US20070048752A1 - Mass tags for quantitative analyses - Google Patents

Mass tags for quantitative analyses Download PDF

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US20070048752A1
US20070048752A1 US11/355,904 US35590406A US2007048752A1 US 20070048752 A1 US20070048752 A1 US 20070048752A1 US 35590406 A US35590406 A US 35590406A US 2007048752 A1 US2007048752 A1 US 2007048752A1
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group
canceled
mass
heterocycloalkyl
heteroaryl
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Inventor
Xiongwei Yan
Pau-Miau Yuan
Sylvia Yuen
Kuo-Liang Hsi
Joe Lam
Krishna Upadhya
Subhakar Dey
Darryl Pappin
Sasi Pillai
Helena Huang
Subhasish Purkayastha
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DH Technologies Pte Ltd
Applied Biosystems Inc
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Applera Corp
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Priority claimed from US11/179,060 external-priority patent/US20060172319A1/en
Application filed by Applera Corp filed Critical Applera Corp
Priority to US11/355,904 priority Critical patent/US20070048752A1/en
Assigned to APPLERA CORPORATION reassignment APPLERA CORPORATION ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: HUANG, HELENA, PAPPIN, DARRYL J., HSI, KUO-LIANG, DEY, SUBHAKAR, LAM, JOE Y., PILLAI, SASI, YUEN, SYLVIA W., PURKAYASTHA, SUBHASISH, UPADHYA, KRISHNA G., YAN, XIONGWEI, YUAN, PAU-MIAU
Priority to CN200780013535.0A priority patent/CN101443664A/zh
Priority to AU2007221425A priority patent/AU2007221425A1/en
Priority to JP2008555337A priority patent/JP2009526998A/ja
Priority to CA002641497A priority patent/CA2641497A1/fr
Priority to PCT/US2007/003990 priority patent/WO2007100506A2/fr
Priority to EP07750802A priority patent/EP1994413A2/fr
Publication of US20070048752A1 publication Critical patent/US20070048752A1/en
Assigned to BANK OF AMERICA, N.A, AS COLLATERAL AGENT reassignment BANK OF AMERICA, N.A, AS COLLATERAL AGENT SECURITY AGREEMENT Assignors: APPLIED BIOSYSTEMS, LLC
Assigned to APPLIED BIOSYSTEMS, INC. reassignment APPLIED BIOSYSTEMS, INC. MERGER (SEE DOCUMENT FOR DETAILS). Assignors: ATOM ACQUISITION CORPORATION
Assigned to APPLIED BIOSYSTEMS, LLC reassignment APPLIED BIOSYSTEMS, LLC MERGER (SEE DOCUMENT FOR DETAILS). Assignors: ATOM ACQUISITION, LLC & APPLIED BIOSYSTEMS INC.
Assigned to APPLIED BIOSYSTEMS INC. reassignment APPLIED BIOSYSTEMS INC. CHANGE OF NAME (SEE DOCUMENT FOR DETAILS). Assignors: APPLERA CORPORATION
Priority to US12/618,452 priority patent/US8501498B2/en
Assigned to DH TECHNOLOGIES PTE. LTD. reassignment DH TECHNOLOGIES PTE. LTD. ASSIGNMENT OF ASSIGNORS INTEREST (SEE DOCUMENT FOR DETAILS). Assignors: APPLIED BIOSYSTEMS, LLC
Assigned to APPLIED BIOSYSTEMS, LLC reassignment APPLIED BIOSYSTEMS, LLC MERGER Assignors: APPLIED BIOSYSTEMS INC.
Assigned to APPLIED BIOSYSTEMS INC. reassignment APPLIED BIOSYSTEMS INC. CHANGE OF NAME Assignors: APPLERA CORPORATION
Assigned to APPLIED BIOSYSTEMS, LLC reassignment APPLIED BIOSYSTEMS, LLC RELEASE BY SECURED PARTY (SEE DOCUMENT FOR DETAILS). Assignors: BANK OF AMERICA, N.A.
Assigned to APPLIED BIOSYSTEMS, INC. reassignment APPLIED BIOSYSTEMS, INC. LIEN RELEASE Assignors: BANK OF AMERICA, N.A.
Abandoned legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D295/00—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms
    • C07D295/16—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms acylated on ring nitrogen atoms
    • C07D295/18—Heterocyclic compounds containing polymethylene-imine rings with at least five ring members, 3-azabicyclo [3.2.2] nonane, piperazine, morpholine or thiomorpholine rings, having only hydrogen atoms directly attached to the ring carbon atoms acylated on ring nitrogen atoms by radicals derived from carboxylic acids, or sulfur or nitrogen analogues thereof
    • C07D295/182—Radicals derived from carboxylic acids
    • C07D295/185—Radicals derived from carboxylic acids from aliphatic carboxylic acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
    • C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
    • C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
    • C07D239/46—Two or more oxygen, sulphur or nitrogen atoms
    • C07D239/52—Two oxygen atoms
    • C07D239/54—Two oxygen atoms as doubly bound oxygen atoms or as unsubstituted hydroxy radicals
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D239/00—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings
    • C07D239/02—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings
    • C07D239/24—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members
    • C07D239/28—Heterocyclic compounds containing 1,3-diazine or hydrogenated 1,3-diazine rings not condensed with other rings having three or more double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
    • C07D239/46—Two or more oxygen, sulphur or nitrogen atoms
    • C07D239/52—Two oxygen atoms
    • C07D239/54—Two oxygen atoms as doubly bound oxygen atoms or as unsubstituted hydroxy radicals
    • C07D239/545—Two oxygen atoms as doubly bound oxygen atoms or as unsubstituted hydroxy radicals with other hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms
    • C07D239/553—Two oxygen atoms as doubly bound oxygen atoms or as unsubstituted hydroxy radicals with other hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, directly attached to ring carbon atoms with halogen atoms or nitro radicals directly attached to ring carbon atoms, e.g. fluorouracil
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/08—Tripeptides
    • C07K5/0802—Tripeptides with the first amino acid being neutral
    • C07K5/0804—Tripeptides with the first amino acid being neutral and aliphatic
    • C07K5/0806—Tripeptides with the first amino acid being neutral and aliphatic the side chain containing 0 or 1 carbon atoms, i.e. Gly, Ala
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
    • C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
    • C07K5/10—Tetrapeptides
    • C07K5/1002—Tetrapeptides with the first amino acid being neutral
    • C07K5/1005—Tetrapeptides with the first amino acid being neutral and aliphatic
    • C07K5/1013—Tetrapeptides with the first amino acid being neutral and aliphatic the side chain containing O or S as heteroatoms, e.g. Cys, Ser
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/06—Linear peptides containing only normal peptide links having 5 to 11 amino acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K7/00—Peptides having 5 to 20 amino acids in a fully defined sequence; Derivatives thereof
    • C07K7/04—Linear peptides containing only normal peptide links
    • C07K7/08—Linear peptides containing only normal peptide links having 12 to 20 amino acids
    • 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
    • G01N33/6848—Methods of protein analysis involving mass spectrometry
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07B—GENERAL METHODS OF ORGANIC CHEMISTRY; APPARATUS THEREFOR
    • C07B2200/00—Indexing scheme relating to specific properties of organic compounds
    • C07B2200/05—Isotopically modified compounds, e.g. labelled
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2458/00—Labels used in chemical analysis of biological material
    • G01N2458/15—Non-radioactive isotope labels, e.g. for detection by mass spectrometry
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/14—Heterocyclic carbon compound [i.e., O, S, N, Se, Te, as only ring hetero atom]
    • Y10T436/142222—Hetero-O [e.g., ascorbic acid, etc.]
    • Y10T436/143333—Saccharide [e.g., DNA, etc.]
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y10—TECHNICAL SUBJECTS COVERED BY FORMER USPC
    • Y10T—TECHNICAL SUBJECTS COVERED BY FORMER US CLASSIFICATION
    • Y10T436/00—Chemistry: analytical and immunological testing
    • Y10T436/24—Nuclear magnetic resonance, electron spin resonance or other spin effects or mass spectrometry

Definitions

  • FIG. 2A is a QTRAPTM 2000 MS analysis of SEQ ID No.: 1 which was alkylated with mass tag (32).
  • FIG. 2B is a QTRAPTM 2000 MS analysis of SEQ ID No.: 2 which was alkylated with mass tag (32).
  • FIG. 3A is a QTRAPTM 2000 MS/MS analysis of SEQ ID No.: 1 which was alkylated with mass tag (32).
