WO2006023398A2 - Reactifs de spectrometrie de masse marques par des isotopes modulaires et procedes d'analyse quantitative d'acides amines, de peptides et de proteines - Google Patents

Reactifs de spectrometrie de masse marques par des isotopes modulaires et procedes d'analyse quantitative d'acides amines, de peptides et de proteines Download PDF

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WO2006023398A2
WO2006023398A2 PCT/US2005/028780 US2005028780W WO2006023398A2 WO 2006023398 A2 WO2006023398 A2 WO 2006023398A2 US 2005028780 W US2005028780 W US 2005028780W WO 2006023398 A2 WO2006023398 A2 WO 2006023398A2
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compound
incorporation
group
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WO2006023398A3 (fr
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Gavin E. Reid
Kade D. Roberts
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Ludwig Institute For Cancer Research
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6842—Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/13—Labelling of peptides
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins

Definitions

  • This invention provides tandem mass spectrometry methods that can be employed in proteome analysis.
  • the present invention also relates to 'modular' stable isotope labelled fixed charge containing compounds that are useful as mass spectrometry (MS) reagents. It is also concerned with methods for the quantitation of amino acids, peptides and proteins using tandem mass spectrometry techniques.
  • proteomics may be broadly defined as (i) the systematic identification of all the gene products i.e., proteins, expressed by a particular cell or tissue type at a given time, (ii) quantitative analysis of the differences in protein abundances observed between two different states of a biological system i.e., such as that encountered between a normal and diseased cell or tissue, (iii) identification and characterization of co- and post-translational protein modifications, and (iv) identification and characterization of the protein complexes and specific protein-protein interactions, involved in the regulation of cellular behaviour [Blackstock, W.P. and Weir, M.P. Trends Biotechnol.
  • MS mass spectrometry
  • sample handling methodologies for protein and peptide purification, separation and analysis, along with sophisticated bioinformatic tools for rapid protein identification and characterization via database interrogation of MS derived data
  • bioinformatic tools for rapid protein identification and characterization via database interrogation of MS derived data
  • the reason for the desired fragmentation pathway often not yielding a dominant product ion may be rationalised by taking into consideration the generally accepted mechanisms and other factors (such as peptide ion charge state and amino acid composition) known to be responsible for the observed fragmentation reactions 5 of protonated peptide ions.
  • the derivatization reagent employed in the strategy mentioned above contains a cleavage 'enhancement' group consisting of a proline residue to promote fragmentation within the label during MS/MS, as well as a guanidino containing 'sensitization' group to promote formation of a characteristic protonated low mass product ion following the fragmentation reaction.
  • the high proton affinity of the guanidino side chains of arginine residues may act to strongly 'sequester' ionizing protons, thereby limiting their ability to be transferred along the peptide backbone to initiate cleavage by 'charge-directed' pathways.
  • the reagents may be selective for reaction with the N-terminal amino groups of amino acids, peptides and proteins, or with amino containing side chains of amino acids such as lysine, or with guanidine containing side chains of amino acids such as arginine or homoarginine, or with thiol containing side chains of amino acids such as cysteine or homocysteine, or with indole containing side chains of amino acids such as tryptophan, or with dehydroalanine or dehydroamino-2-buryric acid amino acids formed by ⁇ -elimination from O-linked phosphorylated or glycosylated serine or threonine, or with peptides or proteins comprising at least one residue of such amino acids.
  • These novel reagents have potential application for the high throughput, sensitive and selective quantitation of these compounds when present in complex mixtures.
  • the present invention provides compounds of formula XMi M 2 + , and XMi M 2 + or salts thereof, wherein:
  • X is a reactive group specific to a functional group contained within an amino acid, peptide or protein, or peptide or protein containing at least one of such amino acid; '+
  • Mi is a linker group between X and M 2 or M 2 , and is isotopically encoded by incorporation of one or more of 2 H, 13 C, 15 N or 18 O;
  • M 2 + is selected from the group consisting of a tertiary alkyl sulfonium ion, a quaternary alkyl ammonium ion or a quaternary alkyl phosphonium ion; and M 2 + is selected from the group consisting of a tertiary alkyl sulfonium ion, a quaternary alkyl ammonium ion or a quaternary alkyl phosphonium ion, and is isotopically encoded by incorporation of one or more of 2 H, 13 C, 15 N or 18 O.
  • the Mi group may be a branched alkyl optionally interrupted or substituted with an alkyl, aryl, substituted alkyl, substituted aryl, amino, amide, acid, ester or thioester.
  • the present invention provides a method for the quantitative . analysis of amino acids, peptides or proteins, the method comprising subjecting: a first amino acid, peptide or protein having a fixed-charge, introduced as a result of derivatization with a compound of formula XM[M 2 + , wherein X and M 2 + are as defined above, and Mi is a linker group between X and M 2 + ; and a second amino acid, peptide or protein having a fixed-charge, introduced as a result of derivatization with a compound of formula XMi M 2 + , wherein X, Mi and M 2 + are as defined above, to dissociation to form product ions that are characteristic of fragmentation occurring at the fixed-charge site.
  • the Mi group may be a branched alkyl optionally interrupted or substituted with an alkyl, aryl, substituted alkyl, substituted aryl, amino, amide, acid, ester or thioester.
  • the present invention provides a method for the quantitative analysis of amino acids, peptides or proteins, the method comprising subjecting: a first amino acid, peptide or protein having a fixed-charge, introduced as a result of derivatization with a compound of formula XM 1 M 2 + , wherein X, Mi and M 2 + are as defined above, and a second amino acid, peptide or protein having a fixed-charge, introduced as a result of derivatization with a compound of formula XMi M 2 + or XMi M 2 + , wherein X, Mi , M 2 + , and M 2 + are as defined above, to dissociation to form product ions that are characteristic of fragmentation occurring at the fixed-charge site.
  • the present invention provides a reagent kit for quantitative analysis of amino acids, peptides or proteins by tandem mass spectrometry, comprising a container containing a compound of formula XMi M 2 + or XMi M 2 of the present invention.
  • the present invention provides a reagent kit for quantitative analysis of amino acids, peptides or proteins by tandem mass spectrometry, comprising a container containing the compounds of formula XMjM 2 + and XMi M 2 + of the present invention.
  • the present invention provides a reagent kit for quantitative analysis of amino acids, peptides or proteins by tandem mass spectrometry, comprising a container containing the compounds of formula XMjM 2 + and XMj M 2 + .
  • the present invention provides a reagent kit for quantitative analysis of amino acids, peptides or proteins by tandem mass spectrometry, comprising a container containing the compounds of formula XMjM 2 + , XMi M 2 + and
  • the present invention also extends to compounds consisting of amino acids, peptides or proteins, that have been derivatized with a compound of formula XMj M 2 + or XM] M 2 + as defined above.
  • the present invention provides a reagent kit comprising a container containing compounds consisting of amino acids, peptides or proteins that have been derivatized with a compound of formula XMi M 2 + or XMj M 2 + as defined above.
  • the present invention provides a compound of formula XMi M 2 + , or a salt thereof,
  • X is a thiol reactive group selected from the group consisting of a halide, a disulfide exchange group or a vinyl group;
  • Mi ' is -RiCH(R 3 )R 2 , where Ri is selected from -(CH 2 ),,-, -Y- or -(CH 2 ) n Y-; R 2 is selected from -(CH 2 J n H, -C 6 H 5 , -CH 2 C 6 H 5 , -NH 2 , -YH, -Y(CH 2 ) n H, -YC 6 H 5 , - YCH 2 C 6 H 5 ; and R 3 is -(CH 2 ),,-, and is isotopically encoded by incorporation of one or more of 2 H, 13 C, 15 N or 18 O; n is from 1 to 3 inclusive;
  • the halide is preferably -Cl, -Br, or - 1.
  • the disulfide exchange group may be selected from -S-S-R' where R' is -C 6 H 5 , 3-carboxyl-4-nitrophenyl, 2,4-dinitrophenyl, 4-nitrophenyl, 2-nitrophenyl, 2-pyridyl, 5-nitropyridyl, 3-nitropyridyl, methanesulfonyl.
  • the present invention provides a compound of fo ⁇ nula XMi M 2 + , or a salt thereof, wherein,
  • M 1 ' is -RiCH(R 3 )R 2 , where Ri is -(CH 2 V; R 2 is -YH, -Y(CH 2 ) n H, -YC 6 H 5 , - YCH 2 C 6 H 5 ; and R 3 is -(CH 2 V, an d is isotopically encoded by incorporation of one or more Of 2 H, 13 C, 15 N or 18 O; n is from 1 to 3 inclusive; Y is CONH; and
  • X and M 2 + are as defined above.
