynamic range and difficult analysis of certain protein classes (e.g. membrane proteins, very acidic or basic proteins, large proteins, etc.) (Gygi, S. P., et al. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 9390-9395; Molloy, M. P., Anal. Biochem. 2000, 280, 1-10), the trend in proteomics is towards the development of non-gel based technologies. One approach is the Multidimensional Protein Identification Technology (MudPIT) (Washburn, M. P. et al. Nat. Biotechnol. 2001 , 19, 242-247) in which peptides are partitioned according to their charge using strong cation exchange chromatography (first dimension), followed by a separation of the collected fractions on reversed phase chromatography (separation based on hydrophobicity) in a second dimension.

Even with the addition of the second separation dimension and the resolving power of mass spectrometry, it still is impossible to detect all proteins in very complex biological samples (complex in terms of protein content and dynamic range). To overcome the shortcomings, one solution is to decrease the sample complexity, using some kind of presorting mechanism, either at protein level or peptide level (often referred to as a 'peptide signature approach'). ICAT (Isotope Coded Affinity Tag) is a quantitative method based on differential labelling and affinity purification of cysteine-containing peptides (Gygi, S. P., et al., Nat. Biotechnol. 1999, 17, 994-999). However, this cysteine-mediated peptide recovery sometimes yields more than one peptide per protein, resulting in an insufficient reduction of the total peptide content to be resolved and analyzed. On the other hand, not all proteins contain a cysteine in their amino acid backbone.

COFRADIC (COmbined FRActional Diagonal Chromatography), as a peptide signature approach, relies on a chromatography-based isolation of subsets of peptides, which are representative for their parent proteins (Gevaert, K., et al., J., MoI. Cell. Proteomics 2002, 1, 896-903). This very powerful and sensitive technique allows the simultaneous identification in serum of both highly abundant and very rare proteins, demonstrating a dynamic range of 10⁹. So far, COFRADIC has been used to isolate representative peptides, including methionyl (Gevaert, K., et al., MoI. Cell. Proteomics 2002, 1, 896-903), cysteinyl (Gevaert, K., et al., Proteomics 2004, 4, 897-908) and N-terminal (Gevaert, K., et al., Nat. Biotechnol. 2003, 21, 566-569) peptides. This approach is not restricted to peptides containing rare amino acids since all post-translational modified peptides that can be specifically altered fall into the application range (e.g. phospho-proteome (Gevaert, K., et al, Proteomics 2005, 5, 3589-3599)). The sorting of N-terminal peptides from a complex mixture is particularly useful because every protein is represented by only one peptide, reducing the sample's complexity to the highest degree.

While COFRADIC provides a detailed protein profile from a complex biological sample, the procedure can be time consuming. For example, a complex peptide mixture derived from the sample is fractionated by a first chromatographic separation. Subsequently, each fraction is subjected to a specific alteration reaction. Each fraction is then re-subjected to a second separation, under conditions identical to those in the first chromatographic step. As a consequence, all fractions collected in the first chromatographic run need to be rerun to achieve the sorting of peptides. Because of the large number of repetitive steps required to arrive at a profile, the procedure lacks high throughput and can be sensitive to minor variations during the sorting process.

The present invention, therefore, aims to overcome the problems of the art by providing a faster and more efficient method for reduction of the complexity of a sample for profiling. By applying the method, typically one peptide per protein is obtained, meaning the sample may be resolved into individual peptides using separation techniques such as high- resolution analytical chromatography.

DESCRIPTION OF THE FIGURES FIG. 1 : scheme depicting a method of the present invention whereby cleaved peptides may be sorted according to interactions with a solid support, the interactions being H- bridges or pi-pi interactions.

FIG. 2: scheme depicting a method of the present invention whereby the sample may be subject to pre-treatment steps to block reactive terminal or side chain amino acid moieties present in the protein. FIG. 3: scheme depicting a method of the invention, whereby cleaved peptides are sorted according to interactions with a solid support, the interactions being H-bridges, and the unbound peptides are subjected to profiling.

FIG. 4: scheme depicting a method of the invention, whereby cleaved peptides are sorted according to interactions with a solid support, the interactions being pi-pi interactions, and the unbound peptides are subjected to profiling.

FIG. 5: illustration of a bead on which a crown ether (host) is immobilized, forming H- bridges with a protonated primary amine, said crown ether immobilized by a linker to the bead.

FIG. 6: illustration of a bead on which an aromatic moiety is immobilized, forming pi-pi interactions with a modified primary amine, said aromatic moiety immobilized by a linker to the bead.

FIGs. 7-10 chromatographic traces of samples demonstrating the sorting capabilities of the invention.

FIG. 11 shows Total Ion Currents (TIC) and Extracted Ion Chromatograms (XIC) of the LC-MS/MS experiments.

FIG. 12 shows the location of the identified sequences within the respective proteins for the combined data of the "reference" and the "PNGase F POST" experiment (binned per

5). The location is determined by the position of the starting amino acid of the identified peptide. The bar graphs correspond to the number of unique sequences observed. The color reflects whether the sequence is N-terminally acetylated ([ight grey) or not (dark grey). Pane A summarizes the data for all unique modified sequences which are considered not to be glycopeptides, whereas pane B plots the same information for the glycopeptides.

SUMMARY OF SOME EMBODIMENTS OF THE INVENTION One embodiment of the invention is a liquid chromatography column having a solid support comprising at least one immobilised crown ether. Another embodiment of the invention is a column as described above, suitable for use in identifying proteins in a complex biological sample.

Another embodiment of the invention is a column as described above, wherein the immobilised crown ether is unsubstituted or substituted.

Another embodiment of the invention is a column as described above, wherein the immobilized crown ether is 18-crown-6-ether.

Another embodiment of the invention is a column as described above, wherein the host compound being the crown ether is immobilized on the solid support using a linker.

Another embodiment of the invention is a column as described above, wherein the linker is (poly)ethylene glycol, a reduced sugar, an acyclic dicarboxylic acid, etc..

Another embodiment of the invention is a column as described above, wherein the solid support is prepared from a native polymer, preferably a cross-linked carbohydrate material.

Another embodiment of the invention is a column as described above, wherein the native polymer material is any of agarose, agar, cellulose, dextran, chitosan, konjac, carrageenan, gellan, alginate.

Another embodiment of the invention is a column as described above, wherein the solid support is prepared from a synthetic polymer or copolymer, preferably a cross-linked synthetic polymer.

Another embodiment of the invention is a column as described above, wherein the synthetic polymer or copolymer is any of styrene or styrene derivatives, divinylbenzene, acrylamides, acrylate esters, methacrylate esters, vinyl esters, vinyl amides.

Another embodiment of the invention is a column as described above, wherein the solid support is prepared from silica. Another embodiment of the invention is a use of a crown ether for preparing a biological sample for protein profiling.

Another embodiment of the invention is a use as described above to identify proteins in a biological sample.

Another embodiment of the invention is a use as described above, wherein the crown ether is substituted or unsubstituted.

Another embodiment of the invention is a use as described above, wherein the crown ether is any as defined herein.

Another embodiment of the invention is a use as described above, wherein the crown ether is immobilised onto a solid support.

Another embodiment of the invention is a use as described above, wherein the solid support is in the form of beads, pellets, resin, small particles, a membrane, a frit, a sintered cake, or a monolith.

Another embodiment of the invention is a use as described above, wherein the solid support is comprised in a chromatography column, a phase extraction cartridge (SPE), magnetic bead, centrifugable or filterable bead.

Another embodiment of the invention is a use as described above, wherein the solid support is prepared from the materials as defined herein.

Another embodiment of the invention is a use as described above, comprising the identification of proteins by sorting peptides in the sample having one or more primary amines, following cleavage of the proteins by a cleavage reagent.

Another embodiment of the invention is a use as described above, wherein the cleavage reagent comprises any of serine protease, threonine protease, cysteine protease, aspartic acid protease, metalloprotease and glutamic acid protease.

Another embodiment of the invention is a use as described above, wherein the cleavage reagent comprises any of Lysobacter enzymogenes endoproteinase Lys-C, Staphylocolococus aureus endoproteinase GIu-C (V8 protease), Pseudomonos tragi endoproteinase Asp-N and clostripain.

Another embodiment of the invention is a use as described above, wherein the cleavage reagent comprises any of Bacillus subtilis subtilisin, procain pepsin and Tritirachium album proteinase K.

Another embodiment of the invention is a use as described above, wherein the cleavage reagent comprises trypsin.

Another embodiment of the invention is a use as described above, wherein the cleavage reagent comprises cyanogen bromide.

Another embodiment of the invention is a use as described above, wherein the peptides having one or more primary amines are N-terminal peptides.

One embodiment of the invention relates to a method of preparing a biological sample for protein profiling, comprising the steps of: pretreating the sample (1 ) with one or more reagents (20) to effect blocking of the primary amines, treating (2) the pretreated sample (1¹) with a cleavage reagent to generate peptides (7) comprising N-terminal primary amines (3, 4, 5), and sorting (9, 10) the peptides (7) by non-covalent interactions using a solid support (11 , 13), wherein the non-covalent interactions are H-bridges or pi-pi (π-π) interactions.

Another embodiment of the invention relates to a method as described above, wherein the cleavage reagent comprises any of serine protease, threonine protease, cysteine protease, aspartic acid protease, metalloprotease and glutamic acid protease.

Another embodiment of the invention relates to a method as described above, wherein the cleavage reagent comprises any of Lysobacter enzymogenes endoproteinase Lys-C, Staphylocolococus aureus endoproteinase GIu-C (V8 protease), Pseudomonos tragi endoproteinase Asp-N and clostripain. Another embodiment of the invention relates to a method as described above, wherein the cleavage reagent comprises any of Bacillus subtilis subtilisin, procain pepsin and Tritirachium album proteinase K.

Another embodiment of the invention relates to a method as described above, wherein the cleavage reagent comprises trypsin.

Another embodiment of the invention relates to a method as described above, wherein the cleavage reagent comprises cyanogen bromide, formic acid or hydroxylamine.

Another embodiment of the invention relates to a method as described above, where the solid support is in the form of beads, pellets, resin, small particles, a membrane, a frit, a sintered cake, or a monolith.

Another embodiment of the invention relates to a method as described above, where the solid support is comprised in a chromatography column, a phase extraction cartridge (SPE), magnetic bead, centrifugable or filterable bead.

Another embodiment of the invention relates to a method as described above, wherein the peptides are sorted by the solid support in a liquid chromatography mode or batch mode.

Another embodiment of the invention relates to a method as described above, comprising the step of blocking the primary amine groups and optionally the cysteine groups of proteins present in the sample prior to treatment with a cleavage reagent, and wherein the solid support (11 - Fig. 3) comprises an immobilized host compound that selectively binds protonated primary amines using H-bridges.

Another embodiment of the invention relates to a method as described above, wherein said host compound is an organic cyclic compound that provides a cylindrical or circular arrangement of hydrogen acceptor atoms at positions and orientations that maximise non- covalent binding with three H-atoms of a protonated primary amine.

Another embodiment of the invention relates to a method as described above, wherein the host compound is a crown ether or a macrolide antibiotic. Another embodiment of the invention relates to a method as described above, wherein the immobilized host compound is 18-crown-6 ether.

Another embodiment of the invention relates to a method as described above, wherein the immobilized 18-crown-6-ether is unsubstituted.

Another embodiment of the invention relates to a method as described above, wherein the immobilized 18-crown-6-ether is substituted.

Another embodiment of the invention relates to a method as described above, wherein the host compound is immobilized on the solid support using a linker.

Another embodiment of the invention relates to a method as described above, wherein the linker is (poly)ethylene glycol, a reduced sugar, an acyclic dicarboxylic acid, etc..

Another embodiment of the invention relates to a method as described above, further comprising the steps of:

- blocking the primary amine groups and optionally cysteine groups of proteins present in the sample prior to treatment with a cleavage reagent, - modifying the N-terminal primary amines of the peptides with at least one aromatic moiety (17 - FIG. 4) prior to sorting (9 - FIG. 4), wherein the solid support (13 - FIG. 4) comprises at least one immobilized aromatic moiety.

Another embodiment of the invention relates to a method as described above, wherein the aromatic moiety used to modify the N-terminal primary amines is an aryl, arylalkyl, heteroaryl or heteroarylalkyl.

Another embodiment of the invention relates to a method as described above, wherein the aromatic moiety immobilized on the solid support is an aryl, arylalkyl, heteroaryl or heteroarylalkyl.

In a further embodiment of the invention, the method is used for the enrichment of glycopeptides, preferably due to the addition of glycan moieties at asparagine (Asn, N), hereafter called N-glycopeptides, or due to the addition of glycan moieties at serine (Ser, S) or threonine (Thr, T), hereafter called O-glycopeptides. Another embodiment of the invention relates to a method as described above, wherein said aryl comprises any of phenyl, biphenylyl, biphenylenyl, 5- or 6-tetralinyl, 1-, 2-, 3-, 4-, 5-, 6-, 7- or 8-azulenyl, 1- or 2-naphthyl, 1-, 2- or 3-indenyl, 1-, 2- or 9-anthryl, 1- 2-, 3-, 4- or 5-acenaphtylenyl, 3-, 4- or 5-acenaphtenyl, 1-, 2-, 3-, 4- or 10-phenanthryl, 1- or 2- pentalenyl, 1 , 2-, 3- or 4-fluorenyl, 4- or 5-indanyl, 5-, 6-, 7- or 8-tetrahydronaphthyl, 1 ,2,3,4-tetrahydronaphthyl, 1 ,4-dihydronaphthyl, dibenzo[a,d]cylcoheptenyl, 1-, 2-, 3-, 4- or 5-pyrenyl.

Another embodiment of the invention relates to a method as described above, wherein said heteroaryl is any of 2- or 3-furyl, 2- or 3-thienyl, 1-, 2- or 3-pyrrolyl, 1-, 2-, 4- or 5- imidazolyl, 1-, 3-, 4- or 5-pyrazolyl, 3-, 4- or 5-isoxazolyl, 2-, 4- or 5-oxazolyl, 3-, 4- or 5- isothiazolyl, 2-, 4- or 5-thiazolyl, 1 ,2,3-triazol-1-, -2-, -4- or -5-yl, 1 ,2,4-triazol-1-, -3-, -4- or -5-yl, 1 ,2,3-oxadiazol-4- or -5-yl, 1 ,2,4-oxadiazol-3- or -5-yl, 1 ,2,5-oxadiazolyl, 1 ,3,4- oxadiazolyl, 1 ,2,3-thiadiazol-4- or -5-yl, 1 ,2,4-thiadiazol-3- or -5-yl, 1 ,2,5-thiadiazol-3- or - 4-yl, 1 ,3,4-thiadiazolyl, 1 - or 5-tetrazolyl, 2-, 3- or 4-pyridyl, 3- or 4-pyridazinyl, 2-, 4-, 5- or 6-pyrimidinyl, 2-, 3-, 4-, 5- 6-2H-thiopyranyl, 2-, 3- or 4-4H-thiopyranyl, 2-, 3-, 4-, 5-, 6- or 7-benzofuryl, 1-, 3-, 4- or 5-isobenzofuryl, 2-, 3-, 4-, 5-, 6- or 7-benzothienyl, 1-, 3-, 4- or 5- isobenzothienyl, 1-, 2-, 3-, 4-, 5-, 6- or 7-indolyl, 2- or 3-pyrazinyl, 1 ,4-oxazin-2- or -3-yl, 1 ,4-dioxin-2- or -3-yl, 1 ,4-thiazin-2- or -3-yl, 1 ,2,3-triazinyl, 1 ,2,4-triazinyl, 1 ,3,5-triazin-2-, - 4- or -6-yl, thieno[2,3-b]furan-2-, -3-, -4-, or -5-yl, 1-, 2-, 4- or 5-benzimidazolyl, 1-, 3-, 4-, 5-, 6- or 7-benzopyrazolyl, 3-, 4-, 5-, 6- or 7-benzisoxazolyl, 2-, 4-, 5-, 6- or 7- benzoxazolyl, 3-, 4-, 5-, 6- or 7-benzisothiazolyl, 2-, 4-, 5-, 6- or 7-benzothiazolyl, 1-, 2- thianthrenyl, 3-, 4- or 5-isobenzofuranyl, 1-, 2-, 3-, 4- or 9-xanthenyl, 1-, 2-, 3- or 4- phenoxathiinyl, 2-, 3-pyrazinyl, 1-, 2-, 3-, 4-, 5-, 6-, 7- or 8-indolizinyl, 2-, 3-, 4- or 5- isoindolyl, 1-, 2-, 3-, 4-, 5-, 6- or 7-indazolyl, 2-, 6-, 7- or 8-purinyl, 4-, 5- or 6-phthalazinyl, 2-, 3- or 4-naphthyridinyl, 2-, 5- or 6-quinoxalinyl, 2-, 4-, 5-, 6-, 7- or 8-quinazolinyl, 1-, 2-, 3- or 4-quinolizinyl, 2-, 3-, 4-, 5-, 6-, 7-, or δ-quinolinyl(quinolyl), 2-, 4-, 5-, 6-, 7- or 8- quinazolyl, 1-, 3-, 4-, 5-, 6-, 7- or δ-isoquinolinyl(isoquinolyl), 3-, 4-, 5-, 6-, 7- or 8- cinnolinyl, 2-, 4-, 6- or 7-pteridinyl, 1-, 2-, 3-, 4- or 9-carbazolyl, 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8- or 9-carbolinyl, 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9- or 10-phenanthridinyl, 1-, 2-, 3- or 4-acridinyl, 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8- or 9-perimidinyl, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9- or 10- (1 ,7)phenanthrolinyl, 1- or 2-phenazinyl, 1-, 2-, 3-, 4-, or 10-phenothiazinyl, 3- or 4- furazanyl, 1-, 2-, 3-, 4-, or 10-phenoxazinyl, azepinyl, diazepinyl, dibenzo[b,f]azepinyl, dioxanyl, thietanyl, oxazolyl dibenzo[a,d]cylcoheptenyl, or additionally substituted derivatives thereof. Another embodiment of the invention relates to a method as described above, wherein the alkyl of an arylalkyl, or heteroarylalkyl is any of methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, te/f-butyl, 2-methylbutyl, pentyl and its isomers, hexyl and its isomers, heptyl and its isomers, and octyl and its isomers.