  • FIG. 4A is a MS analysis of SEQ ID No.: 1, which was alkylated with mass tag (32), using a 4700 Proteomic Analyzer.
  • FIG. 4B is a MS analysis of SEQ ID No.: 3, which was alkylated with mass tag (32), using a 4700 Proteomic Analyzer.
  • FIG. 6B shows a MRM experiment performed on a QTRAPTM 2000 of two samples, in which one sample has been alkylated with mass tag (32) and the other which has been alkylated with mass tag (33), wherein the ratio of the sample label with mass tag (32) to the sample labeled with mass tag (33) is 1:1.
  • FIG. 7C-1 and 7 C- 2 are mass spectra in the MS/MS mode of the sample in FIG. 6C using a 4700 Proteomic Analyzer.
  • FIG. 8 illustrates exemplary formulas of leaving groups (LG) for the alcohol or thiol group of an active ester wherein each G is independently O or S, typically O.
  • FIG. 12 illustrates the synthesis of Mass Tag (3).
  • FIG. 16 illustrates the synthesis of Mass Tag (12).
  • FIG. 20 illustrates the synthesis of Mass Tag (25).
  • FIG. 24 illustrates the synthesis of FmocGly-Ser(Bzl- 13 C 6 ) (29)
  • FIG. 26 illustrates the synthesis of a labeling reagent/mass tag (XX) ((37a)) comprising a thymine nucleobase.
  • FIG. 27A illustrates a known procedure for the synthesis of 6-methyl uracil from which a labeling reagent (mass tag) comprising the 6-methyl uracil nucleobase ((37b)) can be prepared.
  • a labeling reagent mass tag
  • FIG. 27C illustrates various commercially available isotopically substituted versions of urea that can be used in the preparation of isotopically enriched versions of 6-methyl uracil.
  • FIGS. 28A and 28B illustrate various isotopically enriched versions of 6-methyl uracil that can be prepared using the compounds illustrated in FIGS. 27B and 27C in combination with the procedure illustrated in FIG. 27A .
  • Atoms labeled with * are heavy atom isotopes.
  • FIGS. 29 A-E illustrates various isotopically encoded labeling reagents that can be prepared using the procedures and commercially available compounds illustrated in FIGS. 26, 27A , 27 B, 27 C, and isotopically substituted 6-methyl uracils illustrated in FIGS. 28A and 28B .
  • FIG. 30 illustrates the chemical structures of Mass Tag labeled Glu-Fib peptides (38) and (39).
  • FIG. 31 illustrates the chemical structure of the Mass Tag labeled Glu-Fib peptide (40).
  • FIG. 32 illustrates the synthesis of Mass Tag labeled Glu-Fib peptide (41).
  • FIG. 33 illustrates the synthesis of Mass Tag labeled Glu-Fib peptide (42).
  • FIG. 34 illustrates the synthesis of Mass Tag labeled Glu-Fib peptide (43).
  • FIG. 35 illustrates the syntheses of Mass Tags (44) and (45).
  • FIG. 36 illustrates the synthesis of Mass Tag labeled Glu-Fib peptide (46).
  • FIG. 37 illustrates the chemical structures of Mass Tag labeled Glu-Fib peptide (47) and Mass Tag (48)
  • Labeling reagents and labeled analytes can be represented by a compound of the general formula: or a salt form and/or hydrate form thereof, wherein RG can be a reactive group that reacts with an analyte or the reaction product of the reactive group and the analyte.
  • a labeled analyte therefore can have the general formula:
  • the compound can be tethered to a solid support or moieties for linking it to a solid support via S′.
  • the variables RG, RP, X, LK, S′, r, t, and Y are described in more detail below.
  • Non-limiting examples of sources for the analyte, or the sample comprising the analyte include but are not limited to cells or tissues, or cultures (or subcultures) thereof.
  • Non-limiting examples of analyte sources include, but are not limited to, crude or processed cell lysates (including whole cell lysates), body fluids, tissue extracts or cell extracts.
  • Still other non-limiting examples of sources for the analyte include but are not limited to fractions from a separations process such as a chromatographic separation or an electrophoretic separation.
  • Body fluids include, but are not limited to, blood, urine, feces, spinal fluid, cerebral fluid, amniotic fluid, lymph fluid or a fluid from a glandular secretion.
  • processed cell lysate we mean that the cell lysate is treated, in addition to the treatments needed to lyse the cell, to thereby perform additional processing of the collected material.
  • the sample can be a cell lysate comprising one or more analytes that are peptides formed by treatment of the total protein component of a crude cell lysate with a proteolytic enzyme to thereby digest precursor protein or proteins.
  • analyte can include the original analyte and compounds derived therefrom, unless from the context a clearly contrary meaning is intended.
  • the term analyte can apply to a protein as well as to the peptides derived therefrom by digestion of said protein.
  • fragment refers to a product of fragmentation (noun) or the operation of causing fragmentation (verb).
  • mass refers to the absolute mass as well as to the approximate mass within a range where the use of isotopes of different atom types are so close in mass that they are the functional equivalent for the purpose of balancing the mass of the reporter and/or linker moieties (so that the gross mass of the reporter/linker combination is the same within a set or kit of isobaric or isomeric labeling reagents) whether or not the very small difference in mass of the different isotopes types used can be detected.
  • compounds that are “isotopologues” have the same chemical composition but differ in isotopic composition (number of isotopic substitutions), e.g., the methane isotopologues CH 4 , CH 3 D, and CH 2 D 2 .
  • support refers to any solid phase material upon which a labeling reagent or analyte can be immobilized. Immobilization can, for example, be used to label analytes or be used to prepare a labeling reagent, whether or not the labeling occurs on the support.
  • Solid support encompasses terms such as “resin”, “synthesis support”, “solid phase”, “surface” “membrane” and/or “support”.
  • a solid support can be composed of organic polymers such as polystyrene, polyethylene, polypropylene, polyfluoroethylene, polyethyleneoxy, and polyacrylamide, as well as co-polymers and grafts thereof.
  • a solid support can also be inorganic, such as glass, silica, controlled-pore-glass (CPG), or reverse-phase silica.
  • CPG controlled-pore-glass
  • the configuration of a solid support can be in the form of beads, spheres, particles, granules, a gel, a membrane or a surface. Surfaces can be planar, substantially planar, or non-planar.
  • Solid supports can be porous or non-porous, and can have swelling or non-swelling characteristics.
  • a solid support can be configured in the form of a well, depression or other container, vessel, feature or location.
  • a plurality of solid supports can be configured in an array at various locations, addressable for robotic delivery of reagents, or by detection methods and/or instruments.
  • a “library” is a plurality of different compounds (e.g., labeling reagents, mass tags, labeled analytes, or the like), typically 5, 10, 25, 50, 100, 250 or more different compounds.
  • a library is typically configured for ease of sequential, random, and/or parallel access to one, a plurality, and/or all of the different compounds therein.
  • the plurality of different compounds a library can be in the same flask, or can be immobilized on one or more solid supports, or the like.
  • a library can have at least two different compounds immobilized at different locations, e.g., on physically distinct supports (e.g., beads, spheres, particles, granules, or the like) or at addressable locations on the same support (e.g., as a random or regular array on a solid support).
  • the different compounds in a library can be contacted with other compounds (e.g., one or more analytes can be reacted with a library of labeling reagents) or can be analyzed (e.g., a library of labeled analytes can be analyzed), or the like.
  • a library can comprise a plurality of different labeling reagents, wherein each different labeling reagent can be immobilized at a known address in a regular array on a solid support.
  • the library can be used to label a plurality of separate analyte samples with particular labeling reagents by separately spotting (contacting) the analyte samples to each different immobilized labeling reagent, thereby producing a plurality of labeled analytes.
  • a library of labeling reagents can have a different labeling reagent immobilized on each of a plurality of solid particles. The library can be employed by contacting each solid particle with a different analyte sample, whereby a plurality of labeled analytes are immobilized to the solid particles.
  • an “affinity ligand” refers to a molecule that is a member of a molecular recognition system.
  • molecular recognition systems include, for example, an antigen/antibody, an antigen/antibody fragment, an avidin/biotin, a streptavidin/biotin, a protein A/I g or a lectin/carbohydrate.
  • a halo group refers to —F, —Cl, —Br, or —I.
  • alkyl refers to a straight chained or branched C 1 -C 20 hydrocarbon or a cyclic C 3 -C 20 hydrocarbon that is completely saturated.