  • the present invention provides a compound of formula XMi M 2 + , or a salt thereof, wherein, Mi ' is -R 1 CH(R 3 )R 2 , where R 1 is -Y- or -(CH 2 ) n Y-; Ri is -(CH 2 J n H, -NH 2 , - Y(CH 2 ) n H, -YC 6 H 5 , -YCH 2 C 6 H 5 ; and R 3 is -(CH 2 ),,-, and is isotopically encoded by inco ⁇ oration of one or more of 2 H, 13 C, 15 N or 18 O; n is from 1 to 3 inclusive; Y is CONH; and
  • X and M 2 + are as defined above.
  • the present invention provides a compound of formula XMi M 2 + , or a salt thereof, wherein, Mi ' is -R 1 CH(R 3 )R 2 , where R 1 is -(CH 2 )I 1 -; R 2 is -YH, -Y(CH 2 ) n H, -YC 6 H 5 , -
  • R 3 is -(CH 2 ) n -, and is isotopically encoded by incorporation of one or more of 2 H, 13 C, 15 N or 18 O; n is from 1 to 3 inclusive;
  • Y is NHCO; and X and M 2 + are as defined above.
  • the present invention provides a compound of formula XMi M 2 + , or a salt thereof, wherein,
  • Mi ' is -RiCH(R 3 )R 2 , where Ri is -Y- or -(CH 2 ) n Y-; R 2 is -(CH 2 J 11 H, -NH 2 , - Y(CH 2 ) n H, -YC 6 H 5 , -YCH 2 C 6 H 5 ; and R 3 is -(CH 2 ),,-, and is isotopically encoded by incorporation of one or more of 2 H, 13 C, 15 N or 18 O; n is from 1 to 3 inclusive;
  • Y is NHCO
  • X and M 2 + are as defined above.
  • the present invention provides an isotopically labelled compound of formula XMi M 2 + , or a salt thereof,
  • X is a thiol reactive group selected from the group consisting of a halide, a disulfide exchange group or a vinyl group;
  • Mi ' is -RiCH(R 3 )R 2 , where R 1 is selected from -(CHa) n -, -Y- or -(CH 2 ),, Y-; R 2 is selected from -(CH 2 ) ⁇ H, -C 6 H 5 , -CH 2 C 6 H 5 , -NH 2 , -YH, -Y(CH 2 ) n H, -YC 6 H 5 , - YCH 2 C 6 H 5 ; and R 3 is -(CH 2 ) n -, and is isotopically encoded by incorporation of one or more of 2 H, 13 C, 15 N or 18 O; n is from 1 to 3 inclusive;
  • Y is selected from CONH, NHCO and COO;
  • M 2 + is attached to the R 3 group of Mi and is selected from the group consisting of a tertiary alkyl sulfonium ion, -S + CH 3 R", where R" is selected from - CH 2 COC 6 H 5 ,
  • halide is preferably -Cl, -Br, or - 1.
  • the disulfide exchange group may be selected from -S-S-R' where R' is -C 6 H 5 , 3-carboxyl-4-nitrophenyl, 2,4-dinitrophenyl, 4-nitrophenyl, 2-nitrophenyl, 2-pyridyl, 5-nitropyridyl, 3-nitropyridyl, methanesulfonyl.
  • R' is -C 6 H 5
  • 4-nitrophenyl 2-nitrophenyl, 2-pyridyl, 5-nitropyridyl, 3-nitropyridyl, methanesulfonyl.
  • the present invention provides a compound of formula XMiM 2 + , or a salt thereof, wherein, M 1 is -RjCH(R 3 )R 2 , where R x is -(CH 2 ),,-; R 2 is -YH, -Y(CH 2 ) n H, -YC 6 H 5 , - YCH 2 C6H5; and R 3 is -(CH 2 ) n -, and is isotopically encoded by incorporation of one or more of 2 H, 13 C, 15 N or 18 O; n is from 1 to 3 inclusive; Y is CONH; and
  • X and M 2 + are as defined above.
  • the present invention provides a compound of formula XMi M 2 + , or a salt thereof, wherein, Mi ' is -RiCH(R 3 )R 2 , where R 1 is -Y- or -(CHz) n Y-; R 2 is -(CH 2 ) n H, -NH 2 , -
  • Y(CH 2 ) n H, -YC 6 H 5 , -YCH 2 C 6 H 5 ; and R 3 is -(CH 2 V, and is isotopically encoded by incorporation of one or more Of 2 H, 13 C, 15 N or !8 O; n is from 1 to 3 inclusive;
  • Y is CONH; and X and M 2 + are as defined above.
  • the present invention provides a compound of formula XM t M 2 + , or a salt thereof, wherein,
  • Mi ' is -RiCH(R 3 )R 2 , where R 1 is -(CHa) n -; R 2 is -YH, -Y(CH 2 ) n H, -YC 6 H 5 , - YCH 2 CeH 5 ; and R3 is -(CH 2 )I 1 -, and is isotopically encoded by incorporation of one or more Of 2 H, 13 C, 15 N or 18 O; n is from 1 to 3 inclusive;
  • Y is NHCO
  • X and M 2 + are as defined above.
  • the present invention provides a compound of formula XMi M 2 + , or a salt thereof, wherein,
  • Mi ' is -R 1 CH(R 3 )R 2 , where Ri is -Y- or -(CH 2 ) n Y-; R 2 is -(CH 2 ) n H, -NH 2 , - Y(CH 2 ) n H, -YC 6 H 5 , -YCH 2 C 6 H 5 ; and R 3 is -(CH 2 V-, and is isotopically encoded by incorporation of one or more of 2 H, 13 C, 15 N or 18 O; n is from 1 to 3 inclusive;
  • Y is NHCO
  • X and M 2 + are as defined above
  • the present invention provides a compound of formula XMi M 2 + , or a salt thereof,
  • X is an amino reactive group selected from the group consisting of an acid anhydride, an active ester, an acid halide, a sulfonylhalide, a substituted O-methyl isourea, an isocyanate or an isothiocyanate,
  • Mi ' is -RjCH(R 3 )R 2 , where R 1 is selected from -(CH 2 ) n -, -Y- or -(CH 2 ) n Y-; R 2 is selected from -(CH 2 ) n H, -C 6 H 5 , -CH 2 C 6 H 5 , -NH 2 , -YH, -Y(CH 2 ) n H, -YC 6 H 5 , - YCH 2 C O H S ; and R3 is -(CH 2 ) n -, and is isotopically encoded by incorporation of one or more of 2 H, 13 C, 15 N or 18 O; n is from 1 to 3 inclusive;
  • Y is selected from CONH, NHCO, COO and COS;
  • the present invention provides a compound of formula XMi M 2 + , or a salt thereof, wherein,
  • M 1 ' is -RiCH(R 3 )R 2 , where R, is -(CH 2 ) n -; R 2 is -YH, -Y(CH 2 ) n H, -YC 6 H 5 , -YCH 2 C 6 H 5 ; and R 3 is -(CH 2 ) n -, and is isotopically encoded by incorporation of one or more Of 2 H, 13 C, 15 N or 18 O; n is from 1 to 3 inclusive;
  • Y is CONH
  • X and M 2 + are as defined above.
  • the present invention provides a compound of formula XMi M 2 + , or a salt thereof, wherein,
  • Mi ' is -RiCH(R 3 )R 2 , where Ri is -Y- or -(CH 2 ) n Y-; R 2 is -(CHa) n H, -NH 2 , - Y(CH 2 ) n H, -YC 6 H 5 , -YCH 2 C 6 H 5 ; and R 3 is -(CH 2 ),,-, and is isotopically encoded by incorporation of one or more of 2 H, 13 C, 15 N or 18 O; n is from 1 to 3 inclusive;
  • Y is CONH
  • X and M 2 + are as defined above.
  • the present invention provides a compound of formula XMi M 2 + , or a salt thereof, wherein,
  • M 1 ' is -RiCH(R 3 )R 2 , where Ri is -(CH 2 V; Rz is -YH, -Y(CH 2 ) n H, -YC 6 H 5 , -YCH 2 C 6 H 5 ; and R 3 is -(CH 2 ) n -, and is isotopically encoded by incorporation of one or more of 2 H, 13 C, 15 N or 18 O; n is from 1 to 3 inclusive; Y is NHCO; and
  • X and M 2 + are as defined above.
