WO2004111646A1

WO2004111646A1 - Novel ms reagents

Info

Publication number: WO2004111646A1
Application number: PCT/SE2004/000860
Authority: WO
Inventors: Peter James; Jean-Luc Maloisel; Ulrika Carlsson; Philippe Busson; Åsa WÅHLANDER; Ronnie Palmgren
Original assignee: Amersham Bioscience AB
Current assignee: Cytiva Sweden AB
Priority date: 2003-06-19
Filing date: 2004-06-03
Publication date: 2004-12-23
Anticipated expiration: 2005-12-19
Also published as: GB0314209D0

Abstract

The present invention relates to novel MS reagents comprising the following formula: CG-LG1-BG-LG2-RG Wherein CG is Charged Group, LG1 is Linker Group 1, BG is Bridge Group, LG2 is Linker Group 2, RG is Reactive Group, And CG, LG1, BG, LG2, and RG are covalently connected. Preferably the reagent molecule fully or partly contains a label/labels, which is possible to detect, by subsequent analysis such as mass spectroscopy. The invention is to be used for identification and quantification of proteins/ peptides by MS and specifically via the analysis of parent ions. By using two differently labelled reagent molecules the invention enables, for example, relative quantification of protein/peptide in a sample. In a second aspect the invention relates to a kit comprising the reagent molecules. The invention also relates to use of the reagent molecules for analysis of biomolecules.

Description

NOVEL MS REAGENTS

Technical field

The present invention relates to novel mass spectrometry reagents suitable to be used in a fully automated system for, for example, protein expression analysis. The reagents may be used for differential isotopic labelling of whole cell digests and these may be subsequently analysed by multi-dimensional chromatography and/or electrophoresis coupled to mass spectrometry and optionally database searching.

Background to the invention

The study of the protein version of the genome, 'proteomics', must deal with 40,000 or more genes which can be arranged to give some 800,000 proteins (corresponding to some 10⁷ tryptic peptides), which in turn can be modified with over 300 different chemicals. Not only that, proteomics must also define which proteins are being produced in a certain type of cell at a specific time, how they are modified, where they are in the cell and with whom they are in contact and finally and most difficult, what is the function of the protein.

Today, some methods are available for such analyses and determinations. For instance, WOOO/11208 discloses a method in which a protein is derivatised with an isotopically labelled molecule. The labelled protein is captured, digested, released and analysed by mass spectrometry.

WOO 1/74842 (Proteome Systems) teaches a method in which the desired protein sample is subjected to 2D-electrophoresis separation (2D-SDS), specific residues protected be- fore digestion, and derivatisation with a labelled reagent and analysed by mass spectrometry. However, these methods have shown to be limited due to the use of 2D- electrophoresis, which does not allow a separation, which leads to a visualisation of all proteins. Many proteins are incompatible with this method, either being too small or too large, too acidic or alkaline, or just too insoluble. Membrane proteins, which is one of the most important group of proteins, both physiologically and pharmaceutically, are completely underrepresented due to their tendency to aggregate and precipitate during the various steps in 2D electrophoresis. Therefore, these proteins tend to be excluded from labelling and thus also from the analysis. In addition, the method disclosed in WO 01/74842 cannot be used in MS in parent ion-scanning mode, since the reagent described therein is not capable of generating any signature ions.

Further, WO 01/86306 (Purdue Research) relates to a method for protein identification in complex mixtures that utilises affinity selection of constituent proteolytic peptide fragments unique to a protein analyte. These "signature peptides", which are low abundance amino acids such as Cys or Met, act as analytical surrogates for chemical capture of re- agents. Mass spectrometric analysis of the proteolysed mixture permits identification of a protein in a complex sample without purifying the protein or obtaining its composite signature, since the use of "signature peptides" will reduce the complexity of the analysis. However, such "signature peptides" should not be confused with the signature ions required in MS in parent ion mode, which is not possible with the method disclosed in WO 01/86306.

Aebersold et al (American Genomic/Proteomic Technology (Aug. 2001), Vol. 1(1), p. 22-27) discloses isotope-coded affinity tag reagents for quantitative proteomics. However, this method requires the reduction in peptide complexity to be achieved by affinity purification and not by MS in parent ion-scanning mode. Likewise, Goodlet et al (Rapid Communications in MS, 2001, 15, 1214-1221) discloses a chemical tagging of proteins specific to Asp and GIu. The reagents are MS/MS stable, and cannot generate the specific fragment signature ions required in MS in parent ion-scanning mode. Another method which for the same reasons is not useful in parent ion-scanning mode either has been disclosed by Wang et al (Journal of Chromatography A, 924 (2001) 345-357), wherein chemical affinity chromatography is used to reduce sample complexity.