  • alkyl refers to a group that may be substituted or unsubstituted.
  • alkyl can be a straight chained or branched C 1 -C 6 hydrocarbon or a cyclic C 3 -C 6 hydrocarbon that is completely saturated.
  • alkenylene refers to an alkenyl group that has two points of attachment to at least two moieties.
  • alkenylene refers to a group that may be substituted or unsubstituted.
  • alkynyl refers to straight chained or branched C 2 -C 20 hydrocarbons or cyclic C 3 -C 20 hydrocarbons that have one or more triple bonds.
  • alkynyl refers to a group that can be substituted or unsubstituted.
  • alkynyl groups can be straight chained or branched C 2 -C 6 hydrocarbon or cyclic C 3 -C 6 hydrocarbons that have one or more triple bonds.
  • azaalkylene refers to a heteroalkylene wherein at least one X′ is —NR—.
  • azaalkylene refers to a group that can be substituted or unsubstituted.
  • heteroaryl refers to an aromatic heterocycle which comprises 1, 2, 3 or 4 heteroatoms independently selected from nitrogen, sulfur and oxygen.
  • heteroaryl refers to a group that may be substituted or unsubstituted.
  • a heteroaryl may be fused to one or two rings, such as a cycloalkyl, a heterocycloalkyl, an aryl, or a heteroaryl.
  • the point of attachment of a heteroaryl to a molecule may be on the heteroaryl, cycloalkyl, heterocycloalkyl or aryl ring, and the heteroaryl group may be attached through carbon or a heteroatom.
  • Heteroaryl groups may be substituted or unsubstituted.
  • heteroaryl groups include imidazolyl, furyl, pyrrolyl, thienyl, oxazolyl, thiazolyl, isoxazolyl, isothiazolyl, thiadiazolyl, oxadiazolyl, pyridinyl, pyrimidyl, pyrazinyl, pyridazinyl, quinolyl, isoquinolinyl, indazolyl, benzoxazolyl, benzisooxazolyl, benzofuryl, benzothiazolyl, indolizinyl, imidazopyridinyl, pyrazolyl, triazolyl, isothiazolyl, oxazolyl, tetrazolyl, benzimidazolyl, benzothiazolyl, benzoisothiazolyl, benzothiadiazolyl, benzoxadiazoly
  • heteroarylene refers to a heteroaryl group that has at least two points of attachment to at least two moieties.
  • heteroarylene refers to a group that may be substituted or unsubstituted.
  • Suitable substituents for an alkyl, alkylene, alkenylene, alkynylene, heteroalkyl, heteroalkylene, azaalkylene, heterocycloalkyl, heterocycloalkylene, azacycloalkylene, aryl, arylene, arylalkyl, arylalkylene, heteroaryl, heteroarylene, azaarylene, heteroarylalkyl, and heteroarylalkylene groups include any substituent that is stable under the reaction conditions used to label analytes with the mass tags of the invention.
  • substituents for an alkyl, an alkylene, alkenylene, alkynylene, heteroalkyl, heteroalkylene, azaalkylene, heterocycloalkyl, heterocycloalkylene, azacycloalkylene, aryl, arylene, arylalkyl, arylalkylene, heteroaryl, heteroarylene, azaarylene, heteroarylalkyl, and heteroarylalkylene include deuterium, an aryl (e.g., phenyl) group, an arylalkyl (e.g., benzyl) group, a nitro group, a cyano group, a halo (e.g., fluorine, chlorine, bromine and iodine) group, a alkyl (e.g., methyl, ethyl, isopropyl, cyclohexyl, etc.) group, a haloalkyl (e.g., trifluoromethyl
  • Suitable substituents for nitrogen atoms having two covalent bonds to other atoms include, for example, optionally substituted alkyl, optionally substituted cycloalkyl, optionally substituted aliphatic, optionally substituted cycloaliphatic, optionally substituted heterocyclic, optionally substituted benzyl, optionally substituted aryl, optionally substituted heteroaryl, —CN, —NO 2 , —OR a , —C(O)R a , —OC(O)R a , —C(O)OR a , —SR a , —S(O)R a , —SO 2 R a , —SO 3 R a , —N(R a R b ), —C(O)N(R a R b ), —C(O)NR a NR b SO 2 R
  • a nitrogen-containing heteroaryl or non-aromatic heterocycle can be substituted with oxygen to form an N-oxide, e.g., as in a pyridyl N-oxide, piperidyl N-oxide, and the like.
  • salt form includes a salt of a compound (labeling reagent), or a mixture of salts of a compound.
  • zwitterionic forms of a compound are also included in the term “salt form.”
  • Salts of mass tags having an amine, or other basic group can be obtained, for example, by reacting with a suitable organic or inorganic acid, such as hydrogen chloride, hydrogen bromide, acetic acid, perchloric acid and the like.
  • a suitable organic or inorganic acid such as hydrogen chloride, hydrogen bromide, acetic acid, perchloric acid and the like.
  • Compounds with a quaternary ammonium group may also contain a counteranion such as chloride, bromide, iodide, acetate, perchlorate and the like.
  • hydrate form comprises any hydration state of a compound or a mixture of more than one hydration state of a compound.
  • a mass tag of the invention can be a hemihydrate, a monohydrate, a dihydrate, etc.
  • variable “RG” of the labeling reagent or reagents used in the method, mixture, kit and/or composition embodiments can be either a reactive group, e.g., an electrophilic group or a nucleophilic group that is capable of reacting with one or more reactive analytes of a sample, or the reaction product of the reactive group and the analyte.
  • the reactive group can be preexisting or it can be prepared in-situ. In some embodiments, in-situ preparation of the reactive group can proceed in the absence of the reactive analyte and in some embodiments, it can proceed in the presence of the reactive analyte.
  • a carboxylic acid group can be modified in-situ with water-soluble carbodiimide (e.g. 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride; EDC) to thereby prepare an electrophilic group that can be reacted with a nucleophilic group such as an amine group.
  • EDC water-soluble carbodiimide
  • activation of the carboxylic acid group of a labeling reagent with EDC can be performed in the presence of an amine (nucleophilic group) containing analyte.
  • the amine (nucleophilic group) containing analyte can also be added after the initial reaction with EDC is performed.
  • the reactive group can be generated in-situ by the in-situ removal of a protecting group. Consequently, any existing or newly created reagent or reagents that can effect the derivatization of analytes by the reaction of nucleophilic groups and/or electrophilic groups are contemplated by the method, mixture, kit and/or composition embodiments of this invention.
  • the reactive group of a labeling reagent can be an amine reactive group.
  • the amine reactive group can be an active ester.
  • Active esters are well known in peptide synthesis and refer to certain esters that are easily reacted with the N- ⁇ amine of an amino acid under conditions commonly used in peptide synthesis.
  • the amine reactive active ester can be an N-hydroxysuccinimidyl ester, a N-hydroxysulfosuccinimidyl ester, a pentafluorophenyl ester, a 2-nitrophenyl ester, a 4-nitrophenyl ester, a 2,4-dinitrophenylester or a 2,4-dihalophenyl ester.
  • FIG. 8 illustrates exemplary formulas of leaving groups (LG) for the alcohol or thiol group of an active ester wherein each G is independently O or S, but typically O. All of these groups are alcohol or thiol groups known to form active esters in the field of peptide chemistry wherein said alcohol or thiol group is displaced by the reaction of the N- ⁇ -amine of the amino acid with the carbonyl carbon of the ester.
  • active ester e.g. N-hydroxysuccinimidyl ester
  • any suitable labelling/tagging reagent described herein could be prepared using well-known procedures (See: Greg T. Hermanson (1996).
  • silyl reactive moieties can be alkyl substituted silyl halides, such as Y-dimethylsilyl, Y-ditriethylsilyl, Y-dipropylsilyl, Y-diisopropylsilyl, etc.) wherein Y is defined below.
  • the labeling reagents of the invention comprise 2 or more RG groups.
  • a labeling reagent of formula RP—X-LK—(Y—RG) y is provided wherein y is 1-3. In some embodiments, y is 2.
  • the reporter moiety of the labeling reagent or reagents used in the method, mixture, kit and/or composition embodiments is a group that has a unique mass (or mass to charge ratio) that can be determined. Accordingly, each reporter of a set can have a unique gross mass. Different reporters can comprise one or more heavy atom isotopes to achieve their unique mass. For example, isotopes of carbon ( 12 C, 13 C and 14 C), nitrogen ( 14 N and 15 N), oxygen ( 16 O and 18 O) or hydrogen (hydrogen, deuterium and tritium) exist and can be used in the preparation of a diverse group of reporter moieties. Examples of stable heavy atom isotopes include 13 C, 15 N, 18 O and deuterium.