  • the present invention provides a compound of formula XMi M 2 + , or a salt thereof, wherein,
  • Mi' is -RiCH(R 3 )R 2 , where Ri is -Y- or -(CH 2 ) n Y-; R 2 is -(CHj) n H, -NH 2 , - Y(CH 2 ) n H, -YC 6 H 5 , -YCH 2 C 6 H 5 ; and R 3 is -(CH 2 V, and is isotopically encoded by incorporation of one or more of 2 H, 13 C, 15 N or 18 O; n is from 1 to 3 inclusive;
  • Y is NHCO
  • X and M 2 + are as defined above.
  • the present invention provides a compound of formula XMi M 2 + , or a salt thereof,
  • X is an amino reactive group selected from the group consisting of an acid anhydride, an active ester, an acid halide, a sulfonylhalide, a substituted O-methyl isourea, an isocyanate or an isothiocyanate,
  • Mi' is -CH(R 2 )Ri, where Ri is selected from -(CH 2 ) n H, -C 6 H 5 , -CH 2 C 6 H 5 , - NH 2 , -YH, -Y(CH 2 ) n H, -YC 6 H 5 , -YCH 2 C 6 H 5 ; and R 2 is -(CHa) n -, and is isotopically encoded by incorporation of one or more of 2 H, 13 C, 15 N or 18 O; n is from 1 to 3 inclusive;
  • Y is selected from CONH, NHCO, COO and COS;
  • M 2 + is attached to the R 2 group of Mi and is selected from the group consisting of a tertiary alkyl sulfonium ion, -S CH 3 R", where R" is selected from - CH 2 COC 6 H 5 ,
  • the present invention provides a compound of formula XMi M 2 + , or a salt thereof, wherein,
  • Mi' is -CH(R 2 )R 1 , where R, is -(CH 2 X 1 H, -C 6 H 5 , -CH 2 C 6 H 5 , -NH 2 , -YH, - Y(CH 2 )JH,
  • R 2 is -(CH 2 ) n -, and is isotopically encoded by incorporation of one or more of 2 H, 13 C, 15 N or 18 O; n is from 1 to 3 inclusive;
  • Y is CONH
  • X and M 2 + are as defined above.
  • the present invention provides a compound of formula XMi M 2 + , or a salt thereof, wherein, Mi ' is -CH(R 2 )Ri, where R 1 is -(CH 2 ) n H, -C 6 H 5 , -CH 2 C 6 H 5 , -NH 2 , -YH, - Y(CH 2 ) n H,
  • R 2 is -(CHa) n -, and is isotopically encoded by incorporation of one or more Of 2 H, 13 C, 15 N or 18 O; n is from 1 to 3 inclusive;
  • Y is NHCO
  • X and M 2 + are as defined above.
  • the present invention provides a compound of formula XMi M 2 + , or a salt thereof,
  • X is an amino reactive group selected from the group consisting of an acid anhydride, an active ester, an acid halide, a sulfonylhalide, a substituted O-methyl isourea, an isocyanate or an isothiocyanate,
  • M 1 ' is -RiCH(R 3 )R 2 , where Ri is selected from -(CH 2 ) n -, -Y- or -(CHb) n Y-; R 2 is selected from -(CHa) n H, -C 6 H 5 , -CH 2 C 6 H 5 , -NH 2 , -YH, -Y(CH 2 )JH, -YC 6 H 5 , - YCH 2 C 6 H 5 ; and R 3 is -(CH 2 ) n -, and is isotopically encoded by incorporation of one or more Of 2 H, 13 C, 15 N or 18 O; n is from 1 to 3 inclusive;
  • the present invention provides a compound of formula XMi M 2 + , or a salt thereof, wherein,
  • Mi ' is -RiCH(R 3 )R 2 , where R, is -(CH 2 V; R2 is -YH, -Y(CH 2 ) n H, -YC 6 H 5 , -YCH 2 CeH 5 ; and R 3 is -(CH 2 V, and is isotopically encoded by incorporation of one or more of 2 H, 13 C, 15 N or 18 O; n is from 1 to 3 inclusive; Y is CONH; and
  • X and M 2 + are as defined above.
  • the present invention provides a compound of formula XMi M 2 + , or a salt thereof, wherein, Mi ' is -RiCH(R 3 )R 2 , where R, is -Y- or -(CH 2 ) n Y-; R 2 is -(CH 2 ) n H, -NH 2 , -
  • Y(CH 2 ) n H, -YC 6 H 5 , -YCH 2 C 6 H 5 ; and R 3 is -(CH 2 V, and is isotopically encoded by incorporation of one or more Of 2 H, 13 C, 15 N or 18 O; n is from 1 to 3 inclusive;
  • Y is CONH; and X and M 2 + are as defined above.
  • the present invention provides a compound of formula XMi M 2 + , or a salt thereof, wherein,
  • Mi ' is -RiCH(R 3 )R 2 , where R 1 is -(CH 2 V; R2 is -YH, -Y(CH 2 ) n H, -YC 6 H 5 , -YCH 2 C 6 H 5 ; and R 3 is -(CH 2 V, an d is isotopically encoded by incorporation of one or more Of 2 H, 13 C, 15 N or 18 O; n is from 1 to 3 inclusive;
  • Y is NHCO
  • X and M 2 + are as defined above.
  • the present invention provides a compound of formula XMi M 2 + , or a salt thereof, wherein, Mi is -RiCH(R 3 )R 2 , where R 1 is -Y- or -(CH 2 ) n Y-; R 2 is -(CH 2 ) n H, -NH 2 , - Y(CH 2 ) n H, -YC 6 H 5 , -YCH 2 C 6 H 5 ; and R 3 is -(CH 2 ),,-, and is isotopically encoded by incorporation of one or more of 2 H, ' 3 C, 15 N or 18 O; n is from 1 to 3 inclusive;
  • Y is NHCO
  • X and M 2 + are as defined above.
  • the present invention provides a compound of formula XMj M 2 + , or a salt thereof,
  • X is an amino reactive group selected from the group consisting of an acid anhydride, an active ester, an acid halide, a sulfonylhalide, a substituted O-methyl isourea, an isocyanate or an isothiocyanate;
  • Mi ' is -CH(R 2 )Ri, where R 1 is selected from -(CH 2 ) n H, -C 6 H 5 , -CH 2 C 6 H 5 , - NH 2 , -YH, -Y(CH 2 )JH, -YC 6 H 5 , -YCH 2 C 6 H 5 ; and R 2 is -(CH 2 ) n -, and is isotopically encoded by incorporation of one or more Of 2 H, 13 C, 15 N or 18 O; n is from 1 to 3 inclusive;
  • Y is selected from CONH, NHCO, COO and COS; and M 2 + is attached to the R 2 group of Mi and is selected from the group consisting of a tertiary alkyl sulfonium ion, -S + CH 3 R", where R" is selected from - CH 2 COC 6 H 5 ,
  • the present invention provides a compound of formula XMi M 2 + , or a salt thereof, wherein,
  • Mi ' is -CH(R 2 )Ri, where R, is -(CH 2 ) n H, -C 6 H 5 , -CH 2 C 6 H 5 , -NH 2 , -YH, - Y(CH 2 ) n H, -YC 6 H 5 , -YCH 2 C 6 H 5 ; and R 2 is -(CH 2 ) n -, and is isotopically encoded by incorporation of one or more of 2 H, 13 C, 15 N or 18 O; n is from 1 to 3 inclusive;
  • Y is CONH
  • X and M 2 + are as defined above.
  • the present invention provides a compound of formula XMi M 2 + , or a salt thereof, wherein,
  • M 1 ' is -CH(R 2 )Ri, where R 1 is -(CH 2 ) n H, -C 6 H 5 , -CH 2 C 6 H 5 , -NH 2 , -YH, - Y(CH 2 ) n H, -YC 6 H 5 , -YCH 2 C 6 H 5 ; and R 2 is -(CH 2 ) n -, and is isotopically encoded by incorporation of one or more of 2 H, 13 C, 15 N or 18 O; n is from 1 to 3 inclusive;
  • Y is NHCO
  • X and M 2 + are as defined above.