Another embodiment of the invention relates to a method as described above, wherein the aromatic moiety used to modify the N-terminal primary amines is a pi-donor, when the aromatic moiety immobilized on the solid support is a pi-acceptor.

Another embodiment of the invention relates to a method as described above, wherein the aromatic moiety used to modify the N-terminal primary amines is a pi-acceptor, when the aromatic moiety immobilized on the solid support is a pi-donor.

Another embodiment of the invention relates to a method as described above, wherein a pi-acceptor is an aromatic moiety as defined above substituted with at least one electron- withdrawing group.

Another embodiment of the invention relates to a method as described above, wherein the electron-withdrawing group is any of NO₂, NH₃, SO₂OH, CN, CF₃, F, COOH, ⁺NR₃, ⁺NHR₂ or ⁺NH₂R, where R is an alkyl group.

Another embodiment of the invention relates to a method as described above, wherein said aromatic moiety comprises trinitrophenyl and/or pentafluorophenyl.

Another embodiment of the invention relates to a method as described above, wherein a pi-donor is an aromatic moiety as defined above substituted with at least one electron- donating group.

Another embodiment of the invention relates to a method as described above, wherein the electron-donating group is any of OH, OMe or NH₂, NR₂ or NHR, where R is an alkyl group.

Another embodiment of the invention relates to a method as described above, wherein said aromatic moiety comprises p-methoxyphenyl, 4-N,N-dimethylaminophenyl, or phenyl. Another embodiment of the invention relates to a method as described above, wherein said aromatic moiety is immobilized on the solid support or peptide by a linker.

Another embodiment of the invention relates to a method as described above, wherein said linker is (poly)ethylene glycol, a reduced sugar, an acyclic dicarboxylic acid, etc.

Another embodiment of the invention relates to a method as described above, wherein said pretreatment comprises the steps of blocking the cysteine groups followed by blocking the primary amine groups.

Another embodiment of the invention relates to a method as described above, wherein said primary amine groups are blocked, e.g. using N-hydroxysulfosuccinimidyl acetate.

Another embodiment of the invention relates to a method as described above, wherein said cysteine groups are blocked comprising the use of any of iodoacetamide, N- substituted maleimides, acrylamide, N-substituted acrylamide, tris(2- carboxyethyl)phosphine, or 2-vinylpyridine.

Another embodiment of the invention relates to a method as described above, further comprising the step of analytical separation of peptides not captured by the solid support, so providing a protein profile of the sample.

Another embodiment of the invention relates to a method as described above, wherein the analytical separation, preferably chromatography is one-, two-, three-, or higher- dimensional liquid chromatography.

Another embodiment of the invention relates to a kit for preparing a sample for protein profiling comprising one or more of the following components:

- a primary amine blocking reagent as defined above, - a cysteine group blocking reagent as defined above,

- cleavage reagent as defined above,

- host compound as defined above,

- aromatic moiety as defined above,

- solid support as defined above, - solid support as defined above, comprising one or more immobilized aromatic moieties as defined above, - solid support as defined above, comprising one or more immobilized host compounds as defined above,

- said solid support provided in a cartridge.

The inventors further surprisingly found that crown-ether functionalised solid supports such as crown ether-based columns as taught by the present invention can also be advantageously employed for enrichment of glycopeptides, in particular N-glycopeptides. N-glycopeptides typically comprise one or more N-linked glycan moieties, linked to Asn residue(s). More particularly, such N-glycopeptides tend to be recovered and enriched in a flow-through from crown-ether functionalised solid supports. It shall be understood that enrichment of O-glycopeptides, which typically comprise one or more O-linked glycan moieties, linked to Thr or Ser residue(s), will also be recovered and enriched in a flow- through from crown-ether functionalised solid supports.

Hence, in an aspect, the invention also provides a method for preparing a biological sample for protein profiling, comprising the steps of: treating a sample with a cleavage reagent to generate peptides, and sorting the peptides by non-covalent interactions using a solid support functionalised with crown ether, whereby glycopeptides are enriched from said peptides.

Another aspect is a use of a crown ether for preparing a biological sample for protein profiling, comprising the identification of proteins by sorting peptides in the sample having one or more linked glycan groups, following cleavage of the proteins by a cleavage reagent.

It shall be understood that the features relating inter alia to the sources of samples, preparation and pretreatment of samples, cleavage of samples to generate peptides and cleavage agents, crown ethers, solid supports and columns functionalised thereby, and sorting steps using such solid supports and columns, as well as further proteomic analysis such as analytical separation and characterisation of the enriched glycopeptides, as described elsewhere in this specification, also apply to the methods and uses of the above aspects. Optionally but not necessarily, the sample may be pretreated with one or more reagents to effect blocking of the primary amines, whereby N-terminal peptides may be co-isolated.

Preferably, the immobilized crown ether is 18-crown-6 ether, for example substituted or unsubstituted as taught herein. Preferably, it may be immobilized using a linker as taught herein.

In a particular embodiment, the crown-ether (CE) functionalised solid support may further be a cation exchange (CX) solid support. The inventors have found that the combination of CE and WCX (weak cationic exchange) moieties gives particularly effective enrichment of glycopeptides particularly in the flow-through.

The cation exchange solid support can alternatively be a strong cation exchange (SCX) column. Preferably, said WCX solid support is functionalised with one or more acidic moieties having pKa greater than 1 , more preferably greater than 2, even more preferably greater than 3, such as, e.g., between 1 and 7, or between 2 and 7, or between 3 and 6. In a non-limiting embodiment, a pKa of 3 is used.

In a non-limiting embodiment, said WCX or SCX solid support is functionalised with one or more moieties chosen from carboxylate and phosphonate.

In another non-limiting embodiment, said WCX or SCX solid support is functionalised with carboxylate and phosphonate moieties.

Versatility of the platform can further be achieved by adding/removing the N-termini and lysine acetylation step in the sample preparation procedure and by the timing of the deglycosylation step. This way, one can solely target N-terminally acetylated peptides, or N-terminally acetylated peptides and glycopeptides, or glycopeptides and in vivo acetylated peptides only.

In certain embodiments, the methods of the invention can be used for the enrichment of glycopeptides, preferably formed by addition of a glycan group at asparagine (N), serine (S) or threonine (T).

In a further embodiment, no N-terminal acetylation step is performed on the peptide mixture, in order to isolate in vivo glycosylated and N-acetylated peptides. In yet another embodiment, the peptide mixture is additionally pretreated with an N- terminal blocking agent, in order to identify or enrich in vivo glycosylated and in vivo and in vitro N-acetylated peptides.

In a further embodiment, a deglycosylation step is performed on the peptide mixture, in order to eliminate the glycosylated peptides from the analysis and enrich only the in vivo or in vitro N-acetylated peptides.

In a further embodiment, no additional N-acetylation step is performed on the peptide sample, in order to enrich only the in vivo N-acetylated peptides.

DETAILED DESCRIPTION OF THE INVENTION Unless defined otherwise, all technical and scientific terms used herein have the same meaning as is commonly understood by one of skill in the art. All publications referenced herein are incorporated herein by reference thereto. All United States patents and patent applications referenced herein are incorporated by reference herein in their entirety including the drawings.

The articles "a" and "an" are used herein to refer to one or to more than one, i.e. to at least one of the grammatical object of the article. By way of example, "a sample" means one sample or more than one sample.

Throughout this application, the term "about" is used to indicate that a value includes the standard deviation of error for the device or method being employed to determine the value.

The recitation of numerical ranges by endpoints includes all integer numbers and, where appropriate, fractions subsumed within that range (e.g. 1 to 5 can include 1 , 2, 3, 4 when referring to, for example, a number of samples, and can also include 1.5, 2, 2.75 and 3.80, when referring to, for example, concentrations). The recitation of end points also includes the end point values themselves (e.g. from 1.0 to 5.0 includes both 1.0 and 5.0)

Reference is made in the description below to the drawings which exemplify particular embodiments of the invention; they are not at all intended to be limiting. The skilled person may adapt the method and substitute components and features according to the common practices of the person skilled in the art

With reference to FIG. 1 , which gives a broad overview, the method of the present invention relates to preparing a biological sample, i.e. a complex protein mixture, 1 for protein profiling comprising the steps of:

- pretreating the sample 1 with one or more agents 20 to effect blockage of the primary amines,

- treating 2 the pretreated sample V with a cleavage reagent to generate peptides 7 comprising N-terminal primary amines 3, 4, 5, and

- sorting 9, 10 the peptides 7 by non-covalent interactions using a solid support 11 , 13 wherein the non-covalent interactions are H-bridges or pi-pi (π-π) interactions, respectively.

The resulting peptides are sorted into a set that binds to the solid support i.e. set 14 when using pi-pi interactions, and set 12 when using H-bridges. The method also provides a set that does not bind to the solid support i.e. set 16 when pi-pi interactions are used, and set 15 when H-bridges are involved. It would be expected that set 15 and 16 would comprise essentially identical peptides 6 for the same sample 1. Generally, the set 15, 16 of peptides that does not interact with the solid support 11 , 13 is used in further profiling, while the set 12, 14 retained on the solid support 11 , 13 is not used.

Using the method of the invention, the number of peptides derived from each protein in the sample is significantly reduced. The resulting peptides have been found by the inventors to be representative of the proteins present in the more complex biological sample. In most cases a single peptide represents a single protein. The peptides 15, 16 resulting from the method are profiled, meaning they are subjected to analytical separation(s) to determine the size and optionally the quantity of each peptide. From the molecular weight of each peptide and its fragmentation pattern, the corresponding protein present in the original sample is determined; analysis of all or several of the peptides provides a protein profile of the sample.

This method, which employs a single sorting step is a major step forward in high throughput peptide profiling. Since the method produces such a simplified peptide mixture, in most cases the mixture can be resolved using one dimensional liquid chromatography.

Typically, one peptide per protein is obtained, meaning a sample may be resolved into individual peptides. Thus, the method provides a significant time saving over techniques of the art, while also giving excellent resolution. For example, diagonal (2D) chromatographic analysis may entail at least thirteen separations which requires considerable time and hence cost expenditure. Bead related technologies can provide a high throughput, but do not have a good sorting efficiency.

Biological sample

The term "biological sample" as used herein refers to material, in a non-purified or purified form, from biological sources, including but not limited to human, animal, plant, insect, bacterial, viral or other sources. The terms include, for example, a cell, tissue, or organism, or extract thereof. A cell or tissue sample can comprise any cell type or tissue type present in a subject, organism, or biological system. Non-limiting examples of biological fluids include blood, serum, urine, plasma, cerebrospinal fluid (CSF), optic fluid (vitrius), semen, milk, interstitial fluid, saliva, sputum and/or synovial fluid. The sample can include a mixture of cellular and other components, including drug compounds and compositions, excipients, delivery vehicles, and/or assay reagents. The sample can include other drugs, nucleic acid molecules, infectious agents and/or components thereof. The sample can be applied to the method directly or can be processed, extracted, or purified to varying degrees before being used. The sample can be derived from a healthy subject or a subject suffering from a condition, disorder, disease or infection. For example, the subject is a human who has cancer, an inflammatory disease, autoimmune disease, metabolic disease, CNS disease, ocular disease, cardiac disease, pulmonary disease, hepatic disease, gastrointestinal disease, neurodegenerative disease, genetic disease, infectious disease, or viral infection.

Pre-treatment steps

Prior to the treatment 2 with a cleavage reagent, the sample 1 may be reacted with one or more blocking reagents to protect peptide reactive groups that may affect subsequent modification steps or would interact with the solid support. The blocking (protective) group is typically one that, after attachment, is non-reactive under the conditions of the method. Preferably, the reagents effect blockage of the primary amines.

According to one embodiment of the invention, shown in FIG. 2, the sample 21 may be treated with one or more blocking reagents 20, 24, simultaneously or sequentially (depicted), which reagents fall into the following classes: i) modifiers of protein cysteine residues e.g. 24. ii) modifiers of protein primary amines e.g. 20,

Suitable blocking reagents, as well as methods and conditions for attaching the blocking groups will be clear to the skilled person and are generally described in the standard handbooks of organic chemistry, such as Greene and Wuts, "Protective groups in organic synthesis", 3rd Edition, Wiley and Sons, 1999, which is incorporated herein by reference in its entirety.

Using reagent 24 of class i), the cysteine side chains (SH groups) of proteins in the sample 21 may be blocked. The blocking reagent 24 can be any that reacts selectively with cysteine side chains and results in a substituent which is non-reactive in subsequent reactions. Blocking can be performed using any known method. For example, the sample may be treated with reductant dithiothreitol (DDT) or Tris[2-carboxyethyl]phosphine hydrochloride (TCEP. HCI) to quantitatively reduce disulfide bonds and maintain monothiols in reduced state. After reduction of the cysteine bridges, the monthiols are alkylated using iodoacetamide in protein denaturing buffers. This step leads to the derivatization of cysteine side chains. Thus, the proteins present in the mixture may comprise SH-groups as their acetamide derivatives after treatment with blocking reagent of class i). Other blocking reagents, such as N-substituted maleimides, acrylamide, N- substituted acrylamide, 2-vinylpyridine, may alternatively be used.

Using reagent 20 of class ii), primary amines present in amino acid side chains and N- termini of proteins in a sample 21 may be blocked, resulting in a pretreated sample 22 comprising a set of modified proteins. The blocking reagent 20 can be any that reacts with primary amines and results in a substituent which is non-reactive in subsequent steps. The blocking reagent 20 can be substituted once or twice onto each primary amine (i.e. - NH₂ gives -NHX or -NX₂, where X is the substituent introduced by the blocking reagent). An example of a suitable blocking reagent 20 is N-hydroxysulfosuccinimidyl acetate, which leads to acetylation of the primary amine. Other suitable blocking reagents have been extensively described in the art, for example, in Regnier et al., Proteomics 2006, 6, 3968- 3979. The blocking procedure can be applied according to known protocols, such as incubation in buffered phosphate at 30 deg C for 90 minutes. After treatment 2 with a cleavage reagent, a set 28 of peptides is generated comprising peptides with unmodified N-terminal primary amines and peptides 30 with protected N-termini. The latter are a result of blocking at the original protein N-terminus by the non-selective primary amine blocking reagent 20. The treatment with an SH-group blocker 24 preferably occurs prior to treatment with a primary amine blocker 20 (class i)) as depicted in FIG. 2. After treatment with blocking reagent(s), the resulting sample may then optionally be purified, using techniques known per se, such as evaporation of solvent, washing, filtration, and/or chromatographic techniques.