WO 02/48717 relates to an acid-labile isotope-coded extractant and its use in quantitative mass spectrometric analysis of protein mixtures. The reagents used in such method must be thiol specific and MS/MS stable. Thus, this method can not generate any signature fragment ions, and is consequently not useful in MS in parent ion-scanning mode. Finally, Carr et al have described methods for following phosphate loss from phos- phopeptides (Selective detection and sequencing of phosphopeptides at the femtomole level by mass spectrometry, Anal. Biochem. 239(2): 180-92, 1996). The method relies on the generation of a natural signature ion -79 m/z that is due to the loss of phosphate. The occurrence of phosphate can also be followed by the loss of phosphate as a neutral molecule using the neutral loss-scanning mode.

However, often it is desired to compare a cell in two different states, in order to deter- mine the differences on the protein level. In these cases, a problem is to reduce the amount of data obtained by any one of the methods used today, in order to be able to focus on the relevant proteins. Thus, it would be advantageous to provide novel MS reagents with which relevant proteins from different cell states may be studied and compared in a better way.

Accordingly, an object of the invention is to provide a novel MS reagents solving the posed problems.

Summary of the invention In co-pending PCT/EP02/14328 the inventors have developed a method, which meets the demands of the proteomics research society. This method is for labelling of a protein or a polypeptide mixture, which has been extracted from a set of cells, with an isotopic labelled reagent molecule, and analysing it with MS parent ion-scanning. In another preferred embodiment two different sets of cells, representing two different states, are ana- lysed by the method, whereby each set of cells is labelled with different reagent molecules, allowing for a subtractive parent ion or neutral loss scanning. In a first aspect, the present invention relates to improved reagent molecules, and in a second aspect the invention relates to a kit, comprising the reagent molecules. In a last aspect, the invention relates to use of said reagents for analytical and diagnostic pur- poses. In a preferred embodiment, the reagent molecules are designed to be used for identification and quantification of proteins/peptides by MS and specifically via analysis of parent ions.

Thus, the invention relates to a Reagent Molecule of the following formula:

CG-LG1-BG-LG2-RG

wherein CG is Charged Group LGl is Linker Group 1

BG is Bridge Group

LG2 is Linker Group 2

RG is Reactive Group and CG, LGl, BG, LG2, and RG are covalently connected.

By "a Reagent Molecule" is meant a molecule having the ability to covalently react to a specific site in a biomolecule, such as a protein, thereby, if labelled, being used to detect the bound protein in an analysis.

By "specific site" in a protein is meant a chemical group, which can react covalently with a Reagent Molecule. In a preferred embodiment, the specific site can be an amino, a hy- droxyl, a thiol, a vinyl or an allyl group.

By "labelled" is meant that a Reagent Molecule can fully or partly contain a label/labels, which is possible to detect, by subsequent analysis, such as mass spectrometry. In a preferred embodiment, the label is selected from the group that consists of 1H/²D,

¹²C/¹³C/¹⁴C, ¹⁴N/¹⁵N, ¹⁶O/¹⁷O/¹⁸0, ¹⁰BI¹¹B, ³⁵C1/³⁷C1, ⁷⁴Se/⁷⁶Se/⁷⁷Se/⁷⁸Se/⁸⁰Se/⁸²Se, ³²S/³³S/³⁴S/³⁶S, ⁷W¹Br, ²⁸Si/²⁹Si/³⁰Si.

By "Charged Group" is meant a part in the Reagent Molecule, which is positively charged under desired conditions. In a preferred embodiment, the Charged Group is se- lected from the group that consists of positively charged aromatic amines, positively charged alkyl amines, positively charged tertiary amines, positively charged quaternary amines, heterocycle containing one or several positively charged amines, and phosphorous based compounds. In another embodiment, the Charged Group is selected from the group that consists of positively charged solid supports and polymeric structures. In another embodiment, the Charged Group contains one or several labelled groups.