  • the present invention provides a compound of formula XMiM 2 + , or a salt thereof,
  • X is a guanidino specific reactive group selected from the group consisting of a substituted 2,3-butanedione, a substituted 2,4-pentanedione, a substituted glyoxal, or a substituted phenylglyoxal;
  • Mi ' is -R 1 CH(R 3 )R 2 , where R 1 is selected from -(CH 2 ) n -, -Y- or -(CH 2 ) n Y-; R 2 is selected from -(CH 2 ) n H, -C 6 H 5 , -CH 2 C 6 H 5 , -NH 2 , -YH, -Y(CH 2 ) n H, -YC 6 H 5 , - YCH 2 C 6 H 5 ; and R 3 is -(CH 2 ) n -, and is isotopically encoded by incorporation of one or more of 2 H, 13 C, 15 N or 18 O; n is from 1 to 3 inclusive;
  • Y is selected from CONH, NHCO, COO and COS; and M 2 + is attached to the R 3 group of Mi and is selected from the group consisting of a tertiary alkyl sulfonium ion, -S + CH 3 R", where R" is selected from -
  • the present invention provides a compound of formula XMi M 2 + , or a salt thereof, wherein,
  • Mi ' is -RiCH(R 3 )R 2 , where Ri is -(CH 2 ) n -; R 2 is -YH, -Y(CH 2 ) n H, -YC 6 H 5 , -YCH 2 C 6 H 5 ; and R 3 is -(CH 2 ) n -, and is isotopically encoded by incorporation of one or more of 2 H, 13 C, 15 N or 18 O; n is from 1 to 3 inclusive;
  • Y is CONH
  • X and M 2 + are as defined above.
  • the present invention provides a compound of formula XMiM 2 + , or a salt thereof, wherein,
  • Mi' is -RiCH(R 3 )R 2 , where Ri is -Y- or -(CH 2 ) n Y-; R 2 is -(CH 2 ) n H, -NH 2 , - Y(CH 2 ) n H, -YC 6 H 5 , -YCH 2 C 6 H 5 ; and R 3 is -(CH 2 ) n -, and is isotopically encoded by incorporation of one or more of 2 H, 13 C, 15 N or 18 O; n is from 1 to 3 inclusive;
  • Y is CONH
  • X and M 2 + are as defined above.
  • the present invention provides a compound of formula XMi M 2 + , or a salt thereof, wherein,
  • M 1 ' is -RiCH(R 3 )R 2 , where R 1 is -(CH 2 ) n -; R 2 is -YH, -Y(CH 2 ) n H, -YC 6 H 5 , -YCH 2 C 6 H 5 ; and R 3 is -(CH 2 ) n -, and is isotopically encoded by incorporation of one or more Of 2 H, 13 C, 15 N or 18 O; n is from 1 to 3 inclusive;
  • Y is NHCO
  • X and M 2 + are as defined above.
  • the present invention provides a compound of formula XMi M 2 + , or a salt thereof, wherein,
  • M 1 ' is -R 1 CH(R 3 )R 2 , where R 1 is -Y- or -(CH 2 J n Y-; Ra is -(CH 2 ) n H, -NH 2 , -
  • Y(CH 2 ) n H, -YC 6 H 5 , -YCH 2 C 6 H 5 ; and R 3 is -(CH 2 ),,-, and is isotopically encoded by incorporation of one or more Of 2 H, 13 C, 15 N or 18 O; n is from 1 to 3 inclusive;
  • Y is NHCO
  • X and M 2 + are as defined above.
  • the present invention provides a compound of formula XMi M 2 + , or a salt thereof,
  • X is a guanidino specific reactive group selected from the group consisting of a substituted 2,3-butanedione, a substituted 2,4-pentanedione, a substituted glyoxal, or a substituted phenylglyoxal;
  • Mi is -R 1 CH(R 3 )R 2 , where Ri is selected from -(CH 2 J n -, Y- or -(CH 2 J n Y-;
  • R2 is selected from -(CH 2 ) n H, -C 6 H 5 , -CH 2 C 6 H 5 , -NH 2 , -YH, -Y(CH 2 ) n H, -YC 6 H 5 , - YCH 2 C O H 5 ;
  • R 3 is -(CH2)n-, and is isotopically encoded by incorporation of one or more Of 2 H, 13 C, 15 N or 18 O; n is from 1 to 3 inclusive
  • Y is selected from CONH, NHCO, COO and COS;
  • the present invention provides a compound of formula XM 1 M 2 + , or a salt thereof, wherein,
  • Mi' is -RiCH(R 3 )R 2 , where Ri is -(CHa) n -; R 2 is -YH, -Y(CH 2 ) n H, -YC 6 H 5 , -YCH 2 C 6 H 5 ; and R 3 is -(CH 2 ),,-, and is isotopically encoded by incorporation of one or more of 2 H, 13 C, 15 N or 18 O; n is from 1 to 3 inclusive;
  • Y is CONH
  • X and M 2 + are as defined above.
  • the present invention provides a compound of formula XMi M 2 + , or a salt thereof, wherein,
  • Mi' is -RiCH(R 3 )R 2 , where Ri is -Y- or -(CH 2 ) n Y-; R 2 is -(CH 2 ) n H, -NH 2 , -
  • Y(CH 2 ) n H, -YC 6 H 5 , -YCH 2 C 6 H 5 ; and R 3 is -(CH 2 ) n -, and is isotopically encoded by incorporation of one or more of 2 H, 13 C, 15 N or 18 O; n is from 1 to 3 inclusive;
  • Y is CONH
  • X and M 2 + are as defined above.
  • the present invention provides a compound of formula XMi M 2 + , or a salt thereof, wherein,
  • Mi ' is -RiCH(R 3 )R 2 , where Ri is -(CH 2 ),,-; R 2 is -YH, -Y(CH 2 ) n H, -YC 6 H 5 , -YCH 2 C 6 H 5 ; and R 3 is ⁇ (CH 2 ) n -, and is isotopically encoded by incorporation of one or more Of 2 H, 13 C, 15 N Or 18 O; n is from 1 to 3 inclusive;
  • Y is NHCO
  • X and M 2 + are as defined above.
  • the present invention provides a compound of formula XM; M 2 + , or a salt thereof, wherein,
  • Mi ' is -RiCH(R 3 )R 2 , where Ri is -Y- or -(CH 2 ) n Y-; R 2 is -(CH 2 ) n H, -NH 2 , -
  • Y(CH 2 ) n H, -YC 6 H 5 , -YCH 2 C 6 H 5 ; and R 3 is -(CH 2 ),,-, and is isotopically encoded by incorporation of one or more Of 2 H, 13 C, 15 N or 18 O; n is from 1 to 3 inclusive;
  • Y is NHCO
  • X and M 2 + are as defined above.
  • the present invention provides a compound of formula XMi M 2 + , or a salt thereof, for specific reaction with the C2-indole position of the side chain of tryptophan or tryptophan containing proteins or peptides,
  • X is a reactive group specific to the C2-indole position of the side chain of tryptophan selected from a halide or a dimethyl sulfonium ion, wherein the halide is preferably
  • M 1 ' is -RiCH(R 3 )R 2 , where Ri is selected from a -2-hydroxy-5-nitrobenzyI-, or -(2-hydroxy-5-nitrobenzyl)-4-Y- group;
  • R 2 is selected from -(CH 2 ) n H, -C O H 5 , - CH 2 C 6 H 5 , -NH 2 , -YH, -Y(CH 2 ) n H, -YC 6 H 5 , -YCH 2 C 6 H 5 , and R 3 is -(CH 2 ),,-, and is isotopically encoded by incorporation of one or more of 2 H, 13 C, 15 N or 18 O; n is from 1 to 3 inclusive; Y is selected from CONH, NHCO, COO or COS; and
  • the present invention provides a compound of formula XMi M 2 + , or a salt thereof, wherein,
  • M 1 ' is -RiCH(R 3 )R 2 , where Ri is -2-hydroxy-5-nitrobenzyl-;
  • R 2 is -YH, - Y(CH 2 ) n H, -YC 6 H 5 , -YCH 2 C 6 H 5 ; and
  • R 3 is -(CH 2 ) n -, and is isotopically encoded by incorporation of one or more of 2 H, 13 C, 15 N or 18 O; n is from 1 to 3 inclusive;
  • Y is CONH
  • X and M 2 + are as defined above.
  • the present invention provides a compound of formula XMi M 2 + , or a salt thereof, wherein,
  • Mi ' is -RiCH(R 3 )R 2 , where R 1 is -(2-hydroxy-5-nitrobenzyl)-4-Y-; R 2 is - (CH 2 ) n H, -C 6 H 5 , -CH 2 C 6 H 5 , -NH 2 , -YH, -Y(CH 2 ) n H, -YC 6 H 5 , -YCH 2 C 6 H 5 ; and R 3 is -(CHa) n -, and is isotopically encoded by incorporation of one or more Of 2 H, 13 C, 15 N or 18 O; n is from 1 to 3 inclusive;
  • Y is CONH
  • X and M 2 + are as defined above.
  • the present invention provides a compound of formula XMi M 2 + , or a salt thereof, wherein,
  • Mi ' is -RiCH(R 3 )R 2 , where Ri is -2-hydroxy-5-nitrobenzyl-; R 2 is -YH, - Y(CH 2 ) n H,
  • R 3 is -(CH 2 ) n -, and is isotopically encoded by incorporation of one or more Of 2 H, 13 C, 15 N or 18 O; n is from 1 to 3 inclusive;
  • Y is NHCO; and X and M 2 + are as defined above.