Pre-treatment ii) and optionally i) results in a pretreated sample 22 comprising blocked proteins which are cleaved to form a set of peptides 28 (FIGS. 2, 3 and 4). Treatment of this set of peptides 28 with a solid support that induces non-covalent interactions that are H-bridges or pi-pi interactions, allows selection a sub-set of peptides that does not bind to the solid support and which is subjected to profiling.

Further, the inventors have found that glycan modifications proximal to the α-NH₂ terminus of peptides may interfere with [crown ether — H₃N⁺-group] complexation. Also, glycan modifications proximal to primary amino groups appear to at least partly hinder the acetylation of the latter. Therefore, to improve said complexation or said acetylation in embodiments requiring this, and thereby to improve the selectivity of the present sorting methods for N-terminal peptides, a protein or peptide deglycosylation pre-treatment step may also be included, preferably before crown-ether based sorting of the peptides, and more preferably before acetylation or other manners of blocking the primary amino groups of the peptides. Removal of glycan modifications would also prevent enrichment of N- glycopeptides (glycan moiety on Asn residue) by crown-ether-based stationary phases, thereby further improving selectivity of such columns, methods and uses towards N- terminal peptides. Removal of glycan modifications may be achieved using conventional treatments, such as without limitation use of N-Glycosidase F (PNGase F) to remove N- linked glycan modifications, or the likes for O-linked glycan (glycan moiety on Thr or Ser residue) modifications.

In other embodiments, particularly wherein the crown-ether-based columns, methods and uses of the invention aim to enrich for glycopeptides, primary amines need not be blocked. Versatility of the platform can be achieved by adding/removing the N-termini and lysine acetylation step in the sample preparation procedure and by the timing of the deglycosylation step. This way, one can solely target N-terminally acetylated peptides, or N-terminally acetylated peptides and glycopeptides, or glycopeptides and in vivo acetylated peptides only. Cleavage reagent

The pretreated sample 1¹, 22 is subjected to treatment 2 with a cleavage reagent to generate a set of peptides (e.g. 28, FIG. 2) comprising N-terminal primary amines. The set of peptides also comprises peptides 30 having blocked terminal amines. The treatment uses cleavage reagents and methods described in the art such as chemical or enzymatic cleavage or digestion. In a preferred aspect, the cleavage reagent comprises a proteolytic enzyme. Trypsin is a particularly preferred enzyme because it cleaves at the sites of lysine and arginine, yielding charged peptides which typically have a length from about 5 to 50 amino acids and a molecular weight of between about 500 to 5,000 dalton. Such peptides are particularly appropriate for analysis by mass spectroscopy.

A non-limiting list of proteases, which may also be used in this invention, includes serine proteases, threonine proteases, cysteine proteases, aspartic acid proteases, metalloproteases and glutamic acid proteases.

Specific enzymes include, but are not limited to Lysobacter enzymogenes endoproteinase Lys-C, Staphylococcus aureus endoproteinase GIu-C (V8 protease), Pseudomonos tragi endoproteinase Asp-N and clostripain.

Proteases with lower specificity such as Bacillus subtilis subtilisin, procain pepsin and Tritirachium album proteinase K may also be used in this invention.

Alternatively, chemical reagents may also be used to cleave the proteins into peptides. For example, cyanogen bromide may be used to cleave proteins into peptides at methionine residues. In which case the cleavage reagent comprises cyanogen bromide. Chemical fragmentation can also be applied by limited hydrolysis under acidic conditions using formic acid (HCOOH) for example. Alternatively, BNPS-skatole may be used to cleave at the site of tryptophan. Alternatively, hydroxylamine (H₂NOH) may be used.

The conditions for treatment e.g. buffer, temperature, time, can be determined by the person skilled in the art, depending on the enzyme or chemical reagent employed. Where subsequent peptide separation involves the use of crown ethers and particularly 18- crown-6 ethers, cleavage may preferably be performed in conditions substantially free of potassium and ammonium ions, since said ions tend to display affinity for crown ethers and particularly for 18-crown-6 ethers. By means of example and not limitation, cleavage may be performed in a sodium bicarbonate buffer, preferably of relatively low molarity. It will be obvious that cleavage treatment does not necessarily result in all the peptides having an N-terminal primary amine, if the N-terminal primary amine of the native protein has been blocked.

Solid support

The solid support 11 , 13 used in the method is art-recognised and includes any solid support useful for chromatographic separation or solid-phase extraction as described herein. A solid support can be a resin (e.g. a polymer-based material), a hybrid organic/inorganic material, or other solid support forms known to one of ordinary skill in the art. A solid support can be in the form of, e.g., beads, pellets, resin, small particles, a membrane, a frit, a sintered cake, or a monolith or any other form desirable for use. The solid support particles can have, for example, a spherical shape, a regular shape or an irregular shape.

The solid support may be comprised in a chromatography column as a chromatography matrix, in a phase extraction cartridge (SPE), in a magnetic bead, in a centrifugable or filterable bead or in any other known format suitable for separations.

The requirements for a solid support are known in the art, being any that is chemically inert, which exhibits low or no residual non-specific interactions.

The support may be made from an organic or inorganic material. In one embodiment, the support is prepared from a native polymer, such as cross-linked carbohydrate material, e.g. agarose, agar, cellulose, dextran, chitosan, konjac, carrageenan, gellan, alginate, etc.. The native polymer supports are easily prepared and optionally cross-linked according to standard methods, such as inverse suspension gelation (Hjerten, S. Biochim Biophys Acta 1964, 79, 393-398). In an especially advantageous embodiment, the support is a type of relatively rigid but porous agarose, which is prepared by a method that enhances the flow properties of the support, see e.g. US 6,602,990 (Berg) or SE 0402322-2 (Berg et al.) In an alternative embodiment, the support is prepared from a synthetic polymer or copolymer, such as cross-linked synthetic polymers, e.g. styrene or styrene derivatives, divinylbenzene, acrylamides, acrylate esters, methacrylate esters, vinyl esters, vinyl amides, etc.. Such synthetic polymers are easily prepared and optionally cross-linked according to standard methods, see e.g. "Styrene based polymer supports developed by suspension polymerization" (Arshady, R. Chimica e L'lndustria 1988, 70, 70-75). Native or synthetic polymer supports are also available from commercial sources, such as GE Healthcare, Uppsala, Sweden, for example in the form of porous particles. In yet an alternative embodiment, the support is prepared from an inorganic polymer, such as silica. Inorganic porous and non-porous supports are well known in this field, some of which are commercially available . Examples of commercially available matrix materials include, but are not limited those based on silica, polystyrene, sepharose®, sepharopore™, and other variations thereof. The skilled person will choose the matrix material based on the expected unwanted non-specific interactions, capacity, loadability and flow characteristics.

Suitable particle sizes may be in the diameter range of 5-500 μm, such as 10-100 μm, e.g. 20-80 μm. In the case of essentially spherical particles, the average particle size may be in the range of 5-1000 μm, such as 10-500 μm. In a specific embodiment, the average particle size is in the range of 10-200 μm. The skilled person in this field can easily choose the suitable particle size and porosity depending on the process to be used. For example, for a large scale process, for economical reasons, a more porous but rigid support may be preferred to allow processing of large volumes, especially for the capture step. In chromatography, process parameters such as the size and the shape of the column will affect the choice.

The solid support should allow for the immobilization of one or more moieties that interact with peptides (from cleaved proteins) by non-covalent interactions using H-bridges (e.g. modified with crown ethers) or pi-pi interactions (e.g. modified with phenyl or pentafluorophenyl). Methods for immobilization of such moieties to solid supports are well known in this field; see e.g. Immobilized Affinity Ligand Techniques, Hermanson, G. T. et a/, Academic Press, INC, 1992; Combinatorial Chemistry, Eds: Bannwarth, Willi, Hinzen, Berthold, Wiley-VCH.

H-bridges

According to one aspect of the invention, the sorting of peptides by non-covalent interactions uses H-bridges. Reference is made in this aspect to FIG. 3, which depicts the step of sorting 10 a set of peptides 28 obtained by pretreating sample 21 with blocking reagents of classes i) and ii), and cleavage 2 as describe above.

The set 28 of peptides 3, 30, 41 , 42 resulting from the earlier cleavage 2 is applied to the solid support 11. Sorting of the peptides 3, 30, 41 , 42 depends on H-bridges formed with the host attached to the solid support (see FIG. 5). After sorting 10, two sets of peptides result, one set 46 comprising peptides 3, 41 , 42 that interact with the solid support 11 by non-covalent H-bridges, the other set 45 comprising peptides 30 that do not interact with the solid support 11.

The H-bridges preferably involve N-terminal primary amines, generated by the cleavage step. The H-bridges preferably involve N-terminal primary amines that have been protonated. Protonation may be achieved, for example, by lowering the pH of the solution of the peptides that will interact with the host of the solid support to less than 9.0, 8.8, 8.6, 8.4, 8.2, 8.0, 7.8, 7.6, 7.4, 7.2, 7.0 or below. Preferably, the pH of the solution of the peptides applied to the solid support is less than or equal to 7.0.

According to one embodiment of the invention, at least three H-bridges, preferably six are formed between the peptide and the host on the solid support. The H-bridges may be formed between hydrogen atoms (hydrogen donor) present in the peptide N-terminus and hydrogen acceptor atoms present in the host immobilised on the solid support, such as oxygen or nitrogen atoms. The arrangement of hydrogen acceptor atoms in the solid support maximises H-bridging e.g. is in a geometry which permits all the donating hydrogen atoms in the N-terminus to be shared via H-bridges with acceptor atoms in the solid support.

The inventors have found that certain compounds, known as 'hosts' are preferred. These host compounds are typically organic cyclic compounds, that provide a cylindrical or circular arrangement of hydrogen acceptor atoms (e.g. oxygen or nitrogen) at positions and orientations which maximise non-covalent binding with three H-atoms of a protonated primary amine (a guest compound). Typically a host compound will form three or six strong H-bridges with the protonated primary amine. Interaction is most efficient when combination with the guest causes no or very little distortion of the host, or simply put, when the guest fits in the cavity of the preorganized host molecule. These host compounds, when immobilized on the solid support, provide effective sorting of the peptides by non-covalent interactions using H-bridges. One embodiment of the invention is a solid support provided with a host compound having these properties. The host may or may not be substituted. Generally, the substitution refers to a substitution on the crown the ring which is in addition to any covalent attachment (i.e. other substitution) to a solid support. Examples of suitable host compounds include crown ethers and macrolide antibiotics. A particularly strong interaction is observed between substituted or un-substituted ammonium ions and 18-crown-6 molecules; a preferred host compound is, therefore, the 18-crown-6 molecule.

It will be clear to the skilled person that some of the compounds of the invention may exist in the form of different isomers and/or tautomers, including but not limited to geometrical isomers, conformational isomers, and stereochemical isomers (i.e. enantiomers and diastereoisomers) and isomers that correspond to the presence of the same substituents on different positions of the rings present in the compounds of the invention. All such possible isomers, tautomers and mixtures thereof are included within the scope of the invention.

The H-bridge non-covalent interactions preferably use a host compound, immobilized on the solid support, as a hydrogen acceptor. The host compound may be immobilized on the solid support using covalent or non-covalent attachment means. Where covalent attachment is used, the host may be immobilized using, for example, a CNBr-activated sepharose. The use of 2-aminomethyl-18-crown-6 ether is preferred as it possesses a handle to immobilise the host onto a solid support. Non-covalent attachment can be achieved using a binding pair, whereby one pair is attached to the column, and the other is attached to the compound (e.g. streptavidin/biotin, avidin/biotin, Ni²⁺/His6, etc.). The skilled person will choose components of the binding pair that show no or little interference with the intended non-covalent interactions between the host compound and the peptides.

The host compound may be immobilized on the solid support using a linker or spacer compound. These linkers or spacers are well known in the art, and are generally chemically inert insofar as they show little interference with the interaction between the host compound and the primary amine.

The skilled person will be aware of the types of linkers available and the properties of each, and will select the most suitable linker for the intended application. Examples of parameters that may be considered by the skilled person include cyclic or acyclic chain length, presence of hetero atoms and/or functional groups.

Generally, the linker is of sufficient length to avoid steric interference of the solid support with the intended interaction between peptides and the host compound. To avoid non- specific hydrophobic interactions between peptides and the spacer, the linker may have hydrophilic character. Suitable linker arms: (poly)ethylene glycol, reduced sugars, acyclic dicarboxylic acids, etc..

Methods for immobilisation of such linkers to solid supports are well known in this field; see e.g. Immobilized Affinity Ligand Techniques, Hermanson, G. T. et al,, Academic Press, INC, 1992; Combinatorial Chemistry, Eds: Bannwarth, Willi, Hinzen, Berthold, Wiley-VCH.

FIG. 5 depicts 18-crown-6-ether 61 bound to a protonated primary amine 62 via H- bridges. The crown ether 61 is immobilized on a bead 11' using a linker 60, so forming the solid support 11 suitable for capturing protonated primary amines 62.

Commercially available chromatography columns functionalized with a crown ether may be used in the present method as an alternative to (a column prepared from) the solid support described above. These include, but are not limited to:

- Crownpak: primarily used for chiral separations of small molecules, containing primary amines, has a silica support with a particle diameter of 5 μm. The column is functionalized with chiral phenylnaphtalene-substituted crown ethers and can operate in the pH range of 1-9. The column can withstand an organic modifier of maximum 15% CH₃OH (no other modifiers allowed) and can operate within a temperature range of -5°C to 50⁰C and a pressure range of < 150/200 kg/cm₂ (< 147/196 bar) .

- Dionex CS 15: primarily used as a cation exchange column, has a PS/DVB support with particle diameter of 8.5 μm and is medium hydrophilic. The column contains carboxylic acid, phosphonic acid and crown ether functional groups. The column tolerates acidic eluents and can withstand the organic modifier ACN, but not alcohols (CH₃OH). The column can also operate at a temperature of at least 40⁰C.

After sorting 10, the bound peptides 3, 41 , 42 (FIG. 3) may be washed from the solid support 11 , using a solvent with a different ionic or pH composition compared to the solvent applied during sorting conditions. Examples of suitable washing solvents include buffered 1 M saline at pH 7.0, buffered saline (low salt) at pH 9.0, phosphate buffers or any other buffers known in the art, that do not contain primary amines, ammonium, Na⁺, K⁺ or any other molecule that could compete with the analyte for the host. This step regenerates the support which can be used for subsequent preparations. One embodiment of the present is a use of a crown ether for preparing a biological sample for protein profiling. The skilled person will understand that the crown ether may be employed in this way using the steps and materials disclosed herein. Further embodiments are given below.

Another embodiment of the present is a use of a crown ether to identify proteins in a complex biological sample. The crown ether may be any as described herein. It may be substituted or unsubstituted. Preferably the crown ether is 18-crown-6-ether.

Another embodiment of the invention is a use of a crown ether as described above, comprising the identification of proteins by sorting peptides in the sample having one or more primary amines, following enzymatic cleavage of the proteins.

Another embodiment of the invention is a use of a crown ether as described above, wherein the peptides having one or more primary amines are N-terminal peptides.

Another embodiment of the invention is a use of a crown ether as described, wherein the biological sample is any as described elsewhere herein. Another embodiment of the invention is a use of a crown ether as described, wherein the enzymatic cleavage is achieved as described elsewhere herein.

Another embodiment of the invention is a use of a crown ether as described wherein the crown ether is attached to a solid support. The solid support may be any as described herein. Preferably, the solid support is provided in a liquid chromatography column or a Solid-Phase Extraction cartridge format.

