Field of the Invention

The present invention relates to novel mass spectrometry (MS) reagents.

Background of the Invention

The field of research known as proteomics involves the systematic identification of all proteins expressed by a particular cell or tissue type at a given time, quantitative analysis of the differences in protein expression observed between two different cell states, (e.g. normal versus diseased), and identification and characterization of the protein modifications, protein complexes and specific protein-protein interactions involved in the control of cellular function.

Developments in biological mass spectrometry (MS) methods over the past decade have been the major factors enabling proteomics research. In particular, the speed, specificity, and sensitivity of tandem mass spectrometry (MS/MS) methods for peptide sequence analysis make it especially attractive for use in strategies requiring rapid protein identification, characterization and quantitation. However, the task of characterizing the proteome, which in contrast to the static genome is in a state of constant spatial and temporal flux, presents a significant analytical challenge to mass spectrometry approaches, due to the enormous dynamic range, mixture complexity and diversity of transcriptional, translation and post translational protein modifications associated with protein expression throughout the cell cycle.

hi a typical approach to protein identification, individual proteins or simple protein mixtures resolved by electrophoretic or chromatographic methods, or large numbers of proteins present in complex unresolved mixtures are subjected to proteolysis. Then, the resultant peptide mixtures are introduced to the mass spectrometer by on-line chromatography, and following determination of their masses, individual peptides are selected for dissociation by MS/MS. Identification of these selected peptides is then performed by either database analysis of the uninterpreted product ion spectrum, through database searching of a partially derived amino acid "sequence tag", or by "de-novo" sequence analysis. One of the limitations of this general analysis strategy is that typically, peptides observed at high relative abundance are preferentially selected for analysis, such that information regarding the identity of proteins represented as low abundance peptides is commonly not obtained. Although simplification of the peptide mixture prior to mass spectrometric analysis, either by off- or on-line multidimensional chromatography or by affinity purification of selected peptides, as well as the use of dynamic exclusion methods during MS/MS acquisition have been employed to allow greater numbers of distinct peptide ions to be selected throughout the course of an LC/MS/MS experiment, the identification of low abundance proteins in these complex mixtures still remains a significant challenge.

A fundamental aspect of proteomics research is the determination of protein expression levels between two different states of a biological system (i.e., relative quantification of protein levels), such as that encountered between a normal and diseased cell or tissue. In recent years, several approaches for the quantitation of differential protein expression levels between two different cell/tissue states, based on the use of stable isotope labelling and MS analysis, have been developed.

One approach involves the in vivo metabolic incorporation of isotopically depleted (13C-, 15N-, and 2H-depleted) or enriched amino acids (either by uniform labelling with 15N, or by incorporation of selected amino acids containing heavy isotopes (eg., 13C, 15N, 2H)) into a cellular protein population. After isolation of proteins from the cellular matrix, the sample is combined with one incorporating natural isotopes, then the combined protein mixture is subjected to proteolysis and the masses of the peptides determined by MS as described above. By comparing the relative abundances of peptides from the isotopically enriched sample with those from a sample prepared using naturally abundant isotopes, quantitation of any changes in the level of protein expression between the two samples may be obtained. This in vivo labelling approach is limited, however, to those systems where cells can be cultured under conditions suitable for incorporation of the isotopic label.

A second more general strategy involves in vitro chemical derivatization with isotopically enriched labels following isolation of the proteins from the cellular matrix. One method of this type that has received much attention to date is the isotope coded affinity tag (ICAT) technique developed by Aebersold and co-workers and described in WO 00/11208. In that approach, cysteine containing proteins from two different cell/tissue states are reduced and S-alkylated with either naturally abundant (light) or isotopically enriched (heavy) ICAT reagents, respectively, each containing a biotin moiety for subsequent affinity selection of cysteine-containing peptides following proteolysis by streptavidin affinity purification, leading to simplification of the mixture prior to MS analysis.