  • the present invention provides a compound of formula XMi M 2 + , or a salt thereof, wherein,
  • Mi ' is -RiCH(R 3 )R 2 , where Ri is -(2-hydroxy-5-nitrobenzyl)-4-Y-; R 2 is - (CH 2 ) n H,
  • R 3 is -(CH 2 ) n -, and is isotopically encoded by incorporation of one or more Of 2 H, 13 C, 15 N or 18 O; n is from 1 to 3 inclusive;
  • Y is NHCO; and X and M 2 + are as defined above.
  • the present invention provides a compound of formula XMj M 2 + , or a salt thereof, for specific reaction with the C2-indole position of the side chain of tryptophan or tryptophan containing proteins or peptides,
  • X is a reactive group specific to the C2-indole position of the side chain of tryptophan selected from a halide or a dimethyl sulfonium ion, wherein the halide is preferably -Cl, -Br, or - I, and wherein the dimethyl sulfonium ion is preferably - S(CH 3 ) 2 + .
  • Mi is -RiCH(Rs)R 2 , where Ri is selected from a -2-hydroxy-5-nitrobenzyl-, or -(2-hydroxy-5-nitrobenzyl)-4 ⁇ Y- group; R 2 is selected from -(CH 2 ) n H, -C 6 Hs, - CH 2 C 6 H 5 , -NH 2 ,
  • n is from 1 to 3 inclusive;
  • Y is selected from CONH, NHCO, COO or COS;
  • the present invention provides a compound of formula XMi M 2 + , or a salt thereof, wherein,
  • Mi' is -RiCH(R 3 )R 2 , where Ri is -2-hydroxy-5-nitrobenzyl-; R 2 is -YH, - Y(CH 2 ) n H, -YC 6 H 5 , -YCH 2 C 5 H 5 ; and R 3 is -(CH 2 ),,-, and is isotopically encoded by incorporation of one or more of 2 H, 13 C, 15 N or 18 O; n is from 1 to 3 inclusive;
  • Y is CONH
  • X and M 2 + are as defined above.
  • the present invention provides a compound of formula XMi M 2 + , or a salt thereof, wherein,
  • Mi' is -RiCH(R 3 )R 2 , where Ri is -(2-hydroxy-5-nitrobenzyl)-4-Y-;
  • R 2 is - (CH 2 ) n H, -C 6 H 5 , -CH 2 C 6 H 5 , -NH 2 , -YH, -Y(CH 2 ) n H, -YC 6 H 5 , -YCH 2 C 6 H 5 ;
  • R 3 is -(CH 2 ),,-, and is isotopically encoded by incorporation of one or more of 2 H, 13 C, 15 N or 18 O;
  • n is from 1 to 3 inclusive;
  • Y is CONH; and X and M 2 + are as defined above.
  • the present invention provides a compound of formula XMi M 2 + , or a salt thereof, wherein,
  • M 1 ' is -RiCH(R 3 )R 2 , where Ri is -2-hydroxy-5-nitrobenzyl-; R 2 is -YH, - Y(CH 2 ) n H,
  • R 3 is -(CH 2 ) n -, and is isotopically encoded by incorporation of one or more of 2 H, 13 C, 15 N or 18 O; n is from 1 to 3 inclusive;
  • Y is NHCO; and X and M 2 + are as defined above.
  • the present invention provides a compound of formula XMi M 2 + , or a salt thereof, wherein,
  • Mi' is -RiCH(R 3 )R 2 , where Ri is -(2-hydroxy-5-nitrobenzyl)-4-Y-; R 2 is - (CH 2 ) n H,
  • R 3 is -(CH 2 ),,-, and is isotopically encoded by incorporation of one or more of 2 H, 13 C, 15 N or 18 O; n is from 1 to 3 inclusive;
  • Y is NHCO; and X and M 2 + are as defined above.
  • the present invention provides a compound of formula XMi M 2 + , or a salt thereof, for specific reaction with the C2-indole position of the side chain of tryptophan or tryptophan containing proteins or peptides,
  • X is a reactive group specific to the C2-indole position of the side chain of tryptophan selected from a sulfenylhalide, wherein the sulfenylhalide is preferably - SCl, -SBr, or - SI;
  • Mi is -RiCH(Rs)R 2 , where Ri is selected from a -2-nitrophenyl- or -(2- nitrophenyl)-4-Y- group; R 2 is selected from -(CH 2 ) n H, -C 6 H 5 , -CH 2 C 6 H 5 , -NH 2 , -YH, -Y(CH 2 ) n H,
  • -YC 6 H 5 , -YCH 2 C 6 H 5 , and R 3 is -(CH 2 ) n -, and is isotopically encoded by incorporation of one or more of 2 H, 13 C, 15 N or 18 O; n is from 1 to 3 inclusive;
  • the present invention provides a compound of formula XMj M 2 + , or a salt thereof, wherein,
  • Mi ' is -RiCH(R 3 )R 2 , where Ri is -2-nitrophenyl-; R 2 is -YH, -Y(CH 2 ) n H, - YC 6 H 5 ,
  • R 3 is -(CH 2 ) n -. and is isotopically encoded by incorporation of one or more Of 2 H, 13 C, I5 N or iS O; n is from 1 to 3 inclusive; Y is CONH; and X and M 2 + are as defined above.
  • the present invention provides a compound of formula XMi M 2 + , or a salt thereof, wherein,
  • Mi ' is -RiCH(R 3 )R 2 , where Rj is -(2-nitrophenyl)-4-Y-;
  • R 2 is -(CH 2 ) n H, -C 6 H 5 , -CH 2 C 6 H 5 , -NH 2 , -YH, -Y(CH 2 ) n H, -YC 6 H 5 , -YCH 2 C 6 H 5 ;
  • R 3 is -(CH 2 ) n -, and is isotopically encoded by incorporation of one or more Of 2 H, 13 C, 15 N or 18 O; n is from 1 to 3 inclusive; Y is CONH; and
  • X and M 2 + are as defined above.
  • the present invention provides a compound of formula XMi M 2 + , or a salt thereof, wherein, Mi' is -RiCH(R 3 )R 2 , where Ri is -2-nitrophenyl-; R 2 is -YH, -Y(CH 2 ) n H, -
  • R 3 is -(CH 2 ) n -, and is isotopically encoded by incorporation of one or more Of 2 H, 13 Q 15 N Or 18 O; n is from 1 to 3 inclusive; Y is NHCO; and
  • X and M 2 + are as defined above.
  • the present invention provides a compound of formula XMi M 2 + , or a salt thereof, wherein, Mi ' is -R 1 CH(R 3 )R 2 , where R 1 is -(2-nitrophenyl)-4-Y-; R 2 is -(CH 2 ) n H, -C 6 H 5 ,
  • R 3 is -(CH 2 ) n -, and is isotopically encoded by incorporation of one or more of 2 H, 13 C, 15 N or 18 O; n is from 1 to 3 inclusive; Y is NHCO; and X and M 2 + are as defined above.
  • the present invention provides a compound of formula XMi M 2 + , or a salt thereof, for specific reaction with the C2-indole position of the side chain of tryptophan or tryptophan containing proteins or peptides,
  • X is a reactive group specific to the C2-indole position of the side chain of tryptophan selected from a sulfenylhalide, wherein the sulfenylhalide is preferably - SCl, -SBr, or - SI;
  • Mi is -RiCH(R 3 )R 2 , where Ri is selected from a -2-nitrophenyl- or -(2- nitrophenyl)-4-Y- group;
  • R 2 is selected from -(CH 2 ) n H, -C 6 H 5 , -CH 2 C 6 H 5 , -NH 2 , -YH, -Y(CH 2 ) n H, -YC 6 H 5 , -YCH 2 C 6 H 5 , and
  • R 3 is -(CH 2 ) n -, and is isotopically encoded by incorporation of one or more of 2 H, 13 C, 15 N or 18 O;
  • n is from 1 to 3 inclusive;
  • Y is selected from CONH, NHCO, COO or COS; and M 2 + is attached to the R 3 group of Mi and is selected from the group consisting of a tertiary alkyl sulfonium ion, -S + CH 3 R", where R" is selected from - CH 2 COC 6 H 5 ,
  • the present invention provides a compound of formula XMi M 2 + , or a salt thereof, wherein, Mi' is -RiCH(R 3 )R 2 , where Ri is -2-nitrophenyl-; R 2 is -YH, -Y(CH 2 ) n H, -
  • R 3 is -(CH 2 ) n -. and is isotopically encoded by incorporation of one or more of 2 H, 13 C, 15 N or 18 O; n is from 1 to 3 inclusive;
  • Y is CONH
  • X and M 2 + are as defined above.