Pi-Pi interactions

According to one aspect of the invention, the sorting is performed using a solid support wherein the non-covalent interactions are pi-pi (π-π) interactions. Pi-pi interactions refer to the binding interactions when pi-electrons of at least one member of a binding pair are shared between both members of the binding pair. This effect is well known in aromatic ring stacking, where pi-electrons are delocalized, enhancing the affinity between the rings. It is observed, for example, in stacked duplex DNA and RNA structures where pi-pi interactions stabilize the double helix. Pi-pi interactions may be enhanced when they are between an electron donor (pi-donor) and an electron acceptor (pi-acceptor), involving the transfer of electron density from a pi- orbital in the pi-donor to the pi-acceptor. The role of the pi-acceptor is to receive electron density from the pi-donor. The pi-acceptor may have vacant orbitals which can accommodate the electrons donated by the donor.

An example of this kind of donor-acceptor complex formation is provided by the interaction of electron-rich aromatic (pi-donor) and electron-poor aromatic (pi-acceptor) systems. Another example of this type of donor-acceptor complex can be illustrated by the interaction between a metal ion (pi-acceptor) and an olefin (pi-donor). No matter what the nature of the interaction is, the net result is a transfer of pi-orbital electron density from donor to acceptor. Many electron-donor-electron-acceptor complexes are unstable and exist only in solution in equilibrium with their components. The sorting based on pi-pi interactions described herein can make use of such an equilibrium involving a pi-acceptor on a stationary phase and a pi-donor in a mobile phase, or vice versa. In the present invention, interaction between the pi-donor and pi-acceptor can formally be visualized as an electron-rich aromatic system stacked onto an electron-poor aromatic system.

The solid support may be provided with one or more pi-donors or one or more pi- acceptors which will bind compounds in the mobile phase having pi-acceptor or pi-donor groups respectively.

The compounds in the mobile phase that can form an electron-donor-electron-acceptor complex with the stationary phase will be retained longer on the column and will elute later than compounds not capable of forming such a complex.

When the primary amines of internal peptides obtained after tryptic digest are modified with 2,4,6-trinitrobenzenesulfonic acid, peptide fragments are obtained which terminate in a strong pi-acceptor. These fragments will interact strongly with the aromatic rings of a commercially available phenyl column (pi-donor) and can be selectively retained from the mixture. This approach is not restricted to 2,4,6-trinitrobenzene groups. Any aromatic system with strong pi-acceptor properties can be used.

The previous approach may, of course, be inverted. When peptide fragments are equipped with an electron-rich aromatic system (pi-donor), they can be selectively removed with a commercially available pentafluorophenyl (pi-acceptor) column. It should be clear that application of pi-pi interactions for sorting of peptide mixtures is not limited to the examples mentioned above. In fact, any pair of aromatic systems, preferably those capable of forming an electron-donor-electron-acceptor complex can be used for sorting of peptide mixtures and is within the scope of the present invention. Examples of aromatic moieties suitable for immobilization on the solid support or for modifying peptides are given further below.

The pi-pi interactions preferably involve the N-terminal primary amines, generated by the cleavage treatment step 2 (FIG. 1, 2), which have been modified with one or more aromatic moieties. The set of peptides, not captured by the solid support, is subjected to profiling.

FIG. 4 depicts the step of sorting 9 a set 28 of peptides 3, 30, 41 , 42 obtained by pretreating sample 21 with blocking reagents of classes i) and ii), followed by cleavage 2 as describe above. The set of peptides 28 is treated with a reagent 17 that modifies the primary amines present in the peptides, with one or more aromatic moieties. The result is peptides comprising such modified amines 47, 48, 49, and peptides not modified 30. The latter group results from blocking pretreatment as described above, which generates peptides 30 blocked (FIG. 2) at the original protein N-terminus.

When the peptides 30, 47, 48, 49 are applied to the solid support, only those 50 modified with the aromatic moiety bind, while those that do not 51 may be washed away. Typically the loading mobile phase is aqueous in nature comprising a (low) percentage of organic modifier (e.g. ACN or methanol) in order to minimize any unspecific binding of peptides. The skilled person will be aware that the percentages of added modifier, the applied flow rates, temperatures,... are optimised to retain the peptides 50 on the support 13 and to prevent any premature elution of the bound peptides 50. The peptides that do not bind 51 are subject to profiling (see below).

After sorting, the bound peptides 47, 48, 49 may be eluted from the solid support 13, using a solution comprising high percentages of a water miscible solvent with hydrophobic properties such as acetonitrile (ACN), an alcohol (e.g. methanol, ethanol) or other solvents known in the art of reversed phase separation. ACN is preferentially used for fast elution of the bound peptides as it exhibits -next to its hydrophobic action- strong pi-pi interactions itself. This elution step regenerates the support so it can be used for subsequent preparations.

Aromatic moieties The N-terminal primary amines are modified with one or more aromatic moieties 17 (FIG. 4) when pi-pi interactions are used. Similarly, the solid phase is provided with one or more aromatic moieties, able to capture, through pi-pi interactions, the peptides so-modified. Aromatic moieties mostly exhibit pi-orbital character and are suitable for functionalizing the solid phase, or for modifying the peptides.

Preferably, aromatic moieties for modifying N-terminal primary amines include those comprising pi-acceptor groups; such modification is used when the solid phase is provided with pi-donor groups.

Alternatively, preferred aromatic moieties for modifying N-terminal primary amines are those comprising pi-donor groups; such modification is used when the solid phase is provided with pi-acceptor groups.

Pi-acceptors According to one aspect of the invention, the aromatic moiety is an aryl, arylalkyl, aryloxy, heteroaryl, heteroarylalkyl group. Each group may be optionally substituted with at least one (e.g. 2, 3, 4, 5, 6 or more) electron withdrawing group, so forming a pi-acceptor. The substitution refers to a substitution on an aromatic ring that is in addition to any covalent attachment (i.e. other substitution) by the aromatic moiety to a solid support. Electron- withdrawing substituents, such as nitro groups or fluorine atoms, drastically lower the electron density in an aromatic ring, so turning it into a pi-acceptor. Examples of electron- withdrawing substituents include but are not limited to acyl (-COR), nitro (-NO₂), fluorine (- F), and ammonium (-⁺NR₃, -⁺NHR₂, -⁺NH₂R) groups, where R is an alkyl group as described below. Suitable pi-acceptor aromatic moieties are, for example, trinitrobenzene (TNB) and/or pentafluorophenyl.

Pi-donors

According to another aspect of the invention, the aromatic moiety is an aryl, arylalkyl, aryloxy, heteroaryl, heteroarylalkyl, each group being optionally substituted with at least one (e.g. 2, 3, 4, 5, 6 or more) electron donating group, so forming a pi-donor. The substitution refers to a substitution on an aromatic ring that is in addition to any covalent attachment (i.e. other substitution) by the aromatic moiety to a solid support. Examples of electron-donating substituents include, but are not limited to hydroxyl (-OH), methoxy (- OMe) or amino (-NR₂, -NHR) groups, where R is an alkyl group as described below. According to one aspect of the invention, the aromatic moiety is not substituted. A suitable pi-donor aromatic moiety comprises of phenyl, p-methoxyphenyl, 4-N,N- dimethylaminophenyl, etc..

The term "aryl" as used herein by itself or as part of another group refers but is not limited to monocyclic, bicyclic, tricyclic or tetracyclic aromatic hydrocarbon ring systems, containing 1 to 4 rings, at least one of which is aromatic, which are fused together or linked covalently and typically contain 5 to 8 atoms;. The aromatic ring may optionally be fused to one to three additional rings (either cycloalkyl, heterocyclyl or heteroaryl).

Non-limiting examples of aryl comprise phenyl, biphenylyl, biphenylenyl, 5- or 6-tetralinyl, 1-, 2-, 3-, 4-, 5-, 6-, 7- or 8-azulenyl, 1- or 2-naphthyl, 1-, 2- or 3-indenyl, 1-, 2- or 9- anthryl, 1- 2-, 3-, 4- or 5-acenaphtylenyl, 3-, 4- or 5-acenaphtenyl, 1-, 2-, 3-, 4- or 10- phenanthryl, 1- or 2-pentalenyl, 1 , 2-, 3- or 4-fluorenyl, 4- or 5-indanyl, 5-, 6-, 7- or 8- tetrahydronaphthyl, 1 ,2,3,4-tetrahydronaphthyl, 1 ,4-dihydronaphthyl, dibenzo[a,d]cylcoheptenyl, 1-, 2-, 3-, 4- or 5-pyrenyl.

The term "aryloxy" as used herein denotes a group -O-aryl, wherein aryl is as defined above.

The term "heteroaryl" as used herein by itself or as part of another group refers to aryl as defined above in which one or more carbon atoms in one or more of these rings can be replaced by oxygen, nitrogen or sulphur atoms where the nitrogen and sulphur heteroatoms may optionally be oxidized and the nitrogen heteroatoms may optionally be quaternized. Such rings may be fused to an aryl, cycloalkyl, heteroaryl or heterocyclyl ring. An "optionally substituted heteroaryl" refers to a heteroaryl having optionally one or more substituents (for example 1 to 4 substituents, or 1 to 2 substituents), selected from those defined above for substituted aryl.

Non-limiting examples of heteroaryl can be 2- or 3-furyl, 2- or 3-thienyl, 1-, 2- or 3-pyrrolyl, 1-, 2-, 4- or 5-imidazolyl, 1-, 3-, 4- or 5-pyrazolyl, 3-, 4- or 5-isoxazolyl, 2-, 4- or 5-oxazolyl, 3-, 4- or 5-isothiazolyl, 2-, 4- or 5-thiazolyl, 1 ,2,3-triazol-1-, -2-, -4- or -5-yl, 1 ,2,4-triazol-1-, -3-, -4- or -5-yl, 1 ,2,3-oxadiazol-4- or -5-yl, 1 ,2,4-oxadiazol-3- or -5-yl, 1 ,2,5-oxadiazolyl, 1 ,3,4-oxadiazolyl, 1 ,2,3-thiadiazol-4- or -5-yl, 1 ,2,4-thiadiazol-3- or -5-yl, 1 ,2,5-thiadiazol-3- or -4-yl, 1 ,3,4-thiadiazolyl, 1 - or 5-tetrazolyl, 2-, 3- or 4-pyridyl, 3- or 4-pyridazinyl, 2-, 4-, 5- or 6-pyrimidinyl, 2-, 3-, 4-, 5- 6-2H-thiopyranyl, 2-, 3- or 4-4H-thiopyranyl, 2-, 3-, 4-, 5-, 6- or 7-benzofuryl, 1-, 3-, 4- or 5-isobenzofuryl, 2-, 3-, 4-, 5-, 6- or 7-benzothienyl, 1-, 3-, 4- or 5-isobenzothienyl, 1-, 2-, 3-, 4-, 5-, 6- or 7-indolyl, 2- or 3-pyrazinyl, 1 ,4-oxazin-2- or -3-yl, 1 ,4-dioxin-2- or -3-yl, 1 ,4-thiazin-2- or -3-yl, 1 ,2,3-triazinyl, 1 ,2,4-triazinyl, 1 ,3,5-triazin-2-, - 4- or -6-yl, thieno[2,3-b]furan-2-, -3-, -4-, or -5-yl, 1-, 2-, 4- or 5-benzimidazolyl, 1-, 3-, 4-, 5-, 6- or 7-benzopyrazolyl, 3-, 4-, 5-, 6- or 7-benzisoxazolyl, 2-, 4-, 5-, 6- or 7- benzoxazolyl, 3-, 4-, 5-, 6- or 7-benzisothiazolyl, 2-, 4-, 5-, 6- or 7-benzothiazolyl, 1-, 2- thianthrenyl, 3-, 4- or 5-isobenzofuranyl, 1-, 2-, 3-, 4- or 9-xanthenyl, 1-, 2-, 3- or 4- phenoxathiinyl, 2-, 3-pyrazinyl, 1-, 2-, 3-, 4-, 5-, 6-, 7- or 8-indolizinyl, 2-, 3-, 4- or 5- isoindolyl, 1-, 2-, 3-, 4-, 5-, 6- or 7-indazolyl, 2-, 6-, 7- or 8-purinyl, 4-, 5- or 6-phthalazinyl, 2-, 3- or 4-naphthyridinyl, 2-, 5- or 6-quinoxalinyl, 2-, A-, 5-, 6-, 7- or 8-quinazolinyl, 1-, 2-, 3- or 4-quinolizinyl, 2-, 3-, 4-, 5-, 6-, 7-, or 8-quinolinyl(quinolyl), 2-, 4-, 5-, 6-, 7- or 8- quinazolyl, 1-, 3-, 4-, 5-, 6-, 7- or 8-isoquinolinyl(isoquinolyl), 3-, 4-, 5-, 6-, 7- or 8- cinnolinyl, 2-, A-, 6- or 7-pteridinyl, 1-, 2-, 3-, 4- or 9-carbazolyl, 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8- or 9-carbolinyl, 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9- or 10-phenanthridinyl, 1-, 2-, 3- or 4-acridinyl, 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8- or 9-perimidinyl, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9- or 10- (1 ,7)phenanthrolinyl, 1- or 2-phenazinyl, 1-, 2-, 3-, 4-, or 10-phenothiazinyl, 3- or A- furazanyl, 1-, 2-, 3-, 4-, or 10-phenoxazinyl, azepinyl, diazepinyl, dibenzo[b,f]azepinyl, dioxanyl, thietanyl, oxazolyl dibenzo[a,d]cylcoheptenyl, or additionally substituted derivatives thereof.

The term "arylalkyl" by itself or as part of another substituent refers to a group having as alkyl moiety the aforementioned alkyl attached to one of the aforementioned aryl rings. Examples of arylalkyl moieties/groups include benzyl, phenethyl, dibenzylmethyl, methylphenylmethyl, 3-(2-naphthyl)-butyl, and the like.

The term "acyl" by itself or as part of another substituent refers to an alkanoyl group having 2 to 6 carbon atoms or a phenylalkanoyl group whose alkanoyl moiety has 1 to 4 carbon atoms, i.e. a carbonyl group linked to a moiety/group such as, but not limited to, alkyl, aryl. More particularly, the group -COR¹¹, wherein R¹¹ can be selected from aryl or substituted aryl, as defined herein. The term acyl therefore encompasses the group arylcarbonyl (-COR¹¹) wherein R¹¹ is aryl. Said acyl can be exemplified by benzoyl, phenylacetyl, phenylpropionyl and phenylbutynyl.

The term "alkyl" by itself or as part of another substituent, refers to a straight or branched saturated hydrocarbon group joined by single carbon-carbon bonds having 1 to 10 carbon atoms, for example 1 to 8 carbon atoms, for example 1 to 6 carbon atoms, preferably 1 to 4 carbon atoms. When a subscript is used herein following a carbon atom, the subscript refers to the number of carbon atoms that the named group may contain. Thus, for example, Ci_-4 alkyl means an alkyl group of one to four carbon atoms. Examples of alkyl groups are methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, te/f-butyl, 2- methylbutyl, pentyl (e.g. pentyl iso-amyl) and its isomers, hexyl and its isomers, heptyl and its isomers and octyl and its isomers.

Whenever the term "substituted" is used in the present invention, it is meant to indicate that one or more hydrogens on the atom indicated in the expression using "substituted" is replaced with a selection from the indicated group, provided that the indicated atom's normal valency is not exceeded, and that the substitution results in a chemically stable compound, i.e. a compound that is sufficiently robust to survive isolation to a useful degree of purity from a reaction mixture, and attachment to a solid support.

The aromatic moieties may be immobilized on the solid support using covalent or non- covalent attachment means. Methods for covalent attachment of such aromatic moieties to solid supports are well known in this field (see e.g. Immobilized Affinity Ligand Techniques, Hermanson, G. T. et al, Academic Press, INC, 1992; Combinatorial Chemistry, Eds: Bannwarth, Willi, Hinzen, Berthold, Wiley-VCH). Non-covalent attachment can be achieved using a binding pair, whereby one pair is attached to the column, and the other is attached to the compound (e.g. streptavidin/biotin, avidin/biotin, Ni²VHiS₆, etc.). Care is taken so that the treated sample does not bind to the solid support using an interaction other than that with the aromatic moieties. The primary amine present in the peptide or protein may be substituted once or twice with an aromatic moiety. The aromatic moiety may be immobilized on the solid support using a linker or spacer compound. These are well known in the art, and are generally chemically inert insofar as they show little interference with the pi-pi interaction.