Regardless of the approach, it is important to recognise that all of the techniques described above for quantitation of differentially labelled peptides have been based on mass analysis of intact peptide precursor ions. Thus, limitations of the approaches are encountered when; (i) ions of interest are present at low levels (i.e., approaching or below the limit of detection of the mass spectrometer), (ii) the masses of low abundance differentially labeled peptides overlap with other higher abundance components present in the mixture, (iii) separation of the differential labelled "heavy" and "light" peptides occurs during chromatographic fractionation of the peptide mixture, or (iv) the mass spectrometer lacks sufficient resolution to adequately resolve the two labelled components, thereby precluding their detection.

Thus, there is still a need for improved MS reagents to enable (i) selective identification and (ii) differential quantification of proteins/peptides.

Summary of the Invention

The present inventors have developed novel reagents for the selective identification and differential quantitation of peptides containing selected amino acids, using tandem mass spectrometry, having an increase in selectively and sensitivity of several orders of magnitude over existing reagents.

The present invention provides reagents for introducing a fixed charge to a protein/peptide. The fixed-charge may be contained on the side-chain of a selected amino acid, or the side-chain of a selected amino acid residue contained within a protein or peptide. Preferably the selected amino acid residue is methionine. Preferably, the fixed-charge includes a sulfonium ion. - A -

The present invention is based on the fixed charge derivative contained within a peptide ion directing the MS/MS dissociation of peptides containing the fixed charge toward a single predictable fragmentation channel with the formation of a single product ion characteristic of fragmentation occurring at the fixed-charge site, thereby allowing its selective identification from a complex mixture without need for prior resolution or otherwise enrichment of the peptide from a complex mixture prior to its analysis.

The present reagents are suitable to use in methods described in co-pending PCT/US03/36739 which is incorporated herein by reference.

Analysis of derivatized proteins/peptides may be performed by tandem mass spectrometry. The tandem mass spectrometer may be equipped with electrospray ionization (ESI) or matrix assisted laser desorption ionization (MALDI) interfaces to transfer the protein or peptide ion from solution into the gas-phase.

The reagents of the present invention may be used for the selective identification of peptides.

The reagents of the present invention may be used for the differential quantitation of peptide concentrations between two different samples, by incorporation of stable isotope labels to the reagent, to yield a 'light' form (containing only natural isotopes), and a 'heavy' form (containing isotopic or structural labels incorporated into the substituent).

The present inventors have discovered that a mass difference of at least 8 Daltons between 'light' and 'heavy' reagents is desired to enable sensitive and accurate differential quantitative analysis.

The general form of the reagents of the present invention allows a mass difference of at least 8 Daltons and up to 13 Daltons between 'light' and 'heavy' reagents.

The present invention relates to use of substituted acetophenone reagents in MS analysis, especially comparative MS analysis. Preferably, the MS analysis is MS/MS analysis. In a first aspect, the invention relates to a substituted acetophenone, or a salt thereof, or a solvate thereof, having the following formula:

wherein X is Cl, Br, I, sulfonic esters, perchlorate esters and chlorosulfonates, A is H or 2H (D); R1 - R8 are 13C; and R9 is H, 2R (D), 13CH3, 13CH213CH3, 13CH213CH213CH3, 13CH213CO2H, 13CH213CH213CO2H, O13CH3, O13CH213CH3, O13CH213CO2H, O13CH213CH213CO2H, SO213CH3, SO213CH213CH3, SO213CH213CH213CH3, SO213CH213CO2H, SO213CH213CH213CO2H, or SO3H.

The invention also relates to novel acetophenones having the formula above, and wherein X is Cl, Br, I, sulfonic esters, perchlorate esters and chlorosulfonates, A is H; R1 - R8 are C; and R9 is SO2CH2CO2H or SO2CH2CH2CO2H.

A preferred substituted acetophenone has the following formula

13C8 - bromoacetophenone

When used as a MS reagent, this substituted acetophenone gives a mass difference of 8 Daltons compared to the normal isotopic variant.