By "Linker Group 1" is meant a part in the Reagent Molecule which is covalently bonded to the Charged Group and to the Bridge Group. A general schematic structure of a Linker Group 1 is shown below:

-(CRlR2)_n-(XR3R4)_m-

Wherein Rl -R4 are selected from the group that consists of H, halogens, such as F, Cl, Br, I, alkyl groups, such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, aryl group, such as phenyl, diphenyl, naphthyl. n = 0 to 20 m = 0 to 20 X is selected from the elements or atoms that consist of C, O.

All the groups are covalently bonded by single bonds, double bonds or triple bonds. In a preferred embodiment, the Linker Group 1 contains one or several labelled groups.

By "Linker Group 2" is meant a part in the Reagent Molecule which is covalently bonded to the Bridge Group and to the Reactive Group. A general schematic structure of a Linker Group 2 is shown below:

-(CR5R6)_n-(XR7R8)_m- wherein R5-R8 are selected from the group that consists of H, halogens, such as F, Cl, Br, I, alkyl groups such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, aryl group, such as phenyl, diphenyl, naphthyl. n = 0 to 20 m = 0 to 20

X is selected from the elements or atoms that consist of C, O.

All the groups are covalently bonded by single bonds, double bonds or triple bonds.

In a preferred embodiment, the Linker Group 2 contains one or several labelled groups.

By "Bridge Group" is meant a part in the Reagent Molecule, which, after cleavage, allows detection of a unique labelled mass marker in a subsequent analysis, such as mass spectrometry. A general schematic structure of a Bridge Group is shown below:

-(Bridge l)_p-(Bridge 2)_q-

wherein Bridge 1 is selected from the group that consists of ¹²C/¹³C/¹⁴C, ¹⁶O/¹⁷O/¹⁸O, ¹⁴N/¹⁵N, ³²S/³³S/³⁴S/³⁶S, C-O, C=C, SO, SO₂. Bridge 2 is selected from the group that consists of ¹²C/¹³C/¹⁴C, ¹⁶O/¹⁷O/¹⁸0, ¹⁴N/¹⁵N, ³²S/³³S/³⁴S/³⁶S, C=O, C=C, SO, SO₂. ρ= 0 or 1 q= 0 or 1

Bridge 1 and Bridge 2 are covalently connected.

By "Reactive Group" is meant a part of the Reagent Molecule having the ability to react to a specific site on a desired protein as defined earlier, forming a covalent bond. In a preferred embodiment, the Reactive Group is selected from the group that consists of ep- oxy-activated groups, allyl-activated groups, cyanobromide-activated groups, thiol groups, activated esters, carboxylic acids and acid chlorides. Some examples of activated esters are N-hydroxysuccinimide-, />-nitrophenyl-, pentachlorophenyl-, and pentafluoro- phenyl esters.

The coupling of the Reagent Molecule to the specific site can be performed in different ways. In a preferred embodiment, the Reagent Molecule is coupled directly to the specific site. In another embodiment, the coupling of the Reagent Molecule to the specific site is performed in two or several steps. As an example, the part Linker Group 2- Reactive Group is first reacted to the specific site and in a second step the part Charged Group-Linker Group 1 is reacted to form the Reagent Molecule coupled to the specific site (see experimental).

Presently preferred Reagent Molecules according to invention are:

(1 -Methyl-1 H-imidazol-2-ylsulfanyl) (5-Amino-2H-[1 ,2,4]triazol-3-ylsulf -acetic acid 2,5-dioxo-pyrrolidin-1 anyl)-acetic acid 2,5-dioxo-pyrroli -yl ester din-1-yl ester

3-(2,5-Dioxo-pyrrolidin-1-yloxycarb [2-(2 , 5-Dioxo-py rrol id i n- 1 -yloxycar onylmethylsulfanyl)-2-guanidino-pro bonylmethylsulfanyl)-ethyl]-trimeth pionic acid yl-ammonium

(2-Diethylamino-ethylsulfanyl)-acet (2-Dimethylamino-ethylsulfanyl)-ace ic acid 2,5-dioxo-pyrrolidin-1-yl e tic acid 2,5-dioxo-pyrrolidin-1-yl ster ester

(2-Pyrazin-2-yl-ethylsulfanyl)-acet (2-Pyridin-4-yl-ethylsulfanyl)-acet ic acid 2,5-dioxo-pyrrolidin-1-yl e ic acid 2,5-dioxopyrrolidin-1-yl e ster ster