  • a unique reporter can be associated with a sample of interest thereby labeling one or multiple analytes of that sample with a labeling reagent comprising the reporter.
  • information about the reporter can be associated with information about one or all of the analytes of the sample.
  • the reporter need not be physically linked to an analyte when the reporter is determined. Rather, the unique gross mass of the reporter can, for example, be determined in a second mass analysis of a tandem mass analyzer, after ions of the labeled analyte are fragmented to thereby produce daughter fragment ions and detectable reporters.
  • the determined reporter can be used to identify the sample from which a determined analyte originated.
  • the amount of the unique reporter can be used to determine the relative or absolute amount (often expressed as a concentration and/or quantity) of analyte in the sample or samples. Therefore information, such as the amount of one or more analytes in a particular sample, can be associated with the reporter moiety that is used to label each particular sample. Where the identity of the analyte or analytes is also determined, that information can be correlated with information pertaining to the different reporters to thereby facilitate the determination of the identity and amount of each labeled analyte in one or a plurality of samples.
  • one or more calibration standards e.g. an analyte labeled with a specific reporter
  • the reporter either comprises a fixed charge or is capable of becoming ionized. Because the reporter either comprises a fixed charge or is capable of being ionized, the labeling reagent might be isolated or used to label the reactive analyte in a salt or zwitterionic form. Ionization of the reporter facilitates its determination in a mass spectrometer. Accordingly, the reporter can be determined as a ion, sometimes referred to as a signature ion. When ionized, the reporter can comprise one or more net positive or negative charges. Thus, the reporter can comprise one or more acidic groups or basic groups since such groups can be easily ionized in a mass spectrometer.
  • the reporter can comprise one or more basic nitrogen atoms (positive charge) or one or more ionizable acidic groups such as a carboxylic acid group, sulfonic acid group or phosphoric acid group (negative charge).
  • the reporter can comprise a substituted or unsubstituted benzyl ion.
  • the reporter can be selected so that it does not substantially sub-fragment under conditions typical for the analysis of the analyte.
  • the reporter can be chosen so that it does not substantially sub-fragment under conditions of dissociative energy applied to cause fragmentation of both bonds X and Y of at least a portion of selected ions of a labeled analyte in a mass spectrometer.
  • does not substantially sub-fragment we mean that fragments of the reporter are difficult or impossible to detect above background noise when applied to the successful analysis of the analyte of interest.
  • the gross mass of a reporter can be intentionally selected to be different as compared with the mass of the analyte sought to be determined or any of the expected fragments of the analyte.
  • the reporter's gross mass can be chosen to be different as compared with any naturally occurring amino acid or peptide, or expected fragments thereof. This can facilitate analyte determination since, depending on the analyte, the lack of any possible components of the sample having the same coincident mass can add confidence to the result of any analysis. Examples of mass ranges where little background can be expected for peptides can be found in Table 1.
  • the gross mass of a reporter can be less than 250 Daltons. Such a small molecule can be easily determined in the second mass analysis, free from other components of the sample having the same coincident mass in the first mass analysis.
  • the second mass analysis can be performed, typically in a tandem mass spectrometer, on selected ions that are determined in the first mass analysis. Because ions of a particular mass to charge ratio can be specifically selected out of the first mass analysis for possible fragmentation and further mass analysis, the non-selected ions from the first mass analysis are not carried forward to the second mass analysis and therefore do not contaminate the spectrum of the second mass analysis.
  • the sensitivity of a mass spectrometer and the linearity of the detector (for purposes of quantitation) can be quite robust in this low mass range. Additionally, the present state of mass spectrometer technology can allow for baseline mass resolution of less than one Dalton in this mass range. These factors may prove to be useful advancements to the state of the art.
  • the linker moiety represented by LK, LK 1 , LK 2 , LK 3 , LK 4 , LK 5 and LK 6 of the compounds used with the method, mixture, kit and/or composition embodiments links the reporter to the analyte or the reporter to the reactive group depending on whether or not a reaction with the analyte has occurred.
  • the linker can be selected to produce a neutral species when both bonds X and Y are fragmented (i.e. undergoes neutral loss upon fragmentation of both bonds X and Y).
  • the linker can be a very small moiety such as a carbonyl or thiocarbonyl group.
  • the linker can comprise at least one heavy atom isotope and comprise the formula: wherein each R 1 is the same or different and is an alkyl group comprising one to eight carbon atoms which may optionally contain a heteroatom or a substituted or unsubstituted aryl group wherein the carbon atoms of the alkyl and aryl groups independently comprise linked hydrogen, deuterium and/or fluorine atoms.
  • the linker can be a larger moiety such as amino acid or heteroalkyl.
  • the linker can be a polymer or a biopolymer (e.g., a peptide).
  • the linker can be designed to sub-fragment when subjected to dissociative energy levels; including sub-fragmentation to thereby produce one or more neutral fragments of the linker. In some embodiments, only neutral fragments are produced from the linker.
  • the linker moiety can compensate for the difference in gross mass between reporters of labeled analytes from different samples wherein the unique gross mass of the reporter correlates with the sample from which the labeled analyte originated and the aggregate gross mass of the reporter-linker combination is the same for each labeled analyte of a sample mixture regardless of the sample from which it originated.
  • the gross mass of identical analytes in two or more different samples can have the same gross mass when labeled and then mixed to produce a sample mixture.
  • the labeled analytes, or labeling reagent (e.g., mass tags) of a set and/or kit for labeling the analytes can be isomers or isobars.
  • ions of a particular mass to charge ratio taken from the sample mixture
  • selected ions i.e. selected ions
  • identical analytes from the different samples that make up the sample mixture are represented in the selected ions in proportion to their respective concentration and/or quantity in the sample mixture.
  • the linker not only links the reporter to the analyte, it also can serve to compensate for the differing masses of the unique reporter moieties to thereby harmonize the gross mass of the reporter-linker combination in the labeled analytes of the various samples.
  • the linker can act as a mass balance for the reporter in the labeling reagents such that the aggregate gross mass of the reporter-linker combination is the same for all reagents of a set or kit, the greater the number of atoms in the linker, the greater the possible number of different isomeric/isobaric labeling reagents of a set and/or kit.
  • the total number of labeling reagents of a set and/or kit can be two, three, four, five, six, seven, eight, nine, ten or more.
  • the diversity of the labeling reagents of a set or kit is limited only by the number of atoms of the reporter and linker moieties, the heavy atom isotopes available to substitute for the light isotopes and the various synthetic configurations in which the isotopes can be synthetically placed.
  • numerous isotopically enriched basic starting materials are readily available from manufacturers such as Cambridge Isotope Laboratories and Isotec.
  • Such isotopically enriched basic starting materials can be used in the synthetic processes used to produce sets of isobaric and isomeric labeling reagents or be used to produce the isotopically enriched starting materials that can be used in the synthetic processes used to produce sets of isobaric and isomeric labeling reagents.
  • Some examples of the preparation of isobaric labeling reagents suitable for use in a set of labeling reagents can be found in the Examples section, below.
  • the labeling reagents described herein comprise reporters and linkers that are linked through the bond X.
  • the reporter-linker combination can be identical in gross mass for each member of a set and/or kit of labeling reagents.
  • bond X of the reporter-linker combination of the labeling reagents can be designed to fragment, in at least a portion of the selected ions, when subjected to dissociative energy levels thereby releasing the reporter from the analyte. Accordingly, the gross mass of the reporter (as a m/z ratio) and its intensity can be observed directly in MS/MS analysis.
  • tandem mass spectrometers and other mass spectrometers that have the ability to select and fragment molecular ions. Tandem mass spectrometers (and to a lesser degree single-stage mass spectrometers) have the ability to select and fragment molecular ions according to their mass-to-charge (m/z) ratio, and then record the resulting fragment (daughter) ion spectra. More specifically, daughter fragment ion spectra can be generated by subjecting selected ions to dissociative energy levels (e.g. collision-induced dissociation (CID)).
  • CID collision-induced dissociation
  • mass spectrometers may be used in conjunction with a variety of ionization sources, including, but not limited to, electrospray ionization (ESI) and matrix-assisted laser desorption ionization (MALDI).