Another preferred substituted acetophenone is one according to the above formula, wherein the hydrogens on R1 are substituted for 2H (D).

D2,13C8 - bromoacetophenone

When used as a MS reagent, this substituted acetophenone gives a mass difference of 10 Daltons compared to the normal isotopic variant.

A further preferred substituted acetophenone has the following formula O H

13Cg - /'-methyl-bromoacetophenone

When used as a MS reagent, this substituted acetophenone gives a mass difference of 9 Daltons compared to the normal isotopic variant. Yet another further preferred substituted acetophenone is one according to the above formula, wherein the hydrogens on R1 are substituted for 2H (D).

O H

D2j13C9 - /?-methyl-bromoacetophenone

This substituted acetophenone gives a mass difference of 11 Daltons compared to the normal isotopic variant.

A further preferred substituted acetophenone reagent has the following formula O H

This substituted acetophenone reagent gives a mass difference of 10 Daltons compared to the normal isotopic variant.

Yet another further preferred substituted acetophenone is one according to the above formula, wherein the hydrogens on R1 are substituted for H (D).

D2, γ 3 C10 - j9-ethyl-bromoacetophenone

This substituted acetophenone reagent gives a mass difference of 12 Daltons compared to the normal isotopic variant.

A further preferred substituted acetophenone reagent has the following formula

13C1Q - jo-propyl-bromoacetophenone

This substituted acetophenone reagent gives a mass difference of 11 Daltons compared to the normal isotopic variant.

Yet another further preferred substituted acetophenone is one according to the above formula, wherein the hydrogens on R1 are substituted for 2H (D).

D2,13C12 -j>propyl-bromoacetophenone

This substituted acetophenone reagent gives a mass difference of 13 Daltons compared to the normal isotopic variant.

In a second aspect, the invention provides MS reagents comprising substituted acetophenones as described above. Thus, the invention relates to use of these acetophenones as reagents, particularly MS reagents. Preferred MS-reagents are 13C8- bromoacetophenone, 1 T 1 *5 D2, C8 - bromoacetophenone and C9 -j?-methyl-bromoacetophenone. The reagents of the invention may be used for selective identification of proteins/peptides. In one embodiment of the invention, the acetophenones are used for selective identification of proteins/peptides, preferably the corresponding acetophenones with natural isotopes are used for selective identification of proteins/peptides. In another embodiment of the invention, the above acetophenones with stable isotopes are used in combination with the corresponding acetophenones with natural isotopes for quantitative determination of proteins/peptides.

Thus, in a third aspect, the invention provides a kit for quantitative determination of proteins/peptides in a sample, comprising the substituted acetophenone(s) with R1-R8 = 13C, and A=H or D and R9 = H, 13CH3, 13CH213CH3 or 13CH213CH213CH3 as heavy stable isotope labelled reagent(s) and the corresponding substituted acetophenone(s) with R1-R8 = C, and A=H and R9 = H, CH3, CH2CH3 or CH2CH2CH3 as light reagent(s). Alternatively, any of the substituted acetophenones mentioned above with heavy stable isotopes can be used as heavy reagents and the corresponding acetophenones with natural isotopes as light reagents.

A preferred kit comprises the heavy stable isotope labelled reagent 13C8 bromoacetophenone and the light reagent C8 bromoacetophenone. Another preferred kit comprises the heavy stable isotope labelled reagent D2513C8 bromoacetophenone and the light reagent C8 bromoacetophenone.

A further preferred kit comprises the heavy reagent 13C9 - jt?-methyl-bromoacetophenone and the light reagent C9-/?-methyl-bromoacetophenone.

Yet another further preferred kit comprises the heavy reagent D2,1 C9 - /»-methyl- bromoacetophenone and the light reagent C9-p-methyl-bromoacetophenone.

Yet another further preferred kit comprises the heavy reagent 13C10 - p-ethyl- bromoacetophenone and the light reagent C10-/'-ethyl-bromoacetophenone.