  • the present invention provides a compound of formula XMi M 2 + , or a salt thereof, wherein,
  • Mi ' is -RiCH(R 3 )R 2 , where Ri is -(2-nitrophenyl)-4-Y-;
  • R 2 is -(CH 2 ) n H, -C 6 H 5 , -CH 2 C 6 H 5 , -NH 2 , -YH, -Y(CH 2 ) n H, -YC 6 H 5 , -YCH 2 C 6 H 5 ;
  • R 3 is -(CH 2 ) n -, and is isotopically encoded by incorporation of one or more of 2 H, 13 C, 15 N or 18 O; n is from 1 to 3 inclusive;
  • Y is CONH
  • X and M 2 + are as defined above.
  • the present invention provides a compound of formula XMi M 2 + , or a salt thereof, wherein,
  • Mi' is -RiCH(R 3 )R 2 , where Rj is -2-nitrophenyl-; R 2 is -YH, -Y(CH 2 ) n H, - YC 6 H 5 ,
  • R 3 is -(CH 2 ) n -, and is isotopically encoded by incorporation of one or more Of 2 H, 13 C, 15 N or 18 O; n is from 1 to 3 inclusive;
  • Y is NHCO
  • X and M 2 + are as defined above.
  • the present invention provides a compound of formula XMi M 2 + , or a salt thereof, wherein,
  • Mi' is -RiCH(R 3 )R 2 , where R t is -(2-nitrophenyl)-4-Y-;
  • R 2 is -(CH 2 ) n H, -C 6 H 5 , -CH 2 C 6 H 5 , -NH 2 , -YH, -Y(CH 2 ) n H, -YC 6 H 5 , -YCH 2 C 6 H 5 ;
  • R 3 is -(CH 2 ) n -, and is isotopically encoded by incorporation of one or more Of 2 H, 13 C, 15 N or 18 O; n is from 1 to 3 inclusive;
  • Y is NHCO; and
  • X and M 2 1+ are as defined above.
  • the present invention provides a compound of formula XMi M 2 + , or a salt thereof, for specific reaction with dehydroalanine or dehydroamino-2-buty ⁇ c acid formed by ⁇ -elimination from O-linked phosphorylated or glycosylated serine or threonine, or dehydroalanine or dehydroamino-2-butyric acid residues formed by ⁇ -elimination from O-linked phosphorylated or glycosylated serine or threonine containing proteins or peptides respectively,
  • X is a thiol, wherein the thiol is preferably -SH;
  • Mi ' is -RiCH(R 3 )R 2 , where R 1 is selected from -(CH 2 ) n -, -Y- or -(CH 2 ) n Y-; R 2 is selected from -(CH 2 ) n H, -C 6 H 5 , -CH 2 C 6 H 5 , -NH 2 , -YH, -Y(CH 2 ) n H, -YC 6 H 5 , - YCH 2 C 6 H 5 , and R 3 is -(CH 2 ) n -, and is isotopically encoded by incorporation of one or more of 2 H, 13 C, 15 N or 18 O; n is from 1 to 3 inclusive;
  • the present invention provides a compound of formula XMi M 2 + , or a salt thereof, wherein,
  • Mi' is -R 1 CH(R 3 )R 2 , where R, is -(CH 2 ) n -; R 2 is -YH, -Y(CH 2 ) n H, -YC 6 H 5 , -YCH 2 C 6 H 5 ; and R 3 is -(CH 2 ) n -, and is isotopically encoded by incorporation of one or more Of 2 H, 13 C, 15 N or 18 O; n is from 1 to 3 inclusive;
  • Y is CONH
  • X and M 2 + are as defined above.
  • the present invention provides a compound of formula XMi M 2 + , or a salt thereof, wherein,
  • Mi' is -RiCH(R 3 )R 2 , where Ri is -Y- or -(CH 2 ) n Y-; R 2 is -(CHa) n H, -C 6 H 5 , - CH 2 C 6 H 5 , -NH 2 , -YH, -Y(CHz) n H, -YC 6 H 5 , -YCH 2 C 6 H 5 ; and R 3 is -(CH 2 ),,-, and is isotopically encoded by incorporation of one or more of 2 H, 13 C, 15 N or 18 O; n is from 1 to 3 inclusive;
  • Y is CONH
  • X and M 2 + are as defined above.
  • the present invention provides a compound of formula XMi M 2 + , or a salt thereof, wherein,
  • Mi' is -RiCH(R 3 )R 2 , where Ri is -(CHz) n -; R 2 is -YH, -Y(CH 2 ) n H, -YC 6 H 5 , -YCH 2 C 6 H 5 ; and R 3 is -(CH 2 ) n -, and is isotopically encoded by incorporation of one or more Of 2 H, 13 C, 15 N or 18 O; n is from 1 to 3 inclusive; Y is NHCO; and
  • the present invention provides a compound of formula XMi M 2 + , or a salt thereof, wherein, Mi' is -RiCH(R 3 )R 2 , where Ri is -Y- or -(CH 2 ) n Y-; R 2 is -(CH 2 ) n H, -C 6 H 5 , -
  • R 3 is -(CH 2 J n -, and is isotopically encoded by incorporation of one or more of 2 H, 13 C, 15 N or 18 O; n is from 1 to 3 inclusive;
  • Y is NHCO; and X and M 2 + are as defined above.
  • the present invention provides a compound of formula XMi M 2 + , or a salt thereof, for specific reaction with dehydroalanine or dehydroamino-2-butyric acid formed by ⁇ -elimination from O-linked phosphorylated or glycosylated serine or threonine, or dehydroalanine or dehydroamino-2-butyric acid residues formed by ⁇ -elimination from O-linked phosphorylated or glycosylated serine or threonine containing proteins or peptides respectively,
  • X is a thiol, wherein the thiol is preferably -SH;
  • Mi ' is -R]CH(R 3 )R 2 , where R 1 is selected from -(CH 2 ) n -, -Y- or -(CH 2 ) n Y-;
  • R 2 is selected from -(CH 2 ) n H, -C 6 H 5 , -CH 2 C 6 H 5 , -NH 2 , -YH, -Y(CH 2 ) n H, -YC 6 H 5 , -
  • YCH 2 C 6 H 5 , and R 3 is -(CH 2 ) n -, and is isotopically encoded by incorporation of one or more Of 2 H, 13 C, 15 N or 18 O; n is from 1 to 3 inclusive; Y is selected from CONH, NHCO or COO; and
  • M 2 + is attached to the R 3 group of Mi and is selected from the group consisting of a tertiary alkyl sulfonium ion, -S + CH 3 R", where R" is selected from -
  • n 1 to 3
  • -C 6 H 5 -CH 2 C 6 H 5 ; and is isotopically encoded by incorporation of one or more of 2 H, 13 C, 15 N or 18 O.
  • the present invention provides a compound of formula XMi M 2 + , or a salt thereof, wherein,
  • Mi' is -RiCH(R 3 )R 2 , where R 1 is -(CH 2 ) n -; R 2 is -YH, -Y(CH 2 ) n H, -YC 6 H 5 , -YCH 2 C 6 Hs; and R 3 is -(CH 2 ) H -, and is isotopically encoded by incorporation of one or more Of 2 H, 13 C, 15 N or 18 O; n is from 1 to 3 inclusive;
  • Y is CONH
  • X and M 2 + are as defined above.
  • the present invention provides a compound of formula XMiM 2 + , or a salt thereof, wherein,
  • Mi is -R 1 CH(R 3 )R 2 , where Ri is -Y- or -(CH 2 J n Y-; R 2 is -(CHa) n H, -C 6 H 5 , - CH 2 C 6 H 5 , -NH 2 , -YH, -Y(CH 2 ) n H, -YC 6 H 5 , -YCH 2 C 6 H 5 ; and R 3 is -(CH 2 ),,-, and is isotopically encoded by incorporation of one or more of 2 H, 13 C, 15 N or 18 O; n is from 1 to 3 inclusive;
  • Y is CONH
  • X and M 2 + are as defined above.
  • the present invention provides a compound of formula XMi M 2 + , or a salt thereof, wherein,
  • Mi ' is -RiCH(R 3 )R 2 , where Ri is -(CH 2 ) n -; R 2 is -YH, -Y(CH 2 ) n H, -YC 6 H 5 , -YCH 2 C 6 H 5 ; and R 3 is -(CH 2 ) n -, and is isotopically encoded by incorporation of one or more of 2 H, 13 C, 15 N or 18 O; n is from 1 to 3 inclusive; Y is NHCO; and
  • X and M 2 + are as defined above.