  • Ionization sources can be used to generate charged species for the first mass analysis where the analytes do not already possess a fixed charge.
  • Additional mass spectrometry instruments and fragmentation methods include post-source decay in MALDI-MS instruments and high-energy CID using MALDI-TOF (time of flight)-TOF MS.
  • MALDI-TOF time of flight
  • bonds can fragment as a result of the processes occurring in a mass spectrometer.
  • bond fragmentation can be induced in a mass spectrometer by subjecting ions to dissociative energy levels.
  • the dissociative energy levels can be produced in a mass spectrometer by collision-induced dissociation (CID).
  • CID collision-induced dissociation
  • the process of fragmenting bonds by collision-induced dissociation involves increasing the kinetic energy state of selected ions to a point where bond fragmentation occurs.
  • kinetic energy can be transferred by collision with an inert gas (such as nitrogen, helium or argon) in a collision cell.
  • the amount of kinetic energy that can be transferred to the ions is proportional to the number of gas molecules that are allowed to enter the collision cell. When more gas molecules are present, a greater amount of kinetic energy can be transferred to the selected ions, and less kinetic energy is transferred when there are fewer gas molecules present.
  • the dissociative energy level in a mass spectrometer can be controlled. It is also well accepted that certain bonds are more labile than other bonds. The lability of the bonds in an analyte or the reporter-linker moiety depends upon the nature of the analyte or the reporter-linker moiety. Accordingly, the dissociative energy levels can be adjusted so that the analytes and/or the labels (e.g. the reporter-linker combinations) can be fragmented in a manner that is determinable.
  • dissociative energy can be applied to ions that are selected/isolated from the first mass analysis.
  • the extracted ions can be subjected to dissociative energy levels and then transferred to a second mass analyzer.
  • the selected ions can have a selected mass to charge ratio.
  • the mass to charge ratio can be within a range of mass to charge ratios depending upon the characteristics of the mass spectrometer.
  • collision induced dissociation the ions can be transferred from the first to the second mass analyzer by passing them through a collision cell where the dissociative energy can be applied to thereby produce fragment ions.
  • the ions sent to the second mass analyzer for analysis can include all, some, or a portion, of the remaining (unfragmented) selected ions, as well as reporter ions (signature ions) and daughter fragment ions of the labeled analyte.
  • analytes can be determined based upon daughter-ion fragmentation patterns that are analyzed by computer-assisted comparison with the spectra of known or “theoretical” analytes.
  • the daughter fragment ion spectrum of a peptide ion fragmented under conditions of low energy CID can be considered the sum of many discrete fragmentation events.
  • the common nomenclature differentiates daughter fragment ions according to the amide bond that breaks and the peptide fragment that retains charge following bond fission. Charge-retention on the N-terminal side of the fissile amide bond results in the formation of a b-type ion.
  • experimental peptide CID spectra are matched or correlated with ‘theoretical’ daughter fragment ion spectra computationally generated from peptide sequences obtained from protein or genome sequence databases.
  • the match or correlation is based upon the similarities between the expected mass and the observed mass of the daughter fragment ions in MS/MS mode.
  • the potential match or correlation is scored according to how well the experimental and ‘theoretical’ fragment patterns coincide.
  • the constraints on databases searching for a given peptide amino acid sequence are so discriminating that a single peptide CID spectrum can be adequate for identifying any given protein in a whole-genome or expressed sequence tag (EST) database.
  • EST expressed sequence tag
  • daughter fragment ion analysis of MS/MS spectra can be used not only to determine the analyte of a labeled analyte, it can also be used to determine analytes from which the determined analyte originated.
  • identification of a peptide in the MS/MS analysis can be can be used to determine the protein from which the peptide was cleaved as a consequence of an enzymatic digestion of the protein. It is envisioned that such analysis can be applied to other analytes, such as oligonucleotides.
  • X is a bond between an atom of the reporter and an atom of the linker.
  • Y is a bond between an atom of the linker and an atom of either the reactive group or, if the labeling reagent has been reacted with a reactive analyte, the analyte.
  • Bonds X and Y of the various labeling reagents i.e. RP—X-LK—Y—RG
  • Bonds X and Y of the various labeling reagents i.e. RP—X-LK—Y—RG
  • the dissociative energy level can be adjusted in a mass spectrometer so that both bonds X and Y fragment in at least a portion of the selected ions of the labeled analytes (i.e. RP—X-LK—Y-Analyte). Fragmentation of bond X releases the reporter from the analyte so that the reporter can be determined independently from the analyte. Fragmentation of bond Y releases the reporter-linker combination from the analyte, or the linker from the analyte, depending on whether or not bond X has already been fragmented. Bond Y can be more labile than bond X. Bond X can be more labile than bond Y. Bonds X and Y can be of the same relative lability.
  • bond X can be more labile than bond Y. In some embodiments, bond X cleaves and bond Y remains intact. In still other embodiments, bond X cleaves and bond Y cleaves.
  • bonds X and Y can also exist such that fragmentation of bond Y results in the fragmentation of bond X, and vice versa. In this way, both bonds X and Y can fragment essentially simultaneously such that no substantial amount of analyte, or daughter fragment ion thereof, comprises a partial label in the second mass analysis.
  • substantially amount of analyte we mean that less than 25%, and preferably less than 10%, partially labeled analyte can be determined in the MS/MS spectrum.
  • Analytes can be labeled by reacting a functional group of the analyte with the reactive group (RG) of the labeling reagent.
  • the functional group on the analyte can be one of an electrophilic group or a nucleophilic group and the functional group (i.e. the RG or reactive group) of the labeling reagent can be the other of the electrophilic group or a nucleophilic group.
  • the electrophilic group and nucleophilic group can react to form a covalent link between the analyte and the labeling reagent.
  • the labeling reaction can take place in solution.
  • one of the analyte or the labeling reagent can be support bound.
  • the labeling reaction can sometimes be performed in aqueous conditions.
  • Aqueous conditions can be selected for the labeling of biomolecules such as proteins, peptides, nucleotides and oligonucleotides.
  • the labeling reaction can sometimes be performed in organic solvent or a mixture of organic solvents.
  • Organic solvents can be selected for analytes that are small molecules. Mixtures of water and organic solvent or organic solvents can be used across a broad range. For example, a solution of water and from about 60 percent to about 95 percent organic solvent or solvents (v/v) can be prepared and used for labeling the analyte.
  • a solution of water and from about 65 percent to about 80 percent organic solvent or solvents can be prepared and used for labeling the analyte.
  • organic solvents include N,N′-dimethylformamide (DMF), acetonitrile (ACN), and alcohol such as methanol, ethanol, propanol and/or butanol.
  • the pH can be modulated.
  • the pH can be in the range of 4-10.
  • the pH can be outside this range.
  • suitable bases include N-methylmorpholine, triethylamine and N,N-diisopropylethylamine.
  • the proteolytic enzyme trypsin is a serine protease that cleaves peptide bonds between lysine or arginine and an unspecific amino acid to thereby produce peptides that comprise an amine terminus (N-terminus) and lysine or arginine carboxyl terminal amino acid (C-terminus).
  • N-terminus amine terminus
  • C-terminus lysine or arginine carboxyl terminal amino acid
  • the free amine termini of a peptide can be a good nucleophile that facilitates its labeling.
  • Other exemplary proteolytic enzymes include papain, pepsin, ArgC, LysC, V8 protease, AspN, pronase, chymotrypsin and carboxypeptidase C.
  • the sequence of peptides that are produced from degradation of a protein of known sequence can be predicted. With this information, “theoretical” peptide information can be generated. A determination of the “theoretical” peptide fragments in computer assisted analysis of daughter fragment ions (as described above) from mass spectrometry analysis of an actual sample can therefore be used to determine one or more peptides or proteins in one or more unknown samples.
  • TCEP tris[2-carboxyethyl]phosphine
  • MMTS methyl methanethiosulfonate
  • sample processing can include the immobilization of the analytes or analyte precursors to a solid support, whether labeled with a labeling reagent or not.
  • immobilization can facilitate reducing sample complexity.
  • immobilization can facilitate analyte labeling.
  • immobilization can facilitate analyte precursor labeling.
  • immobilization can facilitate selective labeling of a fraction of sample components comprising a certain property (e.g. they comprise or lack cysteine moieties). The immobilization can facilitate two or more of the foregoing.
  • a sample mixture comprising differentially labeled analytes from different samples can be prepared.
  • differentially labeled we mean that each of the labels comprises a unique property that can be identified (e.g. comprises a unique reporter moiety that produces a unique “signature ion” in MS/MS analysis).