Yet another further preferred kit comprises the heavy reagent D2513C10 - p-ethyl- bromoacetophenone and the light reagent Cio-^-ethyl-bromoacetophenone.

Yet another further preferred kit comprises the heavy reagent 13C11 - jσ-propyl- bromoacetophenone and the light reagent Cπ-^-propyl-bromoacetophenone. Yet another further preferred kit comprises the heavy reagent D2513C11 - p-propyl- bromoacetophenone and the light reagent Cπ-p-propyl-bromoacetophenone.

The kit may further comprise one or more containers containing: cysteine disulfide reducing agents, cysteine alkylating reagents, proteases or chemical cleavage agents, and solvents.

The cysteine d isulfide reducing may be: dithiothreitol (DTT), β-mercaptoethanol, tris- carboxyethyl phosphine (TCEP), and/or tributylphosphine (TBP).

The cysteine alkylating reagents may be alkylhalides (e.g. iodoacetic acid, iodoacetamide), vinylpyridine, acrylamide or disulphide-comprising compounds, such as bis-(2-hydroxyethyl) disulphide; bis-(2-hydroxypropyl) disulphide; 3-3-dipropionamidedisulphide; 2-2'- dipyridyldisulphide. Preferably, reagent is DeStreak™.

The proteases or chemical cleavage agents may be trypsin, Endoproteinase Lys-C, Endoproteinase Asp-N, Endoproteinase GIu-C, pepsin, papain, thermolysin, cyanogen bromide, hydroxylamine hydrochloride, 2-[2'-nitrophenylsulfenyl]-3-methyl-3'-bromoindole (BNPS- skatole), iodosobenzoic acid, pentafluoropropionic acid and/or dilute hydrochloric acid.

The solvents may be urea, guanidine hydrochloride, acetonitrile, methanol and/or water.

Detailed description of the invention

Synthesis of1 Cg-bromoacetophenone

1 "X C8 - bromoacetophenone A non-limiting method for the synthesis of one this derivative may be performed using methods known to those skilled in the art via Friedel-Crafts acylation of 13C6 labelled benzene with 13C2 labelled bromoacetyl bromide, prepared from 13C2 labelled bromoacetic acid, to yield 13C8 labelled bromoacetophenone.

For synthesis of the above compound wherein Al and A2 are D in the general formula, the following non-limiting method of synthesis may be used. Synthesis of this derivative may be performed using methods known to those skilled in the art, via Friedel-Crafts acylation of 13C6 labelled benzene with 13C2, D2 labelled acetylbromide, prepared from 13C2, D4 acetic acid or 13C2, D3 acetic acid sodium salt, to yield D2513C8 labelled acetophenone, followed by bromination [Frechet, J.M.J., Farrall, MJ. and Nuyens, LJ. J. Macromol. Sci.-Chem. 1977, All, 507-514.] to yield D2513C8 labelled bromoacetophenone.

Synthesis of 13Cg- p-methyl-bromoacetophenone

13C9 - p-methyl-bromoacetophenone

A non-limiting method for the synthesis of this derivative may be performed using methods known to those skilled in the art via Friedel-Crafts acylation of 13C7 labelled toluene with 13C2 labelled bromoacetyl bromide, prepared from 13C2 labelled bromoacetic acid, to yield 13C9 labelled p-methyl-bromoacetophenone.

For synthesis of the above compound wherein Al and A2 are D in the general formula, the following non-limiting method of synthesis may be used. Synthesis of this derivative may be performed using methods known to those skilled in the art, via Friedel-Crafts acylation of 13C7 labelled toluene with 13C2, D2 labelled acetylbromide, prepared from 13C2, D4 acetic acid or 13C2, D3 acetic acid sodium salt, to yield D2513C9 labelled /7-methyl-acetophenone, followed by bromination to yield D2513C9 labelled p-methyl-bromoacetophenone.

Synthesis Of13Qo or 13Cu labelled reagents according to the invention, where X is Br, Rj - Rs are 13C and R9 is 13CH213CH3 or 13CH213CH213CH3.