The skilled person will be aware of the types of linkers available and the properties of each, and will select the most suitable linker for the intended application. Examples of parameters that may be considered by the skilled person in selecting a linker include chain length, presence of hetero atoms and/or functional groups, cyclic or acyclic structure.

Generally, the linker is of sufficient length to avoid steric interference of the solid support with the intended pi-pi interaction. To avoid non-specific hydrophobic interactions between peptides and the spacer, the linker may have hydrophilic character. Suitable linker examples include (poly)ethylene glycol, reduced sugars, acyclic dicarboxylic acids, etc..

FIG. 6 depicts an aromatic moiety 71 , immobilized on a bead 13¹ using a linker 72, to form the solid support 13 for use in the invention. The solid support 13 interacts 73 with an aromatic moiety 74 attached to the peptide. The figure shows a particular embodiment where the aromatic moieties are substituted phenyls. When the substituents of the aromatic moiety 71 of solid support 13 are electron-withdrawing, said aromatic moiety 71 is a pi-acceptor, and the aromatic moiety 74 of peptide, substituted with electron-donating groups, is a pi-donor (see column A). Conversely, when the substituents of the aromatic moiety 71 of solid support 13 are electron-donating, said aromatic moiety 71 is a pi-donor, and the aromatic moiety 74 of peptide is substituted with electron-donating groups to form a pi-acceptor (see column B).

Methods for immobilisation of such linkers to solid supports are well known in this field (see e.g. Immobilized Affinity Ligand Techniques, Hermanson, G. T. et al,, Academic Press, INC, 1992; Combinatorial Chemistry, Eds: Bannwarth, Willi, Hinzen, Berthold, Wiley-VCH).

Profiling

The sorting step of the present invention provides two sets of peptides - one captured by the solid support, and the other not. Depending on the sample, solid support and interaction, either set of peptides is used in profiling which typically entails analytical separation of the peptides. The present invention is a method for obtaining a protein profile of a biological sample, comprising preparing the biological sample using the method as described above, whereby the set of peptides not captured by the solid support is used for analytical separation.

Analytical separation refers to methods for separating chemical substances for analytical purposes; such methods are widely available in the art. Chromatography is one example of an analytical separation method. The method makes use of the relative rates at which chemical substances are adsorbed from a moving stream of gas or liquid on a stationary substance, which is usually a finely divided solid, a sheet of filter material, or a thin film of a liquid on the surface of a solid. Chromatography is a versatile method that can separate mixtures of compounds even in the absence of detailed previous knowledge of the number, nature, or relative amounts of the individual substances present. The method is widely used for the separation of chemical compounds of biological origin (for example, proteins, fragments of proteins, peptides, amino acids, phospholipids, steroids, etc.) and of complex mixtures of petroleum and volatile aromatic mixtures, such as perfumes and flavours. The most widely used chromatographic technique is high-performance liquid chromatography, in which a pump forces the liquid mobile phase through a high efficiency, tightly packed column at high pressure. Recent overviews of chromatographic techniques are described by Meyer M., 1998, ISBN: 047198373X and Cappiello, A., et al. Mass Spectrom. Rev. 2001 , 20, 88-104, incorporated herein by reference. Other recently developed methods described in the art and novel chromatographic methods coming available in the art can also be used. Some examples of chromatography are reversed phase chromatography (RP), ion exchange chromatography, hydrophobic interaction chromatography, size exclusion chromatography, gel filtration chromatography, and affinity chromatography such as immunoaffinity and immobilized metal affinity chromatography.

According to one aspect of the invention, analytical separation may be one dimensional high performance liquid chromatography (HPLC). This might be performed using, for example, an analytical reversed phase column. The columns and conditions for performing an analytical separation will be known to the skilled person, and is described in Practical HPLC Methodology and Applications, Bidlingmeyer, B. A., John Wiley & Sons Inc., 1993. Chromatography is one of several analytical separation techniques. Electrophoresis and all its variants such as capillary electrophoresis, free flow electrophoresis, etc., is another example of an analytical separation technique. In the latter case, the driving force is an electric field, which exerts different forces on solutes of different ionic charge. The resistive force is the viscosity of the non-flowing solvent. The combination of these forces yields ion mobilities peculiar to each solute. Some examples are sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS-PAGE) and native gel electrophoresis. Capillary electrophoresis methods include capillary gel electrophoresis, capillary zone electrophoresis, capillary electrochromatography, capillary isoelectric focusing and affinity electrophoresis. These techniques are described in An Introduction to Chemistry, McKay, P., Science Seminar, Department of Recovery Sciences, Genentech, Inc., incorporated herein by reference.

Preferably, the analytical separation used to determine the protein profile is one dimensional HPLC, i.e. the protein profile is obtained by a separation using a single chromatographic run. Fractions may be collected during the separation; each fraction may be analysed to arrive at a molecular weight for the peptide(s) in each fraction. Suitable techniques include tandem mass spectrometry. Having obtained the molecular weight and a fragmentation pattern of a peptide, the corresponding protein can be deduced using database searching. The inventors have found that selection of one peptide per protein is obtainable using the present method, thereby providing a rapid and efficient protein profile of the sample.

Alternatively, the analytical chromatography may be multi-dimensional liquid chromatography, e.g. using two, three, or higher dimensions of separation. Preferably it is two-dimensional liquid chromatography.

According to one aspect of the invention, at least one amino acid of the set of peptides is altered before analytical separation in the case of a one dimensional separation, or prior to a second or later separation in the case of a two or multi-dimensional analytical separation. For example, when a two-dimensional analytical separation is utilized (e.g. two dimensional liquid chromatography) altering may proceed only after the first separation and before the second separation.

Altering can be obtained via a chemical reaction or an enzymatic reaction or a combination of a chemical and an enzymatic reaction. A non-limiting list of chemical reactions include alkylation, ac(et)ylation, nitrosylation, oxidation, hydroxylation, methylation, reduction and the like. A non-limiting list of enzymatic reactions includes treating peptides with phosphatases, acetylases, glycosidases or other enzymes which modify co-or post-translational modifications present on peptides. The chemical alteration can comprise one chemical reaction, but can also comprise more than one reaction, such as two consecutive reactions, in order to increase the alteration efficiency. Similarly, the enzymatic alteration can comprise one or more enzymatic reactions.

One aspect of the invention is the method described above, further comprising the step of identifying at least one altered peptide per protein.

One aspect of the invention is the method described above, wherein the identifying step consists of accurate measurement of the mass of the peptides, in particular N-terminal peptides, by tandem mass spectrometry, followed by database searching to trace the peptides back to their parent proteins.

Kit

The present invention also relates to a kit for preparing a sample for protein profiling. The kit may be provided with one or more of the following components: - blocking reagent of class ii), and optionally i) as described above,

- cleavage reagent as described above,

- host compound as described above,

- aromatic moiety as described above,

- solid support as described above, - solid support as described above, comprising one or more immobilized aromatic moieties,

- solid support as described above, comprising one or more immobilized host compounds,

- solid support as described above, provided in a cartridge.

According to a preferred embodiment of the invention, the kit comprises:

- blocking reagent of class ii), and optionally i) as described above,

- cleavage reagent as described above, and

- solid support as described above, comprising one or more immobilized host compounds.

According to a preferred embodiment of the invention, the kit comprises:

- blocking reagent of class ii), and optionally i) as described above, - cleavage reagent as described above, and

- solid support as described above, comprising one or more immobilized aromatic moieties.

EXAMPLES

The invention is exemplified by way of the following non-limiting examples.

Experiment 1 Initial sample preparation, i.e. alkylation of the cysteine residues and the blocking of primary amines (acetylation) is performed as described in Gevaert, K., et al, J. Nat. Biotechnol. 2003, 21, 566-569. Slight adaptations of the currently used N-ter COFRADIC™ tryptic digest procedure may be needed in order not to compromise the subsequent TNBS modification step. To prevent any undesired side reactions when performing the TNBS modification, both the tris(hydroxymethyl)aminomethane buffer and the guanidium hydrochloride (chaotropic agent) are replaced in the tryptic digestion protocol. Sodium bicarbonate buffer (at pH 8.5) is used instead. This allows for the implementation of a one-pot digestion/TNBS-modification approach, eliminating several error-prone transfer (manual pipeting) and loss-sensitive drying (adsorption phenomena) steps. After the TNBS modification of the tryptic digest, the resulting mixture is submitted to a phenyl LC (or a phenyl SPE-cartridge) clean-up. At this point, the TNP-modified internal peptides are selectively retained on the phenyl LC column (phenyl SPE cartridge) whilst the interaction of the acetylated N-terminal peptides (due to aromatic side chains) with the phenyl stationary phase will be less strong (under the applied/optimized chromatographic conditions). This results in a group separation between non-TNP peptides and TNP-peptides. The recovered non-TNP-peptide fraction, still an extremely complex mixture, is separated by means of high efficiency reversed-phase LC, providing an unsurpassed peptide resolution. The separated non-TNP-peptides are collected in 1536 fractions which are submitted to mass spectrometric analysis. The power of the here described platform resides in the fact that only two chromatographic steps are needed, impacting dramatically the achievable throughput compared to a diagonal chromatographic sorting methodology. Experiment 2

Materials & methods: Sample Selection

In order to assess the potential of phenyl LC for the sorting of TNP-peptides vs. non-TNP- peptides, a representative mixture of both non-TNP-peptides and TNP-peptides derived from serum digests is needed. In the well established N-ter COFRADIC™ platform, a serum protein digest sample (containing a mix of N-terminally acetylated peptides (1 ) and internal peptides (2) with free amino groups) is fractionated by RPLC, resulting in 12 fractions of peptides. These fractions are separately subjected, to a TNBS modification step, yielding 12 mixtures containing unaltered acetylated N-terminal peptides (1 ) and TNP-modified internal peptides (3) with increased hydrophobicity. Each fraction is re- chromatographed under identical RPLC conditions (vide supra), and the modified TNP- peptides shift outside of the original collection window due to their increased hydrophobicity. Normally, only peptides eluting in the original collection window, mainly acetylated N-terminal peptides, are used for mass spectrometric analysis, and the shifted TNP-modified peptides are discarded. However, for the purpose of the here described invention, these TNP-modified peptides are the ideal test compounds. Therefore, both the N-terminal peptide fractions as well as the TNP-modified peptides were collected.

Materials & methods: Materials Guanidinium isothiocyanate, guanidinium hydrochloride, hydrogen peroxide, formic acid, disodium hydrogen phosphate (Na₂HPO₄), sodium dihydrogen phosphate monohydrate (NaH₂PO₄-H₂O), boric acid and hydroxylamine were obtained from Merck (Darmstadt, Germany). Ammonium carbonate, ammonium bicarbonate, ammonium hydroxide, 2,4,6- trinitrobenzenesulfonic acid (TNBS), sodium hydroxide and hydrochloric acid were purchased from Fluka (Buchs, Switzerland). Ethanol p. a. 96% was obtained from Sigma- Aldrich (Bornem, Belgium). The BCA protein assay kit, as well as the Slide-A-Lyzer dialysis cassettes and N-hydroxysulfosuccinimidyl acetate (sulfo-NHS acetate) were purchased from Pierce (Erembodegem, Belgium). The PD10 and NAP5 desalting columns were from Amersham Biosciences (Roosendaal, The Netherlands). The spin filters were obtained from Filter Services (Eupen, Belgium). Tris(hydroxymethyl)aminomethane (Tris) was obtained from Biorad (Nazareth, Belgium). HPLC grade water, acetonitrile (ACN) and peptide synthesis grade trifluoroacetic acid (TFA) were purchased from Biosolve (Valkenswaard, The Netherlands). Sequencing grade trypsin was obtained from Promega (Leiden, The Netherlands). The peptide standard mix (Proteomix), containing 5 peptides, and alpha-cyano-4-hydroxycinnamic acid were obtained from LaserBio Labs (Sophia- Antipolis Cedex, France). Blood was obtained from a healthy volunteer and collected in standard serum clotting tubes (BD, Erembodegem, Belgium). Serum was collected after centrifugation at 4,000 rpm for 10 min. For depletion of 6 high-abundant proteins (albumin, alpha-1 -antitrypsin, haptoglobin, IgG, IgA and transferrin) in serum, the MARS depletion system of Agilent Technologies (Waldbronn, Germany) was used. The latter comprises a human high capacity MARS column and a buffer system, containing buffer A and buffer B.

Sample preparation & COFRADIC process

Serum was diluted 1 :4 in buffer A, part of the MARS depletion system, filtered through a spin filter and depleted on a human high capacity MARS column. A rough estimate of the protein concentration of the flow-through fractions was obtained by performing a BCA test. The flow-through fractions were desalted on a PD10 gel filtration column and captured in a 0.1 M ammonium bicarbonate buffer, containing 3M guanidinium isothiocyanate. Four volumes of ice-cold ethanol were added and the mixture was incubated overnight at - 20⁰C. The resulting precipitates were centrifuged for 30 min at 4 100 rpm and the pellet was washed twice with 85% ethanol. The pellet was dissolved in 250 μl_ of a 100 000 molar excess of performic acid and incubated on ice during 45 min. Performic acid was prepared fresh from formic acid and hydrogen peroxide 9:1 (v:v). After incubation, the sample was diluted with water in a 1 :1 ratio (v:v), followed by overnight dialysis against water. At this stage, 20 μL of the solution was used to determine the protein concentration (BCA test). After lyophilization, the sample was redissolved in 900 μL of 100 mM sodium phosphate buffer at pH 8, containing 2M guanidinium isothiocyanate. Solid sulfo-NHS acetate (75 molar excess) was added and the sample was incubated for 90 min at 30⁰C. Next, the sample was treated with hydroxylamine (3.5 molar excess compared to sulfo- NHS acetate) for 20 min at room temperature to deacetylate the serines, threonines and tyrosines that were acetylated during the acetylation step. After this reverse acetylation step, the sample was desalted on a NAP5 column, captured in a 20 mM TrisHCI buffer at pH 7.9 containing 0.2M guanidinium hydrochloride, and digested overnight with trypsin (substrate:trypsin ratio of 50:1 (w:w)) at 37°C. 500 μg portions of the digest were subjected to the primary run of the COFRADIC process (for LC conditions of the primary run, see 'Column and LC conditions' for more details). After the primary run, the collected fractions were dried and subsequently modified with trinitrobenzene sulfonic acid (TNBS). Thereto, the dried fractions were redissolved in 50 μL of 50 mM borate buffer, pH 9.5 and 150 nmol TNBS in 10 μL of 50 mM borate buffer, pH 9.5, was added and each fraction was incubated for 45 min at 37°C. This step was repeated 3 more times, resulting in a total volume of 90 μL. The reaction was stopped by adding 2 μL of 10% aqueous TFA to reach a pH of 2, and the fractions were dried. The samples were dissolved in solvent A for the secondary runs of the COFRADIC process (for LC conditions of the secondary run, see 'Column and LC conditions' for more details). After the secondary runs, the unaltered fractions were collected as a whole, as well as the shifted fractions (TNP-peptides). These samples were dried separately and redissolved in the starting conditions of the phenyl column experiments (see 'Column and LC conditions' for more details). 75 μL of sample was injected on the phenyl column.

LC system (COFRADIC)

A 1100 Agilent LC system (Agilent Technologies, Waldbronn, Germany) equipped with a diode array detector was used. LC fractions were collected using a 1100 series fraction collector from Agilent Technologies. For temperature control of the column, a Polaratherm Series 9000 oven (Selerity Technologies, Salt Lake City, UT, USA) was connected to the HPLC. The latter is equipped with a mobile phase pre-heater and cryo-option. Operation, data collection and analysis were done using the Chemstation software (Agilent Technologies, Waldbronn, Germany).