  • the present invention provides a compound of formula XMi M 2 + , or a salt thereof, wherein, M 1 ' is -R 1 CH(R 3 )R 2 , where Ri is -Y- or -(CH 2 ) n Y-; R 2 is -(CH 2 ) n H, -C 6 H 5 , -
  • R 3 is -(CH 2 ) n -, and is isotopically encoded by incorporation of one or more of 2 H, 13 C, 15 N or 18 O; n is from 1 to 3 inclusive;
  • Y is NHCO; and X and M 2 + are as defined above.
  • the present invention provides a method for the quantitative analysis of amino acids, peptides or proteins, the method comprising subjecting a first amino acid, peptide or protein having a fixed-charge, introduced as a result of derivatization with a compound of formula XMiM 2 + , wherein X and M 2 + are as defined above, and Mi is a linker group having the same structure as Mi below, but containing only naturally abundant isotopes; and a second amino acid, peptide or protein having a fixed-charge, introduced as a result of derivatization with a compound of formula XMiM 2 + , wherein X, Mi and M 2 + are as defined above, to dissociation to form product ions that are characteristic of fragmentation occurring at the fixed-charge site by the loss OfM 2 .
  • the present invention provides a method for the quantitative analysis of amino acids, peptides or proteins, the method comprising subjecting a first amino acid, peptide or protein having a fixed-charge, introduced as a result of derivatization with a compound of formula XMiM 2 + , wherein X and M 2 + are as defined above, and Mi is a linker group having the same structure as M/ below, but containing only naturally abundant isotopes; and a second amino acid, peptide or protein having a fixed-charge, introduced as a result of derivatization with a compound of formula XMiM 2 + or XMi M 2 + , wherein X, M 1 ' , M 2 + , and M 2 + are as defined above, to dissociation to form product ions that are characteristic of fragmentation occurring at the fixed-charge site by the loss of M 2 or M 2 .
  • the compounds of the present invention are suitable for use in the tandem mass spectrometry methods for quantitative analysis described below.
  • the reagents of the invention are employed for the fixed-charge derivatization of an amino acid, peptide or protein, to enable their quantitation via selective and directed fragmentation during MS/MS dissociation.
  • the present invention provides a method for quantitative analysis of amino acids, peptides or proteins, the method comprising: (1) introducing a mixture of amino acids, peptides or proteins containing at least one selected amino acid, peptide or protein, or peptide or protein comprising at least one residue of the selected amino acid, derivatized to contain a fixed-charge using compounds of formula XMiMo + , XMi M 2 + , XMi M 2 + and XMi M 2 + , or salts thereof, as described above;
  • the method of analysis may be used for the identification and/or quantitation of amino acids, peptides or proteins.
  • the amino acid, peptide or protein contains an N-terminal amino group, a cysteine, a homocysteine, a lysine, an arginine, a homoarginine, a tryptophan, a dehydroalanine or a dehydroamino-2 -butyric acid.
  • the method of the latter aspect of the invention may include the preceding step of derivatizing the amino acid, peptide or protein with a compound of formula XMiM 2 + , XMiM 2 + , XMiM 2 1+ and XMi ' M 2 '+ or salts thereof.
  • the method of the invention described above comprises the further step of: (5) determining the identity of the peptide or protein.
  • Step (5) may be performed by first repeating steps (1), (2), and (3) and then subjecting the product ion having the second characteristic mass-to-charge ratio formed by loss from the precursor to a further stage of dissociation to form a series of product ions having a range of mass to charge ratios, for the purpose of determining the amino acid sequence of the peptide or protein and subsequently, the identity of the protein of origin.
  • step (5) may be carried out by use of high resolution mass analyzers to obtain an "accurate mass tag" (i.e., a mass accuracy of, for example, approximately 1-5 ppm) on the product ion detected in step (4).
  • an "accurate mass tag” i.e., a mass accuracy of, for example, approximately 1-5 ppm
  • This coupled with database searching, may be employed for subsequent identification of those peptides found to contain a fixed-charge derivative.
  • the specificity of database searching algorithms can be improved such that unambiguous identification of the protein from which the peptide is derived has been achieved from this information alone.
  • the amino acid, peptide or protein ion may be dissociated by any suitable dissociation method including, but not limited to, collisions with an inert gas (known as collision-induced dissociation (CID or collisionally-activated dissociation (CAD); (ii) collisions with a surface (known as surface-induced dissociation or SID); (iii) interaction with photons (e. g.
  • CID collision-induced dissociation
  • SID surface-induced dissociation
  • photons e.g.
  • ETD electron transfer dissociation
  • tandem mass spectrometry Analysis of the amino acid, peptide or protein ion 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.
  • ESI electrospray ionization
  • MALDI matrix assisted laser desorption ionization
  • the methods of the invention in certain embodiments may also include one or more steps of protein extraction, protein separation, reduction and alkylation of cysteine disulfides and/or protein digestion.
  • the present invention also extends to a reagent kit for quantitative analysis of amino acids, peptides or proteins comprising a compound of the present invention.
  • the kit may also include instructions for use of the compounds of the invention in the quantitative analysis of amino acids, peptides or proteins by mass spectrometry.
  • the reagent kit further may also contain one or more containers containing: cysteine disulfide reducing agents, alkylating agents, proteases or chemical cleavage agents, and/or solvents.
  • the cysteine disulfide reducing agents preferably include dithiothreitol (DTT), mercaptoethanol, tris-carboxyethyl phosphine (TCEP),and/or tributylphosphine (TBP).
  • the cysteine alkylating agents preferably include alkylhalides (e.g. iodoacetic acid, iodoacetamide), vinylpyridine or acrylamide.
  • the proteases or chemical cleavage agents preferably include trypsin, Endoproteinase Lys- C, EndoproteinaseA. sp-N, Endoproteinase GIu-C, pepsin, papain,thermolysin, cyanogen bromide, hydroxylamine hydrochloride ⁇ - [2'-nitrophenylsulfenyl]-3- methyl-3'-bromoindole (BNPS- skatole), iodosobenzoic acid, pentafluoropropionic acidand/or dilute hydrochloric acid.
  • the solvents preferably include urea, guanidine hydrochloride, acetonitrile, methanol and/or water.
  • the invention in another aspect provides methods for providing an internal standard in a mass spectrometer method comprising adding to a sample a predetermined quantity of an isotopically encoded fixed charge derivatized amino acid, peptide or protein described above.
  • Figure 1 Schematic representation of the use of the reagents of the present invention for the 'multiplexed' quantitation of protein abundances observed between different samples in a single neutral loss or precursor ion scan mode MS/MS experiment.
  • Figure 2. Schematic representation of the use of the reagents of the present invention for the 'multiplexed' quantitation of protein abundances observed between different samples in a single product ion scan mode MS/MS experiment.
  • Fiberd-charge includes any charge localised to a specific heteroatom contained within a specific heteroatom contained within the derivatization reagent, by the attachment of any moiety.
  • Fiberd-charge derivatization means the introduction of a fixed-charge as defined above.
  • Protein means any protein, including, but not limited to peptides, enzymes, glycoproteins, hormones, receptors, antigens, antibodies, growth factors, etc., without limitation. Proteins may be endogenous, or produced from other proteins by chemical or proteolytic cleavage. Preferred proteins include those comprised of at least 15-20 amino acid residues.
  • Protein as used herein includes any substance comprising two or more amino acids and includes di-, tri-, oligo and polypeptides etc according to the number of amino acids linked by amide (s) bonds. Peptides may be endogenous, or produced from other peptides or proteins by chemical or proteolytic cleavage. Preferred peptides include those comprised of up to 15-20 amino acid residues.
  • the amino acids are ⁇ -amino acids
  • either the L-optical isomer or the D- optical isomer can be used.
  • the L-isomers are generally preferred.
  • the term "salt thereof includes any suitable counter ion.
  • Non-limiting examples of counter ions are halide ions, for example, chloride, bromide, iodide.
  • the reagents of the present invention may be used for the
  • derivatization of multiple 'diseased' samples is carried out using isotopically distinct labelled alkylation reagents XMi M 2 + , where the Mi 'module' contains an increasing number of isotopically enriched labels (for example 2 H, 13 C, 15 N or 18 O), preferably giving an increase of up to twelve mass units in increments of one, two, three, four or six mass units, compared to the Mi module containing only naturally abundant isotopes, and where the M 2 + module contains only naturally abundant isotopes.
  • the samples are then combined and subjected to tandem mass spectrometry.