In a second aspect, the invention relates to a kit comprising one or more of the above reagent molecules. Preferably, the kit comprises two or more reagent molecules of the same kind but labelled with different labels, wherein the one reagent molecule is labelled with a specific isotope and the other reagent molecule with another isotope of the same atom, and the third reagent molecule, if present, with yet a further isotope etc. Preferably, the label is selected from the group comprising 1H/²D, ¹²C/¹³C/¹⁴C, ¹⁴N/¹⁵N, ¹⁶O/¹⁷O/¹⁸O,

¹⁰BZ¹¹B, ³⁵C1/³⁷C1, ⁷⁴Se/⁷⁶Se/⁷⁷Se/⁷⁸Se/⁸⁰Se/⁸²Se, ³²S/³³S/³⁴S/³⁶S, ⁷⁹Br/⁸¹Br, ²⁸Si/²⁹Si/³⁰Si.

In a third aspect, the invention relates to use of a reagent molecule or kit above to label biomolecules for analysis or identification of biomolecules, such as protein/peptides, and/or their reactions with other compounds in vivo or in vitro. In one embodiment the use is the analysis of protein expression profiles, especially comparison of normal versus different disease states. Thus, the reagents of the invention are also intended for diagnosis of different disease states.

Other objects and advantages of the present invention will appear from the detailed description that follows.

Detailed description of the invention

Advantageously, the present invention utilises reagent molecules that can be produced in two or more forms which confer the ability to distinguish the different forms of reagents and the peptides to which they are linked by mass, but which importantly do not affect the ionisation efficiency of the peptides to which they are linked when subject to mass spectrometry. In one embodiment the sample is treated with a reagent molecule available in different forms that can be distinguished on the basis of mass.

The present invention can be used in a wide variety of applications, such as for example to identify peptides presented by a major histocompatibility complex (MHC) molecule.

Another application where the present method is useful is for the analysis of peptides being carried around or in solution in body fluids such as cerebrospinal and synovial fluids as well as in urine and blood serum. Accordingly, the reagents according to the present invention can be used e.g. in diagnosis of diseases. As discussed above, the use of two or more labelling reagents with different labels allows a determination of relative amounts of proteins in two or more different samples. In particular, the labelling techniques of the present invention may be used to compare protein expression in two different cells. The two different cells may for example be cells of the same type but under different conditions (or states), or they may be cells of a different type (under the same or different conditions). By way of example, a first cell may be treated with an agonist and a second cell untreated, and the expression of one or more proteins in each cell compared. Thus, the present invention may also be used within drug discovery to find new potential drugs affecting biomolecules.

The two conditions could also be cells resting versus cells induced or treated in some manner. Often, differential expression in cells under different conditions can provide useful information on the activity in the cells.

Thus, in an advantageous embodiment of the present invention, the protein-fragment mixture is analysed at a first frequency, thereby generating a first set of cells, and then at a second frequency, thereby generating a second set of cells, followed by an inversion of the intensity values of the second frequency and adding them to the first, whereby a difference spectrum is generated. The second frequency is usually higher man the first, and the analysis is a scanning, such as a parent ion-scanning or a neutral loss scanning.

The invention will now be described with reference to the following examples, which only are intended to exemplify the invention, and not to limit the scope of the invention as defined by the appended claims. All references given below and elsewhere in the pre- sent specification are hereby included herein by reference.

EXPERIMENTAL

Example 1: Reaction of the Reagent Molecule and the specific site in two steps The specific site of the protein (amine group) was modified in two steps to bind the Reagent Molecule, after protection of the Lys side chains and digestion of the protein.

Iodoacetic anhydride was reacted with the free amine group on the peptide to give an iodo functional peptide. This iodo functional peptide was in turn reacted with several different thiol-containing reagents to give the Reagent Molecule tagged peptide. The thiol containing reagents are shown in Figure 1.

Figure 1: Thiol containing reagents coupled to iodo functional peptide.

All thiol-containing reagents were commercially obtained except for compound 7.