A non-limiting method for the synthesis of this derivative may be performed using methods known to those skilled in the art via Friedel-Crafts acylation of 13C6 labelled benzene with 13C2 labelled acetylchloride or 13C3 labelled propanoyl chloride (formed by reaction of 13C3 propionic acid with thionyl chloride) to yield 13C8 labelled acetophenone or 13C9 labelled propiophenone, followed by reduction [Ishimoto, K., Mitoma, Y., Nagashima, S., Tashiro, H., Prakash, G.K.S., Olah, G.A. and Tashiro, M. Chem. Canon. 2003, 4, 514-515.] to yield 13C8 . labelled ethylbenzene or 13C9 labelled propylbenzene, then Friedel-Crafts acylation with 13C2 labelled bromoacetyl bromide, prepared from 13C2 labelled bromoacetic acid, to yield 13C10 labelled />-ethyl-bromoacetophenone or C11 /j-propyl-bromoacetophenone. For synthesis of the above compounds wherein Al and A2 are D in the general formula, the following non-limiting method of synthesis may be used. Synthesis of this derivative may be performed using methods known to those skilled in the art, via Friedel-Crafts acylation of 13C6 labelled benzene with 13C2 labelled acetylchloride or 13C3 labelled propanoyl chloride (formed by reaction of 13C3 propionic acid with thionyl (or oxalyl, phtaloyl) chloride) to yield 13C8 labelled acetophenone or 3C9 labelled propiophenone, followed by reduction to yield 13C8 labelled ethylbenzene or 13C9 labelled propylbenzene, then Friedel-Crafts acylation with 13C2, D2 labelled acetylbromide, prepared from 13C2, D4 acetic acid or 13C2, D3 acetic acid sodium salt, to yield D2513C10 labelled p-ethyl-acetophenone or D2513C11 labelled /j-propyl-acetophenone, followed by bromination to yield D2513C10 labelled /?-ethyl-bromoacetophenone or D2513C11 labelled p-propyl-bromoacetophenone.

Synthesis of13 Cu or 13Cu labelled reagents according to the invention, where X is Br, Rj — Rs are 13C and R9 is 13CH213CO2HOr 13CH213CH213CO2H.

A non-limiting method for the synthesis of these derivatives may be performed using methods known to those skilled in the art via oxidation [Reitsema, R.H. and Allphin, N.L. J. Org. Chem. 1962, 27, 27-28.] Of 13C8 labelled ethylbenzene or 13C9 labelled propylbenzene to yield the 13C8 labelled phenylacetic acid or 13C9 labelled phenylpropionic acid, followed by Friedel-Crafts acylation with 13C2 labelled bromoacetyl bromide, prepared from 13C2 labelled bromoacetic acid, to yield 13C10 labelled p-bromoacetyl-phenylacetic acid Or 13C11 /?- bromoacetyl-phenylpropionic acid.

Synthesis of reagents of the invention where X is Br, R1 -R8 are 13C and R? is O13CHs, O13CH213CH3, O13CH213CO2HOr O13CH213CH213CO2H.

A non-limiting method for the synthesis of these derivatives may be performed using methods known to those skilled in the art via nucleophilic substitution of a 13C1, 13C2, or 13C3 labelled haloalkane or haloalkanoic acid with 13C6 labelled phenol, followed by Friedel-Crafts acylation [Earle, MJ., Seddon, K.R., Adams, CJ. and Roberts, G. Chem. Comm. 1998, 19, 2097-2098.] of the resultant 13C7, 13C8 or 13C9 labelled phenoxyalkane or phenoxyalkanoic acid with 13C2 labelled acetylchloride to yield 13C9 Or 13C11 labelled p-acetyl-phenoxyalkane, or 13C10 or 13C11 labelled /?-acetyl-phenoxyalkanoic acid, followed by bromination to yield 13C9 or 13C10 labelled j>-bromoacetyl-phenoxyalkane, or 13C10 or 13C11 labelled p-bromoacetyl- phenoxyalkanoic acid.