  • Quantitative analysis of the relative peptide concentrations between the 'normal' and 'diseased' samples may then be achieved in a single neutral loss or precursor ion scan mode MS/MS experiment by measurement of the abundances of the isotopically distinct Mi and Mj containing product ions formed by the neutral loss of M 2 , or by measurement of the abundances of the M 2 product ion formed by charged loss of M 2 , respectively, via directed fragmentation occurring at the bonds between the Mi and M 2 + modules.
  • the reagents of the present invention may be used for the 'multiplexed' quantitation of protein abundances observed between different samples in a single product ion scan mode MS/MS experiment, using the 'modular' fixed charge stable isotope labelling approach described below (shown in Figure 2 for reaction with alkylation reagents XM 1 M 2 + and XMjM 2 + ).
  • derivatization of a first 'normal' sample is carried out using an isotopically distinct labelled alkylation reagent XMiM 2 + , where the Mi module contains only naturally abundant isotopes and where the M 2 + 'module' is isotopically enriched (for example with 2 H, 13 C, 15 N or O), preferably giving an increase of up to twelve mass units compared to an M 2 + module containing only naturally abundant isotopes.
  • derivatization of multiple 'diseased' samples may be carried out using (i) the isotopically distinct labelled alkylation reagent XMj M 2 + , where the Mi 'module' is isotopically enriched (for example with 2 H, 13 C, 15 N or 18 O), preferably giving an increase of up to twelve mass units compared to an Mi module containing only naturally abundant isotopes while the M 2 + module contains only naturally abundant isotopes, and (ii) the isotopically distinct labelled alkylation reagents XMi M 2 + , where the Mi 'modules' contain an increasing number of isotopically enriched labels (in increments of one, two, three or four mass units, thereby allowing multiplexed analysis of 12, 6, 4 or 3 'diseased' samples', respectively) compared to that used in the 'normal' sample, while the M 2 + 'modules' contain an equally decreasing number of isotopically enriched labels
  • each of the alkylation reagents employed for labelling both 'normal' and 'diseased' samples are then identical, such that the mass difference between 'normal' and 'diseased' derivatized samples is zero.
  • the samples are then combined and subjected to tandem mass spectrometry.
  • Quantitative analysis of the relative peptide concentrations between the 'normal' and 'diseased' samples may then be achieved in a single product ion scan mode MS/MS experiment by measurement of the abundances of the isotopically distinct Mi and Mi containing product ions formed by neutral loss of M 2 or M 2 , or by measurement of the abundances of the isotopically distinct M 2 and M 2 product ions formed by charged loss of M 2 and M 2 , respectively, via directed fragmentation occurring at the bond between the Mi and M 2 + modules.

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Abstract

L'invention concerne des procédés de spectrométrie de masse en tandem pouvant être utilisés dans une analyse de protéome. L'invention concerne également des composés contenant une charge fixe marquée par des isotopes stables modulaires utilisés comme réactifs de spectrométrie de masse (MS) dans des analyses multiplex. L'invention concerne enfin des méthodes permettant de quantifier des acides aminés, des peptides et des protéines à l'aide de techniques de spectrométrie de masse en tandem.
PCT/US2005/028780 2004-08-16 2005-08-12 Reactifs de spectrometrie de masse marques par des isotopes modulaires et procedes d'analyse quantitative d'acides amines, de peptides et de proteines WO2006023398A2 (fr)

Applications Claiming Priority (4)

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AU2004904613A AU2004904613A0 (en) 2004-08-16 "Modular" isotope labelled mass spectrometry reagents and methods for quantitation of amino acids, peptides and proteins
AU2004904613 2004-08-16
US61190504P 2004-09-21 2004-09-21
US60/611,905 2004-09-21

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7964843B2 (en) 2008-07-18 2011-06-21 The George Washington University Three-dimensional molecular imaging by infrared laser ablation electrospray ionization mass spectrometry
US8067730B2 (en) 2007-07-20 2011-11-29 The George Washington University Laser ablation electrospray ionization (LAESI) for atmospheric pressure, In vivo, and imaging mass spectrometry
US8829426B2 (en) 2011-07-14 2014-09-09 The George Washington University Plume collimation for laser ablation electrospray ionization mass spectrometry
US8901487B2 (en) 2007-07-20 2014-12-02 George Washington University Subcellular analysis by laser ablation electrospray ionization mass spectrometry
US9202678B2 (en) 2008-11-14 2015-12-01 Board Of Trustees Of Michigan State University Ultrafast laser system for biological mass spectrometry
EP2529388B1 (fr) * 2010-01-29 2019-10-23 Micromass UK Limited Réactifs de fragmentation pour une spectrométrie de masse

Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003025576A2 (fr) * 2001-09-14 2003-03-27 Xzillion Gmbh & Co. Kg Marqueurs de masse
WO2004046731A2 (fr) * 2002-11-18 2004-06-03 Ludwig Institute For Cancer Research Methode permettant d'analyser des acides amines, des peptides et des proteines

Patent Citations (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003025576A2 (fr) * 2001-09-14 2003-03-27 Xzillion Gmbh & Co. Kg Marqueurs de masse
WO2004046731A2 (fr) * 2002-11-18 2004-06-03 Ludwig Institute For Cancer Research Methode permettant d'analyser des acides amines, des peptides et des proteines

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
LEAVENS WILLIAM J ET AL: "Derivatization for liquid chromatography/electrospray mass spectrometry: synthesis of tris(trimethoxyphenyl)phosphonium compounds and their derivatives of amine and carboxylic acids." RAPID COMMUNICATIONS IN MASS SPECTROMETRY : RCM. 2002, vol. 16, no. 5, 2002, pages 433-441, XP009058402 ISSN: 0951-4198 *
REID G E ET AL: "Selective Identification and Quantitative Analysis of Methionine Containing Peptides by Charge Derivatization and Tandem Mass Spectrometry" JOURNAL OF THE AMERICAN SOCIETY FOR MASS SPECTROMETRY, ELSEVIER SCIENCE INC, US, vol. 16, no. 7, July 2005 (2005-07), pages 1131-1150, XP004976154 ISSN: 1044-0305 *
ROTH K D W ET AL: "CHARGE DERIVATIZATION OF PEPTIDES FOR ANALYSIS BY MASS SPECTROMETRY" MASS SPECTROMETRY REVIEWS, JOHN WILEY AND SONS, NEW YORK, NY, US, vol. 17, no. 4, 1998, pages 255-274, XP000922702 ISSN: 0277-7037 *
THOMPSON A ET AL: "TANDEM MASS TAGS: A NOVEL QUANTIFICATION STRATEGY FOR COMPARATIVE ANALYSIS OF COMPLEX PROTEIN MIXTURES BY MS/MS" ANALYTICAL CHEMISTRY, AMERICAN CHEMICAL SOCIETY. COLUMBUS, US, vol. 75, 1 March 2003 (2003-03-01), pages 1895-1904, XP002328688 ISSN: 0003-2700 *

Cited By (11)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US8067730B2 (en) 2007-07-20 2011-11-29 The George Washington University Laser ablation electrospray ionization (LAESI) for atmospheric pressure, In vivo, and imaging mass spectrometry
US8299429B2 (en) 2007-07-20 2012-10-30 The George Washington University Three-dimensional molecular imaging by infrared laser ablation electrospray ionization mass spectrometry
US8487246B2 (en) 2007-07-20 2013-07-16 The George Washington University Three-dimensional molecular imaging by infrared laser ablation electrospray ionization mass spectrometry
US8487244B2 (en) 2007-07-20 2013-07-16 The George Washington University Laser ablation electrospray ionization (LAESI) for atmospheric pressure, in vivo, and imaging mass spectrometry
US8809774B2 (en) 2007-07-20 2014-08-19 The George Washington University Laser ablation electrospray ionization (LAESI) for atmospheric pressure, in vivo, and imaging mass spectrometry
US8901487B2 (en) 2007-07-20 2014-12-02 George Washington University Subcellular analysis by laser ablation electrospray ionization mass spectrometry
US7964843B2 (en) 2008-07-18 2011-06-21 The George Washington University Three-dimensional molecular imaging by infrared laser ablation electrospray ionization mass spectrometry
US9202678B2 (en) 2008-11-14 2015-12-01 Board Of Trustees Of Michigan State University Ultrafast laser system for biological mass spectrometry
EP2529388B1 (fr) * 2010-01-29 2019-10-23 Micromass UK Limited Réactifs de fragmentation pour une spectrométrie de masse
US8829426B2 (en) 2011-07-14 2014-09-09 The George Washington University Plume collimation for laser ablation electrospray ionization mass spectrometry
US9362101B2 (en) 2011-07-14 2016-06-07 The George Washington University Plume collimation for laser ablation electrospray ionization mass spectrometry

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