Column and LC conditions (COFRADIC)

Primary and secondary runs were carried out on Zorbax SB300-C18 columns (15 cm x 2.1 mm i.d.; 5 μm particle size, 300A pore size) from Agilent Technologies (Waldbronn, Germany). According to the manufacturer, these columns are stable up to 90⁰C. Stainless steel tubing, with an internal diameter of 0.17 mm and a length of 10.5 cm (Agilent Technologies, Waldbronn, Germany), was used for column coupling. For the COFRADIC run, a linear gradient of 1% B/min at a flow rate of 80 μL/min was used and this at a temperature of 30 deg C. Solvent A consisted of 0.1% TFA, whereas solvent B constituted of 0.1% TFA in 70% ACN.

For the phenyl column experiments, the commercially available Phenomenex Luna phenyl-hexyl (25 cm x 2 mm i.d.; 5 μm particle size, 100A pore size) was used (Bester,

Amstelveen, The Netherlands). The following gradient program was employed, at a flow rate of 80 μL/min and a temperature of 30⁰C:

0-30 min 25 %B

30-45 min 25 %B → 100 %B

45-60 min 100 %B

60-65 min 100 %B → 25 %B 65-80 min 25% B Mobile phase A comprised H₂O:ACN:TFA 98:2:0.1 , while B comprised: H₂O:ACN:TFA 30:70:0.1. To monitor the peptides, the wavelength was set at 214 nm. At 280 nm, the aromatic peptides were monitored and the TNP-modified peptides absorb at a wavelength of 340 nm and 420 nm.

Proof of concept on one COFRADIC fraction

Both an N-terminal fraction and the associated TNP-fraction from a COFRADIC secondary run, were submitted to Phenyl LC sorting. FIG. 7 depicts two separations using a phenyl LC analytical column using an isocratic stage of 25% B followed by a gradient of 25 to 100% B (80) as described in the previous paragraph. One separation (81 ) is 75 μl injection of the acetylated N-terminal fraction which elutes between 10 and 20 minutes 83, measured at 214 nm. The other separation (82) is a 75 μl injection of the corresponding TNP fraction which elutes between 44 and 58 minutes 84, measured at 420 nm. As expected, the TNP-peptides were retained more by the phenyl column, compared to the N-terminally acetylated peptides, indicating the possibility of separating unmodified peptides from TNP-modified peptides on a phenyl LC column.

Experiment 3: Crown ethers - illustrative experiment 1

Initial sample preparation, i.e. alkylation of the cysteine residues and the blocking of primary amines (acetylation) is performed as described in Gevaert, K., et al, J, Nat. Biotechnol. 2003, 21, 566-569. Slight adaptations of the currently used N-ter COFRADIC™ tryptic digest procedure may be needed in order not to compromise the subsequent selection of acetylated N-terminal peptides. In order to prevent undesired enrichment of tryptic buffer components at the lonPac CG15 column (Dionex Corp, Sunnyvale, CA 94085, USA) both the tris(hydroxymethyl)aminomethane buffer and the guanidium hydrochloride (chaotropic agent) are replaced in the tryptic digestion protocol. Sodium bicarbonate (at pH 8.5) is used instead. Prior to injection onto the lonPac CG 15 column the pH of the digest is adapted to pH ~ 5. Compliant with the underlying selection mechanism all peptides that contain no free primary amino groups, i.e. peptides with their lysine- and their N-terminal amino-groups acetylated, will not be retained on the lonPac CG 15 column (under the applied LC-conditions) and will be retrieved in the flow-through. As a result, the sub-set of true N-termini -representative for the serum proteome- is obtained and available for further separation by means of high efficiency reversed phase LC and subsequent mass spectrometric analysis. It has to be noted that -because of the inherent multifunctionality of the used lonPac CG 15 column, i.e. the presence of phosphonate and carboxylate groups next to the immobilized crown ethers- peptides with (several) positive charge(s) will be retained on the column as well and thus be removed from the mixture. Obviously, hisitidine containing peptides (Histidine pKa -6) are susceptible to this extra retention mechanism and this will compromise the attempted subselection. In silico calculations revealed that ca. 23.5% of all hypothetical true N- termini contain a histidine residue, leaving 76.5% of the proteome open for analysis. This sampling pool was found sufficiently large to allow for comprehensive human serum profiling. Column regeneration is achieved by the application of an acid gradient whereby the large excess of H⁺ induces the elution of the bound fraction (i.e. all internal peptides) by means of competition for the active sites. The power of the here described platform resides in the fact that N-terminal peptide selection is achieved in one chromatographic step, impacting dramatically on the achievable throughput compared to a diagonal chromatographic sorting methodology.

Materials Guanidinium isothiocyanate, guanidinium hydrochloride, hydrogen peroxide, formic acid, disodium hydrogen phosphate (Na₂HPC>₄), sodium hydrogen phosphate monohydrate (NaH₂PO₄), boric acid and hydroxylamine were obtained from Merck (Darmstadt, Germany). Ammonium hydrogen carbonate, ammonium bicarbonate, ammonium hydroxide, sodium hydroxide and hydrochloric acid were purchased from Fluka (Buchs, Switzerland). Ethanol p. a. 96% was obtained from Sigma-Aldrich (Bornem, Belgium). The BCA protein assay kit, as well as the Slide-A-Lyzer dialysis cassettes and N- hydroxysulfosuccinimidyl acetate (sulfo-NHS acetate) were purchased from Pierce (Erembodegem, Belgium). The PD10 and NAP5 desalting columns were from Amersham Biosciences (Roosendaal, The Netherlands). The spin filters were obtained from Filter Services (Eupen, Belgium). Trishydroxymethylaminomethane (Tris) was obtained from Biorad (Nazareth, Belgium). Acetonitrile and peptide synthesis grade trifluoroacetic acid (TFA) were purchased from Biosolve (Valkenswaard, The Netherlands). Deionised water was obtained from an in house water purification unit (ENx and Academic MiIIiQ unit, Milipore, Billerica, MA, USA). Sequencing grade trypsin was obtained from Promega (Leiden, The Netherlands). The peptide standard mix (Proteomix), containing 5 peptides, and alpha-cyano-4-hydroxy-cinnamic acid were obtained from LaserBio Labs (Sophia- Antipolis Cedex, France). Blood was obtained from a healthy volunteer and collected in standard serum clotting tubes (BD, Erembodegem, Belgium). Serum was collected after centrifugation at 4,000 rpm for 10 min. For depletion of 6 high abundant proteins (albumin, alpha-1 -antitrypsin, haptoglobin, IgG, IgA and transferrin) in serum, the MARS depletion system of Agilent Technologies (Waldbronn, Germany) was used. The latter comprises a human high capacity MARS column and a buffer system, containing buffer A and buffer B.

Sample preparation Serum was diluted 1 :4 in buffer A, part of the MARS depletion system, filtered through a spin filter and depleted on a human high capacity MARS column. A rough estimate of the protein concentration of the flow-through fractions was obtained by performing a BCA test. The flow-through fractions were desalted on a PD10 gel filtration column and captured in a 0.1 M ammonium bicarbonate buffer, containing 3M guanidinium isothiocyanate. Four volumes of ice-cold ethanol were added and the mixture was incubated overnight at - 20⁰C. The resulting precipitates were centrifuged for 30 min at 4 100 rpm and the pellet was washed twice with 85% ethanol. The pellet was dissolved in 250 μl_ of a 100 000 molar excess of performic acid and incubated on ice during 45 min. Performic acid was prepared fresh from formic acid and hydrogen peroxide 9:1 (v:v). After incubation, the sample was diluted with water in a 1 :1 ratio (v:v), followed by overnight dialysis against water. At this stage, 20 μl_ of the solution was used to determine the protein concentration (BCA test). After lyophilization, the sample was redissolved in 900 μl_ of 100 mM sodium phosphate buffer at pH 8, containing 2M guanidinium isothiocyanate. Solid sulfo-NHS acetate (75 molar excess) was added and the sample was incubated for 90 min at 30⁰C. After deacetylation with ammonium hydroxide (3.5 molar excess compared to sulfo-NHS acetate) for 20 min at room temperature, the sample was desalted on a NAP5 column and captured in a 10 mM NaHCC>3 buffer. The sample was digested overnight with trypsin (substrate:trypsin ratio of 50:1 (w:w)) at 37°C. 200 μg of the resulting digest was made up to 100 μL with 0.1 % HOAc in 50/50 H₂O/can, and injected on the lonPac CS 15 column (for LC conditions of the primary run, see 'Column and LC conditions' for more details). The resulting flow-through was collected in 4 fractions of 500 μL (FIG. 8). From the latter 200 μL aliquots were taken and subsequently dried by means of vacuum centrifugation at 37°C (Centrivap Concentrator, Labconco, Kansas City, Missouri, USA). The dried fractions were redissolved in 22 μL of 0.1% FA in H₂O -of which 20 μL was injected- and further separated by means of a nano-RPLC system (Ultimate 3000, Dionex, Sunnyvale CA, USA) hyphenated with a spotting robot (PROBOT, Dionex, Sunnyvale CA, USA), enabling direct MALDI-plate spotting (for Nano-LC conditions, see 'Column and LC conditions'). Prior to spotting, MALDI-matrix (α-cyano-4-hydroxy-cinnamic acid, recrystallized, LaserBio Labs # M101 , Sophia-Antipolis, France) and MALDI-calibration compounds (Peptide Mix 4 (proteomix), LaserBio Labs # C104) were mixed with the nano- LC column effluent via a T-junction to allow good sample crystallization and accurate mass determinations, both requisite for performant MALDI-MS(/MS) analysis.

LC system A 1100 Agilent LC system (Agilent Technologies, Waldbronn, Germany) equipped with a column heating compartment and multiple wavelength detector was used. LC fractions were collected using a 1 100 series fraction collector from Agilent Technologies. Operation, data collection and analysis were done using the Chemstation software (Agilent Technologies, Waldbronn, Germany).

Column and LC conditions (IonPac CS 15)

N-terminal peptide sorting was carried out on a IonPac CS 15 column (25 cm x 2.1 mm i.d.; 8.5 μm particle size, 100 A pore size) from Dionex (Dionex Corp, Sunnyvale, CA 94085 USA). According to the manufacturer, the column stationary phase is a 55% crosslinked polyethylvinylbenzene-divinylbenzene co-polymer functionalized with carboxylic acids, phosphonic acids and 18-crown-6 ethers, with an ion exchange capacity of 2800 μeq per column. IonPac CS 15 columns are stable up to pH 7, and pressure resistant up to 4000 psi. The IonPac stationary phase is compatible with aqueous solvents, and acetonitrile (0-100%) and tetrahydrofuran (0-20%) are tolerated as organic modifiers.

The following isocratic program was employed for N-terminal peptide sorting, at a flow rate of 80 μL/min and a temperature of 30⁰C: 0-30 min 0.1 % HOAc in 50:50 ACN:H₂O (MQ) Four equal (volume) fractions were collected between 3-28 min, i.e. 500 μL per fraction, for further analysis.

The following gradient program was employed for bound peptide fraction elution, at a flow rate of 80 μL/min and a temperature of 30⁰C: 0-70 min 0 % B → 100 %B

70-80 min 100 %B

80-90 min 0 % B

Mobile phase A comprised 50:50 ACN:H₂O (MQ), while B comprised 1 % TFA in 50:50

ACN:H₂O. To monitor the peptides, the wavelength was set at 214 nm and 280 nm. The following isocratic program was employed for column regeneration, at a flow rate of 80 μL/min and a temperature of 30⁰C: 0-180 min 0.1 % HOAc in 50:50 ACN:H₂O (MQ)

Nano LC conditions and Direct spotting (Nano RPLC)

The 4 sorted N-terminal peptide fractions, prepared as described above, were subjected to nano LC analysis with direct MALDI plate spotting. Briefly, the nano LC analysis involved a 20 μL sample injection and a 3 min pre-concentration via a loading pump at 20 μL/min using 0.1% formic acid (FA) in H₂O as mobile phase on a pre-column (a Dionex C18 PepMap 300 μm i.d. x 5 mm capillary column, packed with C18 PepMap100, 5μm, 100 A). After 3 min, the pre-concentration column is coupled in-line with the analytical nano RP column (a C18 PepMap 75 μm i.d. x 15 cm, packed with C18 PepMap100, 3μm, 100 A), and a 60 min gradient program is run at 0.3μL/min from 4% B to 55% B (with A 0.1% FA in H₂O and B 0.1 % FA in 80:20 ACN:H₂O). The effluent of the nano-RP column was then mixed 1 :4 in a micro T-piece (Probot, Dionex) with MALDI-matrix solution (8mg α-cyano-4-hydroxy-cinnamic acid per mL solvent (0.1% TFA in 70:30 ACN:H₂O)) doped with MALDI calibrant (1 :600). Via the Probot microfraction collector (Dionex) the nano RP- LC gradient is translated into 242 spots on 123 x 81 mm opti-TOF LC/MALDI inserts (Applied Biosystems, Foster City, CA, USA).

MALDI-(MSZMS) analysis

The above prepared MALDI-inserts were analysed via MALDI-MS(/MS) on a 4800 MALDI- TOF/TOF analyzer (Applied Biosystems, Foster City, CA, USA) with the application of instrument settings typical for peptide analysis, which are clear to the skilled person.

Data analysis

The mass data acquired from the two initial lonPac CS 15 flow-through fractions were submitted to the Mascot protein identification search engine (Matrix Science, Boston, MA, USA) with the application of a set of search parameters relevant to the experimental set- up and the used MS instrument, which are clear to the skilled person. Analysis of the resulting peptide identifications reveals that the initial N-terminal peptide sorting is highly efficient, i.e. the majority of the peptides are N-terminally acetylated.

FIG. 8 shows a 214 nm UV trace of a 100 μg digest (prepared as described in the text) corresponding with an isocratic N-terminal peptide sorting run on the lonPac CS 15 column, whereby the collected flow-through (the 4 fractions shown, F1 to F4) contains the peptides of interest, i.e. the N-terminal acetylated peptides.

FIG. 9 shows an overlay of the 214 nm UV traces of the 4 nano-RPLC runs (F1 to F4) corresponding the 4 lonPac CS 15 initial flow-through fractions shown in FIG. 8, which are directly spotted onto the MALDI-targets.

Experiment 4: Crown ethers - illustrative experiment 2

This experiment demonstrates further an N-teromics set-up which enriches deliberately acetylated protein N-terminal peptides from a serum digest. In particular, we demonstrate that peptides with free N-terminal α-NH₂ groups and peptides with α-amino-acetylated groups, can be separated from each other using crown ether based separation, and in particular weak cation exchange/crown ether columns. The peptides of interest, bearing no free primary amines, are significantly enriched in the column's flow through. At the same time it is demonstrated that under the same experimental conditions a favourable co-enrichment of glycopeptides is achieved. The latter is exemplified by confidently identifying N-glycopeptides, which are discernable based on the presence of the consensus motif N-X-S/T, with X not being a proline, in their amino acid sequence.