  • components of the sample mixture can be separated and mass analysis performed on only a fraction of the sample mixture. In this way, the complexity of the analysis can be substantially reduced since separated analytes can be individually analyzed for mass thereby increasing the sensitivity of the analysis process.
  • the analysis can be repeated one or more time on one or more additional fractions of the sample mixture to thereby allow for the analysis of all fractions of the sample mixture.
  • Separation conditions under which identical analytes that are differentially labeled co-elute at a concentration, or in a quantity, that is in proportion to their abundance in the sample mixture can be used to determine the amount of each labeled analyte in each of the samples that comprise the sample mixture provided that the amount of each sample added to the sample mixture is known. Accordingly, in some embodiments, separation of the sample mixture can simplify the analysis whilst maintaining the correlation between signals determined in the mass analysis (e.g. MS/MS analysis) with the amount of the differently labeled analytes in the sample mixture.
  • the separation can be performed by chromatography.
  • chromatography liquid chromatography/mass spectrometry (LC/MS) can be used to effect such a sample separation and mass analysis.
  • LC/MS liquid chromatography/mass spectrometry
  • any chromatographic separation process suitable to separate the analytes of interest can be used.
  • the chromatographic separation can be normal phase chromatography, reversed-phase chromatography, ion-exchange chromatography, size exclusion chromatography or affinity chromatorgraphy.
  • the separation can be performed electrophoretically.
  • electrophoretic separations techniques that can be used include, but are not limited to, 1D electrophoretic separation, 2D electrophoretic separation and/or capillary electrophoretic separation.
  • An isobaric labeling reagent or a set of reagents can be used to label the analytes of a sample.
  • Isobaric labeling reagents are particularly useful when a separation step is performed because the isobaric labels of a set of labeling reagents are structurally and chemically indistinguishable (and can be indistinguishable by gross mass until fragmentation removes the reporter from the analyte).
  • all analytes of identical composition that are labeled with different isobaric labels can chromatograph in exactly the same manner (i.e. co-elute).
  • the eluent from the separation process can comprise an amount of each isobarically labeled analyte that is in proportion to the amount of that labeled analyte in the sample mixture. Furthermore, from the knowledge of how the sample mixture was prepared (portions of samples, an other optional components (e.g. calibration standards) added to prepare the sample mixture), it is possible to relate the amount of labeled analyte in the sample mixture back to the amount of that labeled analyte in the sample from which it originated.
  • the labeling reagents can also be isomeric. Although isomers can sometimes be chromatographically separated, there are circumstances, that are condition dependent, where the separation process can be operated to co-elute all of the identical analytes that are differentially labeled wherein the amount of all of the labeled analytes exist in the eluent in proportion to their concentration and/or quantity in the sample mixture.
  • isobars differ from isomers in that isobars are structurally and chemically indistinguishable compounds (except for isotopic content and/or distribution) of the same gross mass (See for example, FIG. 1 ) whereas isomers are structurally and/or chemically distinguishable compounds of the same gross mass.
  • the labeling of the analytes of a sample can be performed prior to performing sample processing steps. In some embodiments, the labeling of analytes can be performed amongst other sample processing steps. In some embodiments, the labeling of analytes is the last step of sample processing and/or immediately precedes the preparation of a sample mixture.
  • proteomic analysis as a non-limiting example, there are at least several possible workflows that might be used. To aid in understanding of the following discussion a distinction is sometimes made between the precursor protein and the analyte peptide. However, it should be understood that either, or both, of the protein and the peptide can be considered analytes as described herein.
  • the precursor proteins can be digested to peptide analytes that can thereafter be labeled with labeling reagent.
  • the precursor proteins can be labeled with the labeling reagent and then digested to labeled peptide analytes.
  • the precursor proteins can be captured on a solid support, digested and then the support bound peptides can be labeled.
  • the flow through peptides can also labeled.
  • the precursor proteins can be captured on a solid support, labeled and then the support bound protein can be digested to produce labeled peptides.
  • the flow through peptides can also analyzed.
  • additional sample processing e.g. separation steps
  • MS analysis e.g. separation steps
  • the relative quantitation of differentially labeled identical analytes of a sample mixture is possible.
  • Relative quantitation of differentially labeled identical analytes is possible by comparison of the relative amounts of reporter (e.g. intensity, area and/or height of the peak reported) that are determined in the second mass analysis for a selected, labeled analyte observed in a first mass analysis.
  • reporter e.g. intensity, area and/or height of the peak reported
  • each reporter can be correlated with information for a particular sample used to produce a sample mixture
  • the relative amount of that reporter, with respect to other reporters observed in the second mass analysis is the relative amount of that analyte in the sample mixture.
  • the relative amount of the analyte in each sample used to prepare the sample mixture can be back calculated based upon the relative amounts of reporter observed for the ions of the labeled analyte selected from the first mass analysis. This process can be repeated for all of the different labeled analytes observed in the first mass analysis. In this way, the relative amount (often expressed in terms of concentration and/or quantity) of each reactive analyte, in each of the different samples used to produce the sample mixture, can be determined.
  • absolute quantitation of analytes can be determined.
  • a known amount of one or more differentially labeled analytes (the calibration standard or calibration standards) can be added to the sample mixture.
  • the calibration standard can be an expected analyte that is labeled with an isomeric or isobaric label of the set of labels used to label the analytes of the sample mixture provided that the reporter for the calibration standard is unique as compared with any of the samples used to form the sample mixture.
  • corrections to the intensity (or area or height) of the reporter ions can be made, as appropriate, for any naturally occurring, or artificially created, isotopic abundance within the reporters.
  • a more sophisticated example of these types of corrections can also be found in copending and co-owned U.S. Provisional Patent Application Ser. No. 60/524,844, entitled: “Method and Apparatus For De-Convoluting A Convoluted Spectrum”, filed on Nov. 26, 2003.
  • the more care taken to accurately quantify the intensity of each reporter the more accurate will be the relative and absolute quantification of the analytes in the original samples.
  • sample mixtures can be analyzed for the amount of individual analytes in one or more samples.
  • the amount (often expressed in concentration and/or quantity) of those analytes can be determined for the samples from which the sample mixture was comprised. Because the sample processing and mass analyses can be performed rapidly, these methods can be repeated numerous times so that the amount of many differentially labeled analytes of the sample mixture can be determined with regard to their relative and/or absolute amounts in the sample from which the analyte originated.
  • proteomic analysis can be viewed as an experimental approach to describe the information encoded in genomic sequences in terms of structure, function and regulation of biological processes. This may be achieved by systematic analysis of the total protein component expressed by a cell or tissue. Mass spectrometry, used in combination with the method, mixture, kit and/or composition embodiments of this invention is one possible tool for such global protein analysis.
  • kits include one or more of kits, arrays, libraries, mixtures, compounds, labeled analytes, and methods as described in the following sections.
  • RG can refer to the reaction product of an analyte with the nucleophilic groups or electrophilic groups defined for RG, wherein each compound can be a labeled analyte.
  • a plurality of such compounds can be a mixture of labeled analytes, a library of labeled analytes, and the like.
  • the reaction product of an analyte with the nucleophilic groups or electrophilic groups defined for RG is represented by -Analyte.
  • the cleavable linker represented by S′ can be coupled to the solid support at a separate array location on the solid support, the solid support comprising polystyrene, polyethylene, polypropylene, polyfluoroethylene, polyethyleneoxy, polyacrylamide, glass, silica, controlled-pore-glass (CPG), or reverse phase silica, the substrate in the form of a gel, a membrane or a surface, whereby the kit is an array library of the different compounds.
  • the solid support comprising polystyrene, polyethylene, polypropylene, polyfluoroethylene, polyethyleneoxy, polyacrylamide, glass, silica, controlled-pore-glass (CPG), or reverse phase silica, the substrate in the form of a gel, a membrane or a surface, whereby the kit is an array library of the different compounds.
  • RG can be a nucleophilic group or an electrophilic group, whereby the kit is an array library of labeling reagents; or RG can be a reaction product of an analyte with a nucleophilic group or an electrophilic group; whereby the kit is an array library of labeled analytes.
  • the cleavable linker represented by S′ can be coupled to the solid support at a separate solid support bead, sphere, particle, or granule, the solid support comprising polystyrene, polyethylene, polypropylene, polyfluoroethylene, polyethyleneoxy, polyacrylamide, glass, silica, controlled-pore-glass (CPG), or reverse phase silica, whereby the kit is a solid support library of the different compounds.