Succinylation

Succinic anhydride (9mg/ mL) was dissolved in potassium phosphate buffer (200 mM, pH 8.5) containing urea (2M) and pH was rapidly adjusted to 8.5 with NaOH. To this mixture, a solution of pure protein (1-5 mg/ mL) in ddH₂O was added (mol_protei_n/ mol_sl UC- _ci_ni_{c anhyd}ri_de '• 1/ 1000-2000). After 1 hour reaction time, another equimolar fresh batch of succinic anhydride in phosphate buffer was added, and reaction left to proceed for 1 more hour. The reaction was performed at room temperature and the pH continuously checked and adjusted to 8.5.

Digestion

To remove interfering urea, a buffer exchange to potassium phosphate (200 mM, pH 8.6) was performed. The protease GIu-C was added to the solution (protein/ protease : 1/ 20 (w/ w)) and digestion left to proceed at 37 ⁰C, usually overnight.

Preparation of iodofunctional peptide

Iodoacetic anhydride was dissolved in THF (200 mM) and added to the peptide solution 2 times during 1 hour, with 30 min intervals (molp_rotei_n/ mol_iodoacetic a_nhydride : 1/ 200). The reaction has been successfully performed on both ice and at room temperature.

Preparation of reagent molecule tagged peptide

Thiol containing reagent (18 mM in buffer with pH 6-8) is added to the peptide solution (mol_protei_n/ mol_SH-_re_age_nt : 1/ 100) and reaction allowed to proceed for 1-2 hours at 37 ⁰C.

Synthesis of compound 7: Acetate (2-mercapto-ethyl) trimethylammonium

NaOH (0.2 M) (10.6 ml) was added to s-acetylthiocholine iodide (0.604 g, 2.09 mmol) at room temperature under an argon atmosphere. The pH was controlled and found to be pH = 11. After 1.25 h, additional NaOH (0.2 M) (5.3 ml) was added. The pH was controlled and found to be pH = 11. The solution was allowed to proceed overnight. The reaction was stopped and HCl (25 % in water) was added until the pH reached 7. The solution was freeze-dried and submitted to vacuum to give an off-white powder. Methanol (7 ml) was added to the residue and the solution was filtered. The filtrate was concentrated and dried under vacuum to give acetate (2-mercapto-ethyl) trimethylammonium as an off- white solid (531 mg)

¹H NMR (CDCl₃): δ= 1.9 (m, 3H, CH₃COO), 2.9-3.0 (m, 2Η, HS-CH₂-), 3.1 (s, 9Η, (CHs)₃N-) 3.5-3.6 (m, 2Η, (CH₃)₃N-CH₂-) ¹³C NMR (CDCl₃): δ= 18, 24, 54, 70, 180

Example 2: Preparation of Linker Group 2-Reactive Group type compounds These compounds were used in the reaction between Linker Group 2-Reactive Group and the specific site of the protein (amine group) to obtain an iodo functional protein.

Synthesis of N-hvdroxysuccinimide-4-iodobutyric ester

Figure 2: Chemical structure of N-hydroxysuccinimide-4-iodobutyric ester

4-Iodobutyric acid (1 g, 4.67 rnmol) and N-hydroxysuccinimide (0.54 g, 4.69 rnmol) were dissolved in THF (10 ml) in a round-bottom flask (50 ml) equipped with a magnetic stirrer. 1,3-Dicyclohexylcarbodiimide (DCC) (0.96 g, 4.65 mmol) was added to the round-bottom flask and the reaction was allowed to proceed at room temperature for 16h. The DCC urea formed during the reaction was then filtered off on a glass filter (P3) and the solvent evaporated on a rotatory evaporator. The crude product was dissolved in ethyl acetate and the extra precipitate of DCC urea was filtered off on a glass filter (P3). The solvent was then evaporated on a rotatory evaporator. The crude product was then recrys- tallized from a mixture of hexane/ethyl acetate. The product precipitated as a white powder, which was filtered off on a glass filter (P3) and dried under vacuum. 0.90 g of N- hydroxysuccinimide-4-iodobutyric ester was obtained as white crystals. Yield: 62%.