Synthesis of reagents of the invention where X is Br, Rj - Rs are 13C and R? is SO213CH3, SO213CH213CH3, SO213CH213CH213CH3,

A non- limiting method for the synthesis of these derivatives may be performed using methods known to those skilled in the art via reaction of 13C6 labelled bromobenzene with 13C1, 13C2 or 13C3 labelled sodium alkylthiolate [Shaw, J. E. J. Org. Chem. 1991, 56, 3728-3729.], followed by Friedel-Crafts acylation [Abdur Rahim, M., Praveen Rao, P.N., and Knaus, E.E., Bioorg. Med. Chem. Lett. 2002, 12, 2753-2756.; Cutler, R.A., Stenger, RJ., Suter, CM. J. Am. Chem. Soc. 1952, 74, 5475-81.] with 13C2 labelled acetylchloride to yield 13C9, 13Ci0 or 13Cn labelled /7-acetyl-phenylmercaptoalkane, followed by oxidation [Therien, M., Gauthier, J. Y., Leblanc, Y., Leger, S., Perrier, H., Prasit, P. and Wang, Z. Synth. 2001, 12, 1778-1779.] then bromination to yield 13C9, 13Ci0 or 13C11 labelled jc-bromoacetyl-phenylalkylsulfone.

Synthesis of reagents of the invention where X is Br, Rj -Rg are C or 13C and Rρ is SO2CH2CO2H, SO213CH213CO2H, SO2CH2CH2CO2H or SO213CH213CH213CO2H. A non-limiting method for the synthesis of these derivatives may be performed using methods known to those skilled in the art via reaction of bromobenzene or 13C6 labelled bromobenzene with sodium alkylthiolate followed by acid cleavage [Shaw, J. E. J. Org. Chem. 1991, 56, 3728-9.] to yield benzenethiol or 13C6- labelled benzenethiol. Benzenethiol or 13C6- labelled benzenethiol is then reacted with a haloalkanoic acid or 13C1, 13C2 or 13C3 labelled haloalkanoic acid, followed by Friedel-Crafts acylation [Walker, D. and Leib, J. J. Org. Chem. 1963, 28, 3077-82.] with acetylchloride or 13C2 labelled acetylchloride, then oxidation [Webb, K. Tet. Lett. 1994, 35, 3457-60.] to yield p-acetyl-phenylsulfonylalkanoic acid or 13C10 or 13C11 labelled /7-acetyl-phenylsulfonylalkanoic acid, followed by bromination to yield /?-bromoacetyl- phenylsulfonylalkanoic acid or 13C10 or 13C11 labelled /j-bromoacetyl-phenylsulfonylalkanoic acid.

Preparation of Phenylthiopropionic acid.

Benzenethiol (0.045mol, 4.65 mL) was added to a solution of NaOH (0.95 mol, 3.8g) in H2O (40 mL) with stirring at room temp, which was followed by the addition of 3-Chloropropionic acid (0.5 mol, 5.4g). The resulting mixture was refluxed for 4hrs with stirring. Upon cooling to room temp, the solution was acidified to <pH 2 with cone HCl. The white precipitate was filtered and dried at the pump. Yield = 7.76g (94%)

Preparation of 4-Acetylphenylthiopropionic acid.

Acetyl chloride (0.0264mol, 1.86 mL) was slowly added to a mixture of Aluminium chloride (0.0901, 12g) in carbon disulfide (40 mL) with stirring and cooling in an ice/water bath, which was followed by the gradual addition of Phenylthiopropionic acid. The mixture became quite thick and difficult to stir so Nitrobenzene (10 mL) was added to help ease the stirring. The reaction mixture was left to stir overnight (~16 hrs) at room temp. The reaction was quenched (destroy excess AlCl3) by the slow addition of 2.5M HCl (70-80 mL) with cooling in an ice/water bath. The white precipitate was filtered, redissolved in ethyl acetate and washed with water (1 x 60 mL). The ethyl acetate was dried over Na2SO4 and removed in vacuo to yield 4- Acetylphenylthiopropionic acid (5g, 89%) as a pale yellow solid. Preparation of^Acetylphenylsulfonylpropionic acid.