  • RG can be a nucleophilic group or an electrophilic group, whereby the kit is a solid support library of labeling reagents; or RG can be a reaction product of an analyte with a nucleophilic group or an electrophilic group; whereby the kit is a solid support library of labeled analytes.
  • the cleavable linker represented by S′ can be coupled to a different affinity ligand selected from the group consisting of an antigen, an antibody, an antibody fragment, an avidin, biotin, streptavidin, a protein A, a lectin, and a carbohydrate, whereby the kit is an affinity ligand library.
  • RG can be a nucleophilic group or an electrophilic group, whereby the kit is an affinity ligand library of labeling reagents; or RG can be a reaction product of an analyte with a nucleophilic group or an electrophilic group; whereby the kit is an affinity ligand library of labeled analytes.
  • compounds in the kits, arrays, libraries, labeled analyte mixtures, and methods, and the isotopically enriched compound can be further represented by one of Structural Formulas I-S′to VI-S′ or I to VI: or isotopologues thereof.
  • the variables r, s, S′, X, and X are as described above or as further detailed below and can be subject to the corresponding provisos above.
  • LK 5 can be a linking moiety represented by structural formula E (it will be understood that the points of attachment to the remainder of the labeling reagent are identified in the structure by the wavy line): RP 5 —X-LK 5 —Y—RG V wherein:
  • At least one compound can be represented by structural formula D:
  • RP 1 can be a reporter group represented by structural formula A (it will be understood that the point of attachment to the remainder of the labeling reagent is identified in the structure by the wavy line):
  • At least one of RP 1 and LK 1 can be isotopically enriched with one or more heavy atom isotopes, for example, RP 1 .
  • both RP 1 and LK 1 can each be isotopically enriched with one or more heavy atom isotopes.
  • each of RP 1 and LK 1 comprise at least two heavy atom isotopes.
  • each of RP 1 and LK 1 each comprise at least three heavy atom isotopes.
  • n is 2 whereby Ring A can be a six membered ring.
  • either of the Z groups in the ortho or para positions of Ring A can be C-T-R 3 .
  • either of the Z groups in the ortho or para positions of Ring A can be C—NHC(O)—R 3 or C—NHSO 2 —R 3 and each R 3 can be independently an optionally substituted alkyl group.
  • n is 2 and each Z is independently CH or CR 2 , and thus RP 1 can be represented by Structural Formula A-1:
  • At least one atom in formula A is isotopically enriched with a heavy atom isotope.
  • one of the methylene units of LK 1 can be replaced by an optionally substituted azaalkylene, azacycloalkylene, or azaarylene.
  • At least one compound can be represented by a structural formula selected from: wherein the symbol “*” next to a carbon atom can indicate that the carbon can be a 13 C isotope and the symbol “*” next to a nitrogen atom can indicates that the nitrogen can be a 15 N isotope.
  • At least one compound can be represented by structural formula I-1:
  • At least one compound can be represented by a structural formula selected from:
  • At least one compound can be represented by a structural formula selected from: wherein R 8 can be a valence bond, an alkylene, or —(CH 2 ) s —(O—CH 2 CH 2 ) p —(CH 2 ) s —; p can be 1, 2, 3, or 4; and each s can be independently 0, 1, 2, or 3.
  • the compound can be represented by structural formula II or II-S′, wherein RP 2 can be a reporter group represented by structural formula B
  • At least one of RP 2 and LK 2 can be isotopically enriched with one or more heavy atom isotopes, for example, RP 2 .
  • both RP 2 and LK 2 can each be isotopically enriched with one or more heavy atom isotopes.
  • each of RP 2 and LK 2 comprise at least two heavy atom isotopes.
  • each of RP 2 and LK 2 comprise at least three heavy atom isotopes.
  • LK 2 can be as defined for the various embodiments of LK 1 .
  • the reporter group is of formula B wherein n is 1 and each W is O.
  • a reporter group of formula B-2 is provided: wherein each R 2 is as defined above and herein.
  • the compound can be represented by structural formula II-d:
  • the compound can be represented by structural formula II-e:
  • At least one atom in RP 1 is isotopically enriched with a heavy atom isotope.
  • the compound can be represented by structural formula III or III-S′, wherein RP 3 can be a reporter group represented by structural formula C:
  • At least one of RP 3 and LK 3 can be isotopically enriched with one or more heavy atom isotopes, for example, RP 3 .
  • both RP 3 and LK 3 can each be isotopically enriched with one or more heavy atom isotopes.
  • each of RP 3 and LK 3 comprise at least two heavy atom isotopes.
  • each of RP 3 and LK 3 each comprise at least three heavy atom isotopes.
  • LK 3 can be as defined for the various embodiments of LK 1 .
  • the reporter group is of formula C wherein R x and R y are taken together to form Ring C′.
  • a reporter group of formula C′′ is provided: wherein q is 0-6 and Ring C is as defined as above and herein.
  • the reporter group can be represented by C wherein Ring C′′ is heterocycloalkyl and q is 2, 3 or 4.
  • a reporter group of formula C-1 is provided:
  • At least one atom in formula C-1 is isotopically enriched with a heavy atom isotope. Is some embodiments, at least one atom in formula C-1 is isotopically enriched with two heavy atom isotopes.
  • the above-described structures for the reporter group C require that the linker not be a substituted or unsubstituted acetic acid moiety that is N-alkylated to the nitrogen atom through bond X.
  • the compound can be represented by structural formula III-c: wherein q can be an integer from 0 to 6 and LK can contain a carbonyl.
  • the compound can be represented by a structural formula selected from:
  • RP 4 and LK 4 can be each independently a heteroaryl or heterocycloalkyl, or a linear or branched aliphatic or heteroaliphatic group substituted or interrupted with a heteroaryl or heterocycloalkyl, wherein
  • At least one of RP 4 and LK 4 can be isotopically enriched with one or more heavy atom isotopes, for example, RP 4 .
  • both RP 4 and LK 4 can each be isotopically enriched with one or more heavy atom isotopes.
  • each of RP 4 and LK 4 comprise at least two heavy atom isotopes.
  • each of RP 4 and LK 4 each comprise at least three heavy atom isotopes.
  • At least one of RP 4 or LK 4 can comprise an optionally substituted piperazinyl, or a linear or branched aliphatic or heteroaliphatic group substituted or interrupted with piperazinyl, or in some embodiments, RP 4 can be an optionally substituted piperazinyl, for example, N-methyl piperazinyl.
  • At least one of RP 4 or LK 4 can comprise an optionally substituted nucleobase (e.g., optionally substituted purinyl or pyrimidinyl), or a linear or branched aliphatic or heteroaliphatic group substituted or interrupted with an optionally substituted nucleobase.
  • LK 4 can be an optionally substituted nucleobase, or a linear or branched aliphatic or heteroaliphatic group substituted or interrupted with an optionally substituted nucleobase.
  • the compound can be represented by a structural formula selected from: A bond drawn across a ring, as above, indicates that the bond can be attached to any substitutable atom in that ring; a bond drawn across two rings can be attached to any substitutable atom in either of those two rings.
  • the group R 5 can be —C(J) 2 -C(O)—, —C(J) 2 -C(S)—, —C(J) 2 -C(NH)—, or —C(J) 2 -C(NR z )—, wherein R z is an alkyl group comprising one to eight carbon atoms that may optionally contain a heteroatom or optionally substituted aryl group wherein the carbon atoms of the alkyl and aryl groups independently comprise linked hydrogen, deuterium and/or fluorine atoms; and each J is the same or different and is H, deuterium (D), Rz, ORz, SRz, NHRz, N(Rz) 2 , fluorine, chlorine, bromine or iodine.
  • R z is an alkyl group comprising one to eight carbon atoms that may optionally contain a heteroatom or optionally substituted aryl group wherein the carbon atoms of the alkyl and aryl groups independently
  • R 6 and R7 can each independently be alkyl, alkenyl, alkynyl, aryl, heteroaryl, arylalkyl, heteroarylalkyl, heteroalkyl, heterocycloalkyl, —R 3 , -T-R 3 , ribose, deoxyribose, or phosphate, wherein each R 3 is independently hydrogen, deuterium, alkyl, alkenyl, alkynyl, aryl, arylalkyl, heteroalkyl, heterocycloalkyl, heteroaryl, or heteroaralkyl.