¹H NMR (CDCl₃): S= 2.28 (m, 2H₅ 1-CH₂-CH₂-CH₂-COO-), 2.78 (t, 2H, 1-CH₂-CH₂- CH₂-COO-), 2.85 (s, 4Η, -CO-CH₂-CH₂-CO-), 3.29 (t, 2Η, 1-CH₂-CH₂-CH₂-COO-)

¹³C NMR (CDCl₃): <J= 3.83, 25.58, 28.14, 31.81, 167.56, 168.98

Synthesis of N-hvdroxysuccinimide-3-iodoτ>ropionic ester

Figure 3: Chemical structure of N-hydroxysuccinimide-S-iodopropionic ester

3-Iodopropionic acid (1 g, 5 mmol) and N-hydroxysuccinimide (0.57 g, 5 mmol) were dissolved in THF (10 ml) in a round-bottom flask (25 ml) equipped with a magnetic stirrer. 1,3-Dicyclohexylcarbodiimide (DCC) (1.03 g, 5 mmol) was added to the round- bottom flask and the reaction was allowed to proceed at room temperature for 16h. The DCC urea formed during the reaction was then filtered off on a glass filter (P3) and the solvent evaporated on a rotatory evaporator. The crude product was dissolved in ethyl acetate and the extra precipitate of DCC urea was filtered off on a glass filter (P3). The solvent was then evaporated on a rotatory evaporator. The crude product was then recrys- tallized from a mixture of hexane/ethyl acetate. The product precipitated as a white powder, which was filtered off on a glass filter (P3) and dried under vacuum. 1.00 g of N- hydroxysuccinimide-3-iodopropionic ester was obtained as white crystals. Yield: 67%

¹H NMR (CDCl₃): «5= 2.85 (s, 4H₅ -CO-CH₂-CH₂-CO-), 3.28 (m, 2Η, 1-CH₂-CH₂-COO- ), 3.38 (m, 2Η, 1-CH₂-CH₂-COO-)

¹³C NMR (CDCl₃): (5= 25.57, 35.51, 166.58, 168.73

Example 3: Fragmentation results of the thiol containing reagents coupled to iodo functional peptide.

The tagged peptide was prepared by coupling of the Reagent Molecule synthesized ac- cording to Example 1 to the specific site of the peptide (amine group)

The fragmentation nomenclature is shown in Figure 4.

Figure 4: Fragmentation nomenclature

Some of the fragmentation results obtained with tagged peptide are shown in Table 1.

Table 1 : Fragmentation results obtained with some of the Reagent Molecules showed in Example 1

Reagent names:

(2) 2-Mercapto- 1 -methylimidazole

(3) 3 -Amino- 5 -Mercapto- 1 ,2 ,4-triazole

(5) Guanyl-cys-OH

O) Acetate(2-mercapto-ethyl)trimethylammonium

(8) 2-Diethylaminoethanethiol hydrochloride

(9) Dimethylaminoethanethiol hydrochloride

(12) Pyrazine ethanethiol

(14) 4-Pyridyl-ethylmercaptan

The above reagents have been transformed to NHS-activated reagents as claimed. The last two, namely (2-pyrazin-2-yl-ethylsulfanyl)-acetic acid 2,5 dioxo-pyrrolidin-1-yl- ester and (2-pyridin~4-yl-ethylsulfanyl)-acetic acid 2,5 dioxo-pyrrolidin-1-yl-ester are the most preferred.

Claims

1. A reagent molecule having the following formula:

CG-LG1-BG-LG2-RG

wherein CG is Charged Group LGl is Linker Group 1 BG is Bridge Group

LG2 is Linker Group 2

RG is Reactive Group and CG₅ LGl, BG, LG2, and RG are covalently connected.

2. A reagent molecule according to claim 1 which contains at least one label, which is possible to detect by subsequent analysis.

3. A reagent molecule according to claim 2, wherein the label is distinguished on the ba- sis of mass, and wherein the isotopic label is at least one atom selected from the group comprising 1H/²D, ¹²C/¹³C/¹⁴C, ¹⁴N/¹⁵N, ¹⁶O/¹⁷O/¹⁸0, ¹⁰BZ¹¹B₅ ³⁵C1/³⁷C1, ⁷⁴Se/⁷⁶Se/⁷⁷Se/⁷⁸Se/⁸⁰Se/⁸²Se, ³²S/³³S/³⁴S/³⁶S, ⁷W¹Br, ²⁸Si/²⁹Si/³⁰Si.

4. A reagent molecule according to claim 1, 2 or 3, wherein the Charged Group is selected from the group that consists of positively charged aromatic amines, positively charged alkyl amines, positively charged tertiary amines, positively charged quaternary amines, heterocycle containing one or several positively charged amines, phosphorous based compounds, positively charged solid supports and polymeric structures, and wherein the Charged Group optionally contains one or several labelled groups.