4-Acetylphenylthiopropionic acid (0.0089moL 2g) was added to a solution of NaOH (O.lOβmol, 0.424g) in milli-Q H2O (22 niL) and the mixture was stirred for 20 mins. NaHCO3 (0.07 lmol, 6g) was added, followed by the addition of Acetone(0. lmol, 7.5 niL). Oxone solution (7.13g Oxone®(from Aldrich) in 10"4M EDTA (26.5 niL)) was then added dropwise over 5min. The resulting mixture was stirred at room temp for 1 hr. Sodium Hydrogen sulfite (4.4g) was added to quench the reaction. After stirring for a further 15 mins, the reaction solution was acidified (<pH 2) with cone HCl. THF (20 mL) was added to redissolve the precipitate, followed by the addition of Ethyl acetate (30 mL). The organic layer was separated, washed with water (1 x 40 mL) then dried over Na2SO4. The solvent was removed invacuo to yield 4-Acetylphenylsulfonylpropionic acid (1.7g, 74%) as an off white solid.

Preparation of^Bromoaectylphenylsulfonylpropionic acid

Polyvinylpyridinium tribromide resin was added to a solution of 4- Acetylphenylsulfonylpropionic acid (0.00196 mol, 0.5g) in THF (20 mL). The resulting mixture was stirred at room temp overnight (~16 hrs). The mixture was filtered and the THF removed at atmosphere under a stream of nitrogen. The bright orange residue was dissolved in ethyl acetate (20 mL), which was then washed with water (1 x 20 mL) and dried over Na2SO4. The Ethyl acetate was removed at atmosphere under a stream of nitrogen. The remaining solid was washed with Diethyl ether (15 mL), filtered and dried at the pump to yield 4-bromoacetyl- phenylsulfonylpropionic acid (0.44g, 69%) as a white solid.

Synthesis of reagents of the invention where X is Br, Rj — Rs are 13C and R9 is SO3H

A non- limiting method for the synthesis of these derivatives may be obtained using methods known to those skilled in the art via reaction of 13C6 benzene with 2,4- dinitrophenylsulfenyl chloride [Buess, CM. and Kharasch, N. J. Am. Chem. Soc. 1950, 72, 3529-3532.; Kharasch, N. and Swidler, R. J. Org. Chem. 1954, 19, 1704-1707] to yield 13C6- phenyl 2,4-dinitrophenyl sulfide, followed by Friedel-Crafts acylation [Szmant, H.H. and Irwin, D.A. J Am. Chem. Soc. 1956, 78, 4386-4389] using 13C2 acetic acid and trifluoroacetic anhydride in the presence of BF3 to yield the/7-acetylbenzene 2,4-dinitrophenyl sulphide, then oxidization to yield the p-acetylbenzene 2,4-dinitrophenyl sulfone, then cleaved with methanolic alkali to yield the p-acetylbenzene 2,4-dinitrophenyl sulfinic acid, then further oxidised to the sulfonic acid and then brominated.

Use of the reagents of the invention

The reagents of the invention are intended for the side chain fixed-charge derealization of peptides containing methionine to enable selective identification and differential quantitation of peptides by directed and selective fragmentation during MS/MS dissociation. The rationale for using fixed-charge derivatives of methionine containing peptides is based on the idea that approaches for selective protein identification and differential quantitation of protein expression should be specific for the detection of peptides containing amino acids that are rare, thereby limiting the number of peptides required to be analyzed, yet providing comprehensive coverage of proteins in the sequence databases.

The improved reagents according to the invention give increased mass differences compared to known reagents. This is especially useful for differential quantification between two samples. The increased mass difference provides higher sensitivity and enables improved analysis of low abundant proteins/peptides.