Sample Preparation

70 μl_ of a crude human serum sample (healthy male) was diluted 1 :4 in proprietary "buffer A", part of the Multiple Affinity Removal System (MARS) (Agilent, Santa Clara, CA). "Buffer "A" was supplemented with protease inhibitor tablets (Roche, Basel, Switzerland) in a ratio of 1 :300 ml. buffer. After filtration (0.22 μm, 14000 rpm; Costar Spin-X Centrifuge Tube filters) (Cole-Parmer, Vernon Hills, IL) the sample was depleted in 2 consecutive runs on a human MARS human-6 column (Agilent) per the manufacturer's instruction, effectively removing 6 high-abundant serum proteins. Albumin and IgG depletion efficiency was tested via Western Blotting. The 2 flow throughs (2 ^* 1 ml.) were pooled and the protein concentration was roughly determined to be -0.5 mg/mL by measuring the absorbance at 280 nm (Evolution 3000, Thermo Electron, Waltham, MA). The protein sample was subsequently concentrated 3x using a Vivaspin filter with a MWCO of 3000 Da (Vivascience, Littleton, MA) to yield a protein concentration of -1.5 mg/mL for the sample, as confirmed by BCA (Pierce, Rockford, IL). Protein denaturation was effected by adding guanidine hydrochloride (Merck, Whitehouse Station, NJ) up to a final concentration of 3M. The resulting protein mixture was reduced during a 10 min incubation step at 30⁰C with a 25 molar excess tris(2-carboxyethyl)-phosphine (Pierce). Subsequently, the protein cysteinyl residues were alkylated for 60 min with a 50 molar excess of iodoacetamide (Sigma-Aldrich, Buchs SG, Switzerland) at 30⁰C. A buffer exchange to 1.4M guanidinium chloride in 50 mM sodium phosphate pH = 8, was performed on a PD10 column (Amersham Biosciences, Uppsala, Sweden) according to the manufacturer's guidelines. The resulting 3.5 ml. effluent volume was concentrated to 2.0 ml. by vacuum centrifugation (Centrivap Concentrator, Labconco, Kansas City, MO). Then, the protein content was acetylated for 90 min at 30⁰C with a 75 molar excess of sulfo-N-hydroxysuccinimidyl acetate (Pierce). A deacetylation, for 20 min at room temperature, with a 3.5 molar excess hydroxylamine compared to sulfo-N- hydroxysuccinimidyl acetate, was performed to deprotect acetylated serine, threonine and tyrosine side chains. Following the reverse acetylation step, the sample was again desalted on a PD10 column and captured in a 20 mM NaHCC>3 pH = 8 buffer. The final protein concentration was measured to be 0.49 mg/mL (BCA). The modified protein material was then heated for 5 min at 99°C and digested overnight at 37°C after adding trypsin (Promega Corporation, Madison, Wl) in a substrate:trypsin ratio of 50:1 (w:w). Before storage at -20⁰C the sample volume was reduced by vacuum concentration to obtain a concentration of -2 mg/mL (extrapolation of the BCA results) and an associated buffer concentration of 80 mM NaHCO₃ (pH = 8).

For the actual experiments, 2 aliquots of 125 μl_ (-250 μg peptide material) were taken and concentrated to dryness by means of vacuum centrifugation and stored at -20°C till sorting. After WCX-CE sorting (vide infra) 1/3 of the 4 flow through fractions was combined into 1 sample, which was dried by evaporation under reduced pressure (vacuum centrifuge). One of the two combined flow throughs was further processed: after reconstitution in 100 μl_ 0.02M sodium phosphate pH = 8, 1 unit of PNGase F was added, followed by an overnight incubation at 37°C. After deglycosylation the sample was again dried by vacuum centrifugation. This sample will be referred to as "PNGase F POST in the remainder of the text. The other non-deglycosylated sample will be termed "reference".

Weak Cation Exchange-Crown Ether (WCX-CE) operation (sorting)

The WCX-CE column used was an lonPac CS 15 cation-exchange column (Dionex, Amsterdam, The Netherlands) of 2 mm i.d. x 250 mm length, containing a 8 μm particulate resin of 55% crosslinked ethylvinylbenzene/divinylbenzene, functionalised with phosphonate, carboxylate and crown ether groups. The WCX-CE separations were performed on an Agilent 1 100 series HPLC system (Agilent Technologies, Waldbronn, Germany) equipped with a multiple wavelength detector and an 1 100 series fraction collector. Operation, data collection and analysis were done using the Chemstation software (Agilent). An additional 400 μl_ stainless steel seat capillary (Agilent) was mounted on the injector allowing for large volume injections (500 μl_). All chromatographic steps were performed at room temperature with a flow rate of 100 μL/min, and the UV absorbances at 214 and 280 nm were recorded. All solvents, i.e. water (H₂O) and acetonitrile (ACN), and additives, i.e. formic acid (FA) and trifluoroacetic acid (TFA) for WCX-CE operation and nano LC operation (vide infra) were purchased from the same supplier in the appropriate grade (Biosolve, Valkenswaard, The Netherlands)

The samples, i.e. reference and PNGase F POST were reconstituted in 510 μl_ of the loading solvent (0.025:49.975:50 (v/v) FA/H₂O/ACN). Three discrete chromatographic steps were implemented:

(i) A sample loading step comprising a 500 μl_ in-flow injection of the sample followed by an ioscratic 45 min of the loading solvent. Sample flow troughs were collected in 4 fractions: 4.5 -14.5 min (1000 μl_), 14.5 - 24.5 min (1000 μl_), 24.5 - 29.5 min (500 μl_) and 29.5 - 39.5 min (1000 μl_). The corresponding UV traces at 214 and 280 nm are respectively shown in Figure 10 A & B.

(ii) An elution step of 90 min in which the bound peptide fraction was released from the WCX-CE column by a 70 min linear gradient from 100% 50:50 (v/v) H₂O/ACN (A) to 100% 1 :49:50 (v/v) TFA/H₂O/ACN (B) followed by a 10 min isocratic section of 100% B and a 10 min reverse gradient to 100% A. Exemplary UV-traces are also shown in Figure 10 C.

(iii) A 3h equilibration step using the loading solvent (0.025:49.975:50 (v/v) FA/H₂O/ACN), after which the next sample could be applied. Following this sorting step 1/3 of the 4 flow through fractions of each run was pooled. One pool was dried completely (vacuum centrifugation; reference) and the other pool was processed further as described above {PNGase F POST).

Reversed Phase nano LC-MS/MS analysis

Further analysis was performed by a reversed phase nano-LC separation coupled on-line with a QqTOF mass spectrometer via an electrospray ionisation (ESI) interface. An Agilent 1200 series nano-HPLC system (Agilent) was fitted with a column switching set-up comprising a 300 μm i.d. x 5 mm C18 reversed phase (5 μm, 300SB Zorbax) precolumn (Agilent) and a custom made 100 μm i.d. x 120 cm analytical nano RP-column (C18, 5 μm, 300SB Zorbax). The 2 samples were reconstituted in 100 μl_ 0.1% (v/v) FA in H₂O of which 40 μl_ was injected. The precolumn was loaded at 20 μL/min with 0.1 :99.9 (v/v) FA/H₂O. After 5 min, the sample was transferred to the nano RP-column. The analytical chromatography involved a binary solvent system, i.e. 0.1 :99.9 (v/v) FA/H₂O (solvent A) and 0.1 :19.9:80 (v/v) FA/H₂O/ACN (solvent B), and a flow rate of 350 nL/min was used. Peptide elution was achieved by applying a linear gradient from 10%B to 65%B in 400 min (initialised at sample injection), followed by a rinsing (65%B to 90%B (400 - 401 min), 90%B (401 - 416 min)) and a re-equilibration section (90%B to 10% B (416 - 417 min) and 10%B (417-480 min)). The column was directly joined to a PicoTip™ ESI-emitter (silica, distal coated, 360/20 μm o.d., 10 μm i.d.) (New objective, Woburn, MA) by means of a stainless steel zero dead volume connection (Agilent), via which the electrospray voltage was applied to the column effluent. The emitter assembly was fitted on a Nanospray™ stage (Applied Biosystems/MDS SCIEX, Foster City, CA) mounted on a QSTAR^® Elite Hybrid LC/MS/MS system (Applied Biosystems/MDS SCIEX). The mass spectrometer was operated in the information dependent analysis (IDA) mode. The following instrument parameters were used: a positive ESI voltage of +2000 V, a declustering potential of 55 V and a curtain gas pressure of 20 psi. The IDA criteria adopted for precursor ion selection were: a m/z range of 300-1500, a 1 s accumulation time, and selection of the 2 most intense 2⁺ or 3⁺ charged signals per scan for fragmentation, if exceeding a set threshold of 40 cps. Selected precursor ion masses were then excluded for 600s. For the product ions spectra acquisitions a m/z range of 70-1500 was set. Optimal collision energy values were automatically determined as well as spectrum quality: automatic MS/MS accumulation was enabled with a maximum of 3s. Mass spectrometric data was collected during the entire nano-LC run.

MS/MS data analysis and Modified sequence Categorization

The collected MS/MS spectral data were converted to Mascot generic files (mgf) using the Analyst QS 2.0 software plug-in (mascot.dll; Matrix Science/Applied Biosystems/MDS SCIEX). The Mascot™ search algorithm (Matrix Science Inc., Boston, MA, US) was run with Swiss-Prot 54.2 as database, holding 17170 human protein sequences. To accommodate the extensive protein processing encountered in serum samples, the spectra were searched using no-enzyme search settings. All real database searches were complemented with a search against its random counterpart to calculate the false discovery rate (FDR). MS and MS/MS tolerance was set to 0.1 Da, and charges up to 3⁺ were allowed. In total, 9 modifications were preset: 1 fixed i.e., carbamidomethylation of cysteine, and 8 variable i.e., acetylation of lysine and the N-terminus, deamidation of asparagine and glutamine, oxidation of methionine to its sulfoxide derivative, pyro- carbamidomethyl formation on N-terminal cysteine (pyro-cmc) and pyro-glutamate formation of N-terminal glutamine and glutamic acid (pyroglu Q/E). Only peptides ranking #1 with scores above the identity probability threshold were withheld. Spectra that had multiple peptide hits above the probability threshold were regarded as unidentified. A protein is reported only if it was represented by at least 1 unequivocally assigned peptide. For all identified modified sequences titration curves were calculated according to Shimura algorithm (Shimura et al. Analytical Chemistry 2002, 74, 1046-53), using an automated (perl) script providing pi information as well as the net charge for all pH values of 0.1 to 14 in increments of 0.1. Typical amino acid pKa's were used (http://vwtfwjnnovagen.se/custorn-peptide-synthesis/peptide-property-calculator/peptide- βrop_.θrtyr_.cMculatoi:TOles_Λasβ, Innovagen, Lund, Sweden), except where modifications affect the value (pyro-glutamate formation on N-terminal glutamic acid and N-terminal glutamine, deamidation of asparagine and glutamine, acetylation of lysine, N-terminal acetylation). To classify a modified sequence as a N-glycopeptide, the peptide has to contain the consensus motif N-X-S/T, with X not being a proline, together with the PNGase F mediated conversion of asparagine to aspartic acid (cf. deamidation of N to D). Visualization of the data was done by means of Spotfire^® DecisionSite^® 9.0 (Tibco Spotfire, Goteborg, Sweden).

Analysis of a WCX/CE flow through ("reference" sample)

One third of each of the 4 collected flow through fractions of the reference sample was pooled, dried, reconstituted and further analysed by a 1.2m nano RPLC-ESI-MS/MS setup. From this analysis, 341 modified sequences were identified with an adopted database search strategy which was tailored to (i) account for the specific chemistry applied and (ii) to deal with in vivo processing events typical for the serum proteome. An important enrichment of N-terminally blocked peptides was achieved: 67.75% (231/341 ) of the identified modified sequences were N-terminally blocked.

Analysis of the "PNGase F POST" sample: Co-enrichment of glycopeptides

While the reference experiment was promising in terms of sorting efficiency, only a rather low number of modified sequences was identified in spite of the high number of good quality MS/MS spectra generated.

Manual inspection of the MS/MS data revealed that an important fraction of the unidentified spectra had m/z signals corresponding with low-molecular-weight glycopeptide-marker ions. These oxonium marker ions correspond to N-acetylhexosamine (m/z 204), which is considered to be indicative for both N- and O-glycans, and N- acetylneuraminic acid minus H₂O (m/z 274), which is indicative for sialoglycopeptides (Wuhrer et al. Journal of Chromatography B-Analytical Technologies in the Biomedical and Life Sciences 2007, 849, 115-28). In the left panes of Figure 11 , the Total Ion Current (TIC) of the combined MS/MS experiments and the there from extracted Ion Chromatograms (XIC) of the diagnostic oxonium ions are plotted. These traces indicate that (i) glycopeptides can constitute an important fraction of the WCX/CE flow-through, and (ii) especially sialoglycopeptides are well represented.

To confirm the glycopeptide co-enrichment in the WCX/CE flow through, a second WCX/CE pre-purification was performed on the same serum digest. However, prior to further nano-RPLC-ESI-(MS/MS) analysis the flow through was incubated with N- glycosidase F (PNGase F), to remove all types of asparagine bound N-glycans.

These deglycosylated peptides should be easier to identify. The right panes in Figure 11 present the same information for the PNGase F POST experiment as for the reference experiment in the left panes. Compared with the traces of the reference experiment, both the intensity and the peak density (~#) of the extracted ion chromatograms of the diagnostic glycopeptide oxonium ions decreased, although there is clearly room for further optimisation of the deglycosylation step. From this observation one can learn that indeed N-glycopeptides were co-enriched in the WCX/CE flow through.

Following LC-MS analysis and sequence identification, the N-glycan stripped peptides can be discerned from other peptides based on the fact that (i) N-glycosylation sites generally fall into the Asn-XXX-Ser/Thr sequence motif in which XXX denotes any amino acid except proline and (ii) additionally the PNGase F cleavage involves an asparagine to aspartic acid conversion (N<deamidation (NQ)>). Modified sequences that comply with these 2 criteria were considered as originating from N-glycopeptides.

Using the same database search strategy the PNGase F post experiment led to 408 identified sequences, i.e. compared to the reference experiment -20% more sequences were identified. Surprisingly, about 50% of the identified modified sequences correspond to a N-glycopeptide, i.e. 201 out of 408 (49.26%). Furthermore, the percentage of sequences with free α-NH₂ termini is significantly higher in the N-glycopeptide fraction (127/201 ; 83.08%) than in the reference data set (110/341 ; 32.26%) and the non-glycopeptide fraction (56/207; 27.05%). Hence, glycan modifications proximal to the α-NH₂ terminus of peptides may interfere with [crown ether - H₃N⁺-group] complexation thereof.

Given the effective co-enrichment of N-glycopeptides within the WCX/CE flow the inventors also contemplate that a similar experimental set-up but without the N-terminal blocking step (e.g., without acetylation) would lead to a highly efficient N-glycopeptide enrichment approach. Preliminary results (not shown) prove that in such experiment -80% of the retrieved sequences were N-glycopeptides, whereas most of the remainder sequences corresponded to in vivo acetylated protein N-termini.

Conclusions In total, the reference and the PNGase F Post experiment led to the identification of 102 different protein groups, of which 37 were identified from both the true N-teromics data (reference experiment and the non-glycopeptide fraction in the PNGase F POST experiment) and the N-glycopeptide enrichment.

39 proteins were unique to the N-teromics data, whereas 26 were only represented in the glycopeptide enriched fraction. In Figure 12 the position of the identified sequences in their parent proteins is summarised for the non-glycopeptides and the glycopeptides (based on the N-terminal amino acid position; up to position 200). It is clear from pane A in Figure 12 that many of the acetylated non-glycopeptides are positioned at the beginning of their proteins. Most of these peptides are not located at position 1 because the loss of any signal peptides is not corrected for. This confirms the merits of a WCX/CE flow through analysis in an N-teromics context: a preferential enrichment of the true N-termini of proteins is obtained. The acetylated sequences retrieved from other positions in the proteins point to in vivo processing events, information that is also considered relevant in a biomarker context. The glycopeptides show, as expected, a more uniform distribution in terms of their positions within the proteins (Figure 12, pane B).

N-terminally acetylated peptides also can be indicative for in vivo processing of the serum proteins. Often such in vivo processing involves aminopeptidase activity (Sanderink et al. Clinical Chemistry 1988, 34, 1422-26) resulting in N-terminally ragged sequences. The N- teromics approach here applied exposes such processing events because it selects for N- terminally acetylated peptides. An example is given in Table 3. However, within the glycopeptide enriched fraction, groups of unacetylated N-terminally ragged sequences were identified (Table 3). This could indicate that the presence of glycan modifications close to free α-NH₂ groups (in vivo) impairs the acetylation reaction. Therefore, a slightly adapted N-teromics workflow, wherein prior to acetylation the proteins are deglycosylated, could improve the acetylation efficiency. At the same time, these data show that glycopeptide (co-)enrichment can expose in vivo protein processing events.