5. A reagent molecule according to one or more of the above claims, wherein the Linker Group 1 has the following formula:

-(CRlR2)_n-(XR3R4)_m- wherein R1-R4 are selected from the group that consists of H, halogens, such as F,

Cl, Br, I, alkyl groups, such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, aryl group, such as phenyl, diphenyl, benzyl, naphtyl. n = 0 to 20 m = 0 to 20

X is selected from the group that consists of C, O; and wherein all the groups are covalently bonded by single bonds, double bonds or triple bonds and Linker Group 1 optionally contains one or several labelled groups.

6. A reagent molecule according to one or more of the above claims, wherein the Linker

Group 2 has the following formula:

-(CR5R6)_n-(XR7R8)_m-

wherein R5-R8 are selected from the group that consists of H, halogens, such as F,

Cl, Br, I, alkyl groups such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, aryl group, such as phenyl, diphenyl, benzyl, naphtyl n = 0 to 20 m = 0 to 20

X is selected from the group that consists of C, O; and wherein all the groups are covalently bonded by single bonds, double bonds or triple bonds and Linker Group 2 optionally contains one or several labelled groups.

7. A reagent molecule according to one or more of the above claims, wherein the Bridge

Group has the following formula:

-(Bridge l)_p-(Bridge 2)_q-

wherein Bridge 1 is selected from the group that consists of ¹²C/¹³C/¹⁴C, ¹⁶O/¹⁷O/¹⁸O,

¹⁴N/¹⁵N, ³²S/³³S/³⁴S/³⁶S, C-O, C=C, SO, SO₂. Bridge 2 is selected from the group that consists of ¹²C/¹³C/¹⁴C, ¹⁶O/¹⁷O/¹⁸0, ¹⁴N/¹⁵N, ³²S/³³S/³⁴S/³⁶S, C=O, C=C₅ SO₅ SO₂ p= O or 1 q= O or 1 and wherein Bridge 1 and Bridge 2 are covalently connected.

8. A reagent molecule according to one or more of the above claims, wherein the Reactive Group is selected from the group that consists of epoxy-activated groups, allyl- activated groups, cyanobromide-activated groups, thiol groups, activated esters, carbox- ylic acids and acid chlorides.

9. A reagent molecule according to one or more of the above claims, which selected from

3-(2,5-Dioxo-pyrrolidin-1-yloxycarb [2-(2,5-Dioxo-pyrrolidin-1-yloxycar onylmethylsulfanyl)-2-guanidino-pro bonylmethylsulfanyl)-ethyl]-trimeth pionic acid yl-ammonium

(2-Pyrazin-2-yl-ethylsulfanyl)-acet (2-Pyridin-4-yl-ethylsulfanyl)-acet ic acid 2,5-dioxc-pyrrolidin-i-yl e ic acid 2,5-dioxo-pyrrc-lidin-i-yl e ster ster

10. A kit comprising one or more of the reagent molecules according to claim 9.

11. A kit according to claim 10, wherein the kit comprises two reagent molecules of the same kind but labelled with different labels, wherein the first reagent molecule is labelled with one specific isotopic label and the second reagent molecule with a another isotopic label of the same atom.

12. A kit according to claim 11, wherein the label is selected from the group comprising

1H/²D, ¹²C/¹³C/¹⁴C, ¹⁴N/¹⁵N, ¹⁶O/¹⁷O/¹⁸0, ¹⁰BZ¹¹B₅ ³⁵C1/³⁷C1, ⁷⁴Se/⁷⁶Se/⁷⁷Se/⁷⁸Se/⁸⁰Se/⁸²Se, ³²S/³³S/³⁴S/³⁶S, ⁷⁹BrZ⁵¹Br, ²⁸Si/²⁹Si/³⁰Si.

13. Use of a reagent molecule according to one or more of claims 1 -9 or a kit according to one or more of claims 10-12 to label biomolecules for analysis of biomolecules and/or their reactions with other compounds in vivo and in vitro.

14. Use according to claim 13, for analysis of protein expression profiles.

15. Use according to claim 13 or 14, for diagnosis of different disease states.

16. Use according to claim 13 for drug discovery.

17. Use according to any of claims 13-16 wherein the analysis is by mass spectrometry.