Table 3. The left column shows some exemplary N-terminally acetylated sequences of Alpha-1-acid glycoprotein 2 [Precursor] (Swiss-Prot entry P19652). Compliant with the applied N-teromics approach the observed N-terminal ragging is indicative for some in vivo aminopeptidase activity. Within the right column a set of sequences derived from the Platelet basic protein [Precursor] (Swiss-Prot entry P02775) is given. These sequences demonstrate no N-terminal acetylation, yet they were co-enriched within the WCX/CE flow through because they were N-glycosylated during the sorting step. Interestingly, some N- terminal ragging is also apparent from these sequences. To the best of our knowledge no protocol related reasons account for this N-terminal ragging, implicating these sequences also reflect some in vivo processing events:

Alpha-1-acid glycoprotein 2 [Precursor] Platelet basic protein [Precursor]

Position 44, 45, ...—62 Position 56, 59...—81

Ac-NLAKGKEESLDSDLYAELR NH₂-SVQEIQATFFYFTPNKTEDTIFLR

Ac-LAKGKEESLDSDLYAELR NH₂-EIQATFFYFTPNKTEDTIFLR

Ac-AKGKEESLDSDLYAELR NH₂-ATFFYFTPNKTEDTIFLR

Ac-GKEESLDSDLYAELR NH₂-TFFYFTPNKTEDTIFLR

Ac-EESLDSDLYAELR NH₂-FFYFTPNKTEDTIFLR

NH₂-FYFTPNKTEDTIFLR

NH₂-YFTPNKTEDTIFLR

NH₂-FTPNKTEDTIFLR NH₂-TPNKTEDTI FLR

It shall be appreciated that versatility of the platform can be achieved by adding/removing the N-termini and lysine acetylation step in the sample preparation procedure and by the timing of the deglycosylation step. This way, one could solely target N-terminally acetylated peptides, or N-terminally acetylated peptides and glycopeptides, or glycopeptides and in vivo acetylated peptides only.

Claims

1. A method of preparing a biological sample for protein profiling, comprising the steps of: pretreating the sample (1 ) with one or more reagents (20) to effect blocking of the primary amines,

- treating (2) the pretreated sample (1¹) with a cleavage reagent to generate peptides (7) comprising N-terminal primary amines (3, 4, 5), and sorting (9, 10) the peptides (7) by non-covalent interactions using a solid support (11 , 13), wherein the non-covalent interactions are H-bridges or pi-pi (π-π) interactions.

2. A method according to claim 1 , wherein the cleavage reagent comprises trypsin.

3. A method according to any of claims 1 or 2, where the solid support is in the form of beads, pellets, resin, small particles, a membrane, a frit, a sintered cake, or a monolith.

4. A method according to claim 3, where the solid support is comprised in a chromatography column, a phase extraction cartridge (SPE), magnetic bead, centrifugable or filterable bead.

5. Method according to any of claims 1 to 4 wherein the peptides are sorted by the solid support in a liquid chromatography mode or batch mode.

6. Method according to any of claims 1 to 5, comprising the step of blocking the primary amine groups and optionally the cysteine groups of proteins present in the sample prior to treatment with a cleavage reagent, and wherein the solid support (11 - Fig. 3) comprises an immobilized host compound that selectively binds protonated primary amines using H- bridges.

7. Method according to claim 6, wherein said host compound is an organic cyclic compound that provides a cylindrical or circular arrangement of hydrogen acceptor atoms at positions and orientations that maximise non-covalent binding with three H-atoms of a protonated primary amine.

8. Method according to claim 6 or 7, wherein the host compound is a crown ether or a macrolide antibiotic.

9. Method according to claim 8, wherein the immobilized host compound is 18-crown-6 ether.

10. A method according to claim 9, wherein the immobilized 18-crown-6-ether is unsubstituted or substituted.

1 1. A method according to any of claims 6 to 10, wherein the host compound is immobilized on the solid support using a linker, preferably wherein the linker is (poly)ethylene glycol, a reduced sugar, or an acyclic dicarboxylic acid.

12. Method according to any of claims 1-11 , for the enrichment of glycopeptides.

13. Method of claim 12, wherein the glycosylation is due to glycan moieties situated at asparagine, serine or threonine.

14. Method according to any of claims 1 to 5 or 12-13, further comprising the steps of:

15. Method according to claim 14wherein the aromatic moiety used to modify the N- terminal primary amines is an aryl, arylalkyl, heteroaryl or heteroarylalkyl.

16. Method according to claim 14 or 15, wherein the aromatic moiety immobilized on the solid support is an aryl, arylalkyl, heteroaryl or heteroarylalkyl.

17. Method according to claim 15or 16, wherein said aryl comprises any of phenyl, biphenylyl, biphenylenyl, 5- or 6-tetralinyl, 1-, 2-, 3-, 4-, 5-, 6-, 7- or 8-azulenyl, 1- or 2- naphthyl, 1-, 2- or 3-indenyl, 1-, 2- or 9-anthryl, 1- 2-, 3-, 4- or 5-acenaphtylenyl, 3-, 4- or 5-acenaphtenyl, 1-, 2-, 3-, 4- or 10-phenanthryl, 1- or 2-pentalenyl, 1 , 2-, 3- or 4-fluorenyl, 4- or 5-indanyl, 5-, 6-, 7- or 8-tetrahydronaphthyl, 1 ,2,3,4-tetrahydronaphthyl, 1 ,4- dihydronaphthyl, dibenzo[a,d]cylcoheptenyl, 1-, 2-, 3-, 4- or 5-pyrenyl.

18. Method according to claim 17, wherein said heteroaryl is any of 2- or 3-furyl, 2- or 3- thienyl, 1-, 2- or 3-pyrrolyl, 1-, 2-, 4- or 5-imidazolyl, 1-, 3-, 4- or 5-pyrazolyl, 3-, 4- or 5- isoxazolyl, 2-, 4- or 5-oxazolyl, 3-, 4- or 5-isothiazolyl, 2-, 4- or 5-thiazolyl, 1 ,2,3-triazol-1-, -2-, -4- or -5-yl, 1 ,2,4-triazol-1 -, -3-, -4- or -5-yl, 1 ,2,3-oxadiazol-4- or -5-yl, 1 ,2,4-oxadiazol- 3- or -5-yl, 1 ,2,5-oxadiazolyl, 1 ,3,4-oxadiazolyl, 1 ,2,3-thiadiazol-4- or -5-yl, 1 ,2,4- thiadiazol-3- or -5-yl, 1 ,2,5-thiadiazol-3- or -4-yl, 1 ,3,4-thiadiazolyl, 1- or 5-tetrazolyl, 2-, 3- or 4-pyridyl, 3- or 4-pyridazinyl, 2-, 4-, 5- or 6-pyrimidinyl, 2-, 3-, 4-, 5- 6-2H-thiopyranyl, 2-, 3- or 4-4H-thiopyranyl, 2-, 3-, 4-, 5-, 6- or 7-benzofuryl, 1-, 3-, 4- or 5-isobenzofuryl, 2-, 3-, 4-, 5-, 6- or 7-benzothienyl, 1-, 3-, 4- or 5-isobenzothienyl, 1-, 2-, 3-, 4-, 5-, 6- or 7-indolyl, 2- or 3-pyrazinyl, 1 ,4-oxazin-2- or -3-yl, 1 ,4-dioxin-2- or -3-yl, 1 ,4-thiazin-2- or -3-yl, 1 ,2,3- triazinyl, 1 ,2,4-triazinyl, 1 ,3,5-triazin-2-, -4- or -6-yl, thieno[2,3-b]furan-2-, -3-, -4-, or -5-yl, 1-, 2-, 4- or 5-benzimidazolyl, 1-, 3-, 4-, 5-, 6- or 7-benzopyrazolyl, 3-, 4-, 5-, 6- or 7- benzisoxazolyl, 2-, 4-, 5-, 6- or 7-benzoxazolyl, 3-, 4-, 5-, 6- or 7-benzisothiazolyl, 2-, 4-, 5- , 6- or 7-benzothiazolyl, 1-, 2-thianthrenyl, 3-, 4- or 5-isobenzofuranyl, 1-, 2-, 3-, 4- or 9- xanthenyl, 1-, 2-, 3- or 4-phenoxathiinyl, 2-, 3-pyrazinyl, 1-, 2-, 3-, 4-, 5-, 6-, 7- or 8- indolizinyl, 2-, 3-, 4- or 5-isoindolyl, 1-, 2-, 3-, 4-, 5-, 6- or 7-indazolyl, 2-, 6-, 7- or 8-purinyl, 4-, 5- or 6-phthalazinyl, 2-, 3- or 4-naphthyridinyl, 2-, 5- or 6-quinoxalinyl, 2-, 4-, 5-, 6-, 7- or 8-quinazolinyl, 1-, 2-, 3- or 4-quinolizinyl, 2-, 3-, 4-, 5-, 6-, 7-, or δ-quinolinyl(quinolyl), 2- , 4-, 5-, 6-, 7- or 8-quinazolyl, 1-, 3-, 4-, 5-, 6-, 7- or δ-isoquinolinyl(isoquinolyl), 3-, 4-, 5-, 6-, 7- or 8-cinnolinyl, 2-, 4-, 6- or 7-pteridinyl, 1-, 2-, 3-, 4- or 9-carbazolyl, 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8- or 9-carbolinyl, 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9- or 10-phenanthridinyl, 1-, 2-, 3- or 4- acridinyl, 1-, 2-, 3-, 4-, 5-, 6-, 7-, 8- or 9-perimidinyl, 2-, 3-, 4-, 5-, 6-, 7-, 8-, 9- or 10- (1 ,7)phenanthrolinyl, 1- or 2-phenazinyl, 1-, 2-, 3-, 4-, or 10-phenothiazinyl, 3- or 4- furazanyl, 1-, 2-, 3-, 4-, or 10-phenoxazinyl, azepinyl, diazepinyl, dibenzo[b,f]azepinyl, dioxanyl, thietanyl, oxazolyl dibenzo[a,d]cylcoheptenyl, or additionally substituted derivatives thereof.

19. Method according to any of claims 15 to 18, wherein the alkyl of an arylalkyl, or heteroarylalkyl is any of methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert- butyl, 2-methylbutyl, pentyl and its isomers, hexyl and its isomers, heptyl and its isomers, and octyl and its isomers.

20. Method according to claim 14, wherein:

(a) the aromatic moiety used to modify the N-terminal primary amines is a pi-donor, when the aromatic moiety immobilized on the solid support is a pi-acceptor; or (b) the aromatic moiety used to modify the N-terminal primary amines is a pi-acceptor, when the aromatic moiety immobilized on the solid support is a pi-donor.

21. Method according to claim 20, wherein a pi-acceptor is an aromatic moiety as defined in any of claims 15 to 19 substituted with at least one electron-withdrawing group.

22. Method according to claim 21 , wherein the electron-withdrawing group is any of NO₂, NH₃, SO₂OH, CN, CF₃, F, COOH, ⁺NR₃, ⁺NHR₂ or ⁺NH₂R, where R is an alkyl group.

23. Method according to claim 22, wherein said aromatic moiety comprises trinitrophenyl and/or pentafluorophenyl.

24. Method according to claim 20, wherein a pi-donor is an aromatic moiety as defined in any of claims 15 to 19 substituted with at least one electron-donating group.

25. Method according to claim 24, wherein the electron-donating group is any of OH, OMe or NH₂, NR₂ or NHR, where R is an alkyl group.

26. Method according to claim 25, wherein said aromatic moiety comprises p- methoxyphenyl, 4-N,N-dimethylaminophenyl, or phenyl.

27. Method according to any of claims 13 to 26, wherein said aromatic moiety is immobilized on the solid support or peptide by a linker, preferably wherein said linker is (poly)ethylene glycol, a reduced sugar, or an acyclic dicarboxylic acid.

28. Method according to any of claims 1 to 27, wherein said pretreatment comprises the steps of blocking the cysteine groups followed by blocking the primary amine groups.

29. Method according to claim 28, wherein said primary amine groups are blocked using N-hydroxysulfosuccinimidyl acetate.

30. Method according to claim 28 or 29, wherein said cysteine groups are blocked comprising the use of any of iodoacetamide, N-substituted maleimides, acrylamide, N- substituted acrylamide, tris(2-carboxyethyl)phosphine, or 2-vinylpyridine.

31. Method according to any of claims 1 to 30, further comprising the step of analytical separation of peptides not captured by the solid support, so providing a protein profile of the sample.

32. Method according to claim 31 , wherein the analytical separation is one-, two-, three-, or higher-dimensional liquid chromatography.

33. A kit for preparing a sample for protein profiling comprising one or more of the following components: - a primary amine blocking reagent, preferably as defined in claim 29,

- a cysteine group blocking reagent, preferably as defined in claim 30,

- cleavage reagent as defined in any of claims 1 or 2,

- host compound as defined in any of claims 7 to 10,

- aromatic moiety as defined in any of claims 15 to 26, - solid support as defined in any of claims 3, 4, 13, 27,

- solid support as defined in claim 3 or 4, comprising one or more immobilized aromatic moieties as defined in any of claims 15 to 27,

- solid support as defined in claim 3 or 4, comprising one or more immobilized host compounds as defined in any of claims 7 to 10, - said solid support provided in a cartridge.

34. A liquid chromatography column having a solid support comprising at least one immobilised crown ether.

35. Column according to claim 34, suitable for use in identifying proteins in a complex biological sample.

36. Column according to claim 34 or 35, wherein the immobilised crown ether is unsubstituted or substituted.

37. Column according to any of claims 34 to 36, wherein the immobilized crown ether is 18-crown-6-ether.

38. Column according to any of claims 34 to 37, wherein the crown ether is immobilized on the solid support using a linker, preferably wherein the linker is (poly)ethylene glycol, a reduced sugar, or an acyclic dicarboxylic acid.

39. Column according to any of claims 34 to 38, wherein the solid support is prepared from a native polymer, preferably a cross-linked carbohydrate material, more preferably wherein the native polymer material is any of agarose, agar, cellulose, dextran, chitosan, konjac, carrageenan, gellan, alginate.

40. Column according to any of claims 34 to 38, wherein the solid support is prepared from a synthetic polymer or copolymer, preferably a cross-linked synthetic polymer, preferably wherein the synthetic polymer or copolymer is any of styrene or styrene derivatives, divinylbenzene, acrylamides, acrylate esters, methacrylate esters, vinyl esters, vinyl amides.

41. Column according to any of claims 34 to 38, wherein the solid support is prepared from silica.

42. Use of a crown ether for preparing a biological sample for protein profiling.

43. Use of a crown ether to identify proteins in a biological sample.

44. Use according to claim 42 or 43, wherein the crown ether is substituted or unsubstituted.

45. Use according to any of claims 42 to 44, wherein the crown ether is any as defined in claim 34 or 35.

46. Use according to any of claims 42 to 45, wherein the crown ether is immobilised onto a solid support.

47. Use according to claim 46, wherein the solid support is in the form of beads, pellets, resin, small particles, a membrane, a frit, a sintered cake, or a monolith.

48. Use according to claim 46 or 47, wherein the solid support is comprised in a chromatography column, a phase extraction cartridge (SPE), magnetic bead, centrifugable or filterable bead.

49. Use according to any of claims 46 to 48, wherein the solid support is prepared from the materials as defined in any of claims 39 to 41.

50. Use according to any of claims 42 to 49, comprising the identification of proteins by sorting peptides in the sample having one or more primary amines, following cleavage of the proteins by a cleavage reagent.

51. Use according to claim 50, wherein the cleavage reagent comprises trypsin.

52. Use according to any of claims 50 or 51 , wherein the peptides having one or more primary amines are N-terminal peptides.

53. Use according to any of the claims 42 - 52, for the enrichment of glycopeptides

54. Use according to claim 53, wherein the glycopeptides are formed by addition of glycan group at asparagine, serine or threonine.

55. Use according to claim 53 or 54, wherein no N-terminal acetylation step is performed on the peptide mixture, in order to isolate in vivo glycosylated and N-acetylated peptides.

56. Use according to claim 53 or 54, wherein additionally the peptide mixture is pretreated with an N-terminal blocking agent, in order to identify or enrich in vivo glycosylated and in vivo and in vitro N-acetylated peptides.

57. Use according to claims 42 to 52, wherein a deglycosylation step is performed on the peptide mixture, in order to eliminate the glycosylated peptides from the analysis and enrich only the in vivo or in vitro N-acetylated peptides.

58. Use according to claim 57, wherein no additional N-acetlyation step is performed on the peptide sample, in order to enrich only the in vivo N-acetylated peptides.