  • the analyte to be determined can be labeled by reacting the analyte with a disclosed compound, e.g., the compounds as represented by one of Structural Formulas I-S′ to IV-S′ or I to IV, wherein RG is a reagtive group that is a nucleophilic group or electrophilic group.
  • a disclosed compound e.g., the compounds as represented by one of Structural Formulas I-S′ to IV-S′ or I to IV, wherein RG is a reagtive group that is a nucleophilic group or electrophilic group.
  • the labeled analyte, the analyte itself, one or more fragments of the analyte and/or fragments of the label can be determined by mass analysis.
  • analytes from different samples can be differentially isotopically labeled (i.e. isotopically coded) with unique labels that are chemically isomeric or isobaric (have equal mass) and that identify the sample from which the analyte originated.
  • the differentially labeled analytes are not distinguished in MS mode of a mass spectrometer because they all have identical (gross) mass to charge ratios.
  • dissociative energy levels such as through collision induced dissociation (CID)
  • the labels can fragment to yield unique reporters that can be resolved by mass (mass to charge ratio) in a mass spectrometer.
  • the relative amount of reporter observed in the mass spectrum can correlate with the relative amount of a labeled analyte in the sample mixture and, by implication, the amount of that analyte in a sample from which it originated.
  • the relative intensities of the reporters i.e. signature ions
  • the reporter information can be used to measure the relative amount of an analyte or analytes in two or more different samples that were combined to form a sample mixture.
  • absolute amounts (often expressed as concentration and/or quantity) of an analyte or analytes in two or more samples can be derived if calibration standards for the each analyte, for which absolute quantification is desired, are incorporated into the sample mixture.
  • this invention pertains to a method comprising reacting each of two or more samples, each sample containing one or more reactive analytes, with a different labeling reagent of a set of labeling reagents wherein the different labeling reagents of the set each comprise the formula: RP—X-LK—Y—RG. Consequently, one or more analytes of each sample are labeled with the moiety “RP—X-LK—Y—” by reaction of a nucleophilic group or electrophilic group of the analyte with the electrophilic or nucleophilic reactive group (RG), respectively, of the different labeling reagents.
  • RG is a reactive group the characteristics of which have been previously described.
  • RP is a reporter moiety the characteristics of which have been previously described.
  • the gross mass of each reporter can be different for each reagent of the set.
  • LK is a linker moiety the characteristics of which have been previously described.
  • the gross mass of the linker can compensate for the difference in gross mass between the reporters for the different labeling reagents such that the aggregate gross mass of the reporter-linker combination is the same for each reagent of the set.
  • X is a bond between an atom of the reporter and an atom of the linker.
  • the sample mixture can therefore comprise a complex mixture wherein relative amounts of the same and/or different analytes can be identified and/or quantitated, either by relative quantitation of the amounts of analyte in each of the two or more samples or absolutely where a calibration standard is also added to the sample mixture.
  • the mixture can then be subjected to spectrometry techniques wherein a first mass analysis can be performed on the sample mixture, or fraction thereof, using a first mass analyzer. Ions of a particular mass to charge ratio from the first mass analysis can then be selected.
  • the selected ions can then be subjected to dissociative energy levels (e.g. collision-induced dissociation (CID)) to thereby induce fragmentation of the selected ions.
  • dissociative energy levels e.g. collision-induced dissociation (CID)
  • bonds X and/or Y can be fragmented in at least a portion of the selected ions.
  • Fragmentation of both bonds X and Y can cause fragmentation of the reporter-linker moiety as well as cause release the charged or ionized reporter from the analyte. Ions subjected to dissociative energy levels can also cause fragmentation of the analyte to thereby produce daughter fragment ions of the analyte. The ions (remaining selected ions, daughter fragment ions and charged or ionized reporters), or a fraction thereof, can then be directed to a second mass analyzer.
  • a second mass analysis can be performed on the selected ions, and the fragments thereof.
  • the second mass analysis can determine the gross mass (or m/z) and relative amount of each unique reporter that is present at the selected mass to charge ratio as well as the gross mass of the daughter fragment ions of at least one reactive analyte of the sample mixture.
  • the daughter fragment ions can be used to identify the analyte or analytes present at the selected mass to charge ratio. For example, this analysis can be done as previously described in the section entitled: “Analyte Determination By Computer Assisted Database Analysis”.
  • certain steps of the process can be repeated one or more times.
  • ions of a selected mass to charge ratio from the first mass spectrometric analysis can be treated to dissociative energy levels to thereby form ionized reporter moieties and ionized daughter fragment ions of at least some of the selected ions, as previously described.
  • a second mass analysis of the selected ions, the ionized reporter moieties and the daughter fragment ions, or a fraction thereof, can be performed.
  • the gross mass and relative amount of each reporter moiety in the second mass analysis and the gross mass of the daughter fragment ions can also be determined. In this way, the information can be made available for identifying and quantifying one or more additional analytes from the first mass analysis.
  • the whole process can be repeated one or more times. For example, it may be useful to repeat the process one or more times where the sample mixture has been fractionated (e.g. separated by chromatography or electrophoresis). By repeating the process on each sample, it is possible to analyze all the entire sample mixture. It is contemplated that in some embodiments, the whole process will be repeated one or more times and within each of these repeats, certain steps will also be repeated one or more times such as described above. In this way, the contents of sample mixture can be interrogated and determined to the fullest possible extent.
  • analytes may be subjected to dissociative energy levels using a laser source and the resulting fragments recorded following post-source decay in time-of-flight or tandem time-of-flight (TOF-TOF) mass spectrometers.
  • TOF-TOF tandem time-of-flight
  • bond X can be more or less prone to, or substantially equal to, fragmentation as compared with fragmentation of bonds of the analyte (e.g. an amide (peptide) bond in a peptide backbone).
  • bond Y can be more or less prone to fragmentation as compared with fragmentation of bonds of the analyte (e.g. an amide (peptide) bond in a peptide backbone).
  • the linker for each reagent of the set is neutral in charge after the fragmentation of bonds X and Y (i.e. the linker fragments to produce a neutral loss of mass and is therefore not observed in the MS/MS spectrum).
  • the position of bonds X and Y does not vary within the labeling reagents of a set, within the labeled analytes of a mixture or within the labeling reagents of a kit.
  • the reporter for each reagent of the set does not substantially sub-fragment under conditions that are used to fragment the analyte (e.g. an amide (peptide) bond of a peptide backbone).
  • bond X is less prone to fragmentation as compared with bond Y.
  • bond Y is less prone to fragmentation as compared with bond X.
  • bonds X and Y are of approximately the same lability or otherwise are selected such that fragmentation of one of bonds X or Y results in the fragmentation of the other of bonds X or Y.
  • the method of the invention comprises: reacting two or more samples, each sample comprising one or more analytes, with a different labeling reagent to thereby produce two or more differently labeled samples each comprising one or more labeled analytes, and mixing two or more of the labeled samples, or a portion thereof, and optionally one or more calibration standards to thereby produce the mixture comprising analytes labeled with the labeling reagents described herein.
  • each sample used to produce the mixture was labeled with a labeling reagent comprising a unique reporter that can be used to identify the analyte and quantify it relative or absolute amount in the mixture and/or in the sample from which it originated.
  • the labeling reagents or “isobaric mass tags” can be represented by any of Structural Formulas I w , I # , I-S′ to IV-S′ or I to IV, typically one of I to IV, wherein RG represents a nucleophilic group or an electrophilic group, and the remaining variables are as described above for the compounds.
  • the method of the invention comprises reacting two or more samples, each sample comprising one or more reactive analytes, with a set of isobaric mass tags to thereby produce two or more differentially labeled samples each comprising one or more labeled analytes, and mixing two or more of the differentially labeled samples, or a portion thereof, and optionally one or more calibration standards to thereby produce a sample mixture.
  • bond Y links the linker to the analyte; at least one of RP (respectively represented by RP 1 , RP 2 , RP 3 , RP 4 , RP 5 , and RP 6 in the various formula) and LK (respectively represented by LK 1 , LK 2 , LK 3 , LK 4 , LK 5 and LK 6 in the various formula) can be isotopically enriched with one or more heavy atom isotopes; upon reaction of the isobaric mass tag with an analyte, each mass tag can add the same mass to the analyte; and upon fragmentation, RP (respectively represented by RP 1 , RP 2 , RP 3 , RP 4 , RP 5 and RP 6 in the various formula) of each isobaric mass tag can yield a signature ion having a different mass from the signature ions of the other isobaric
  • the analytes from a sample can be reacted with the solid support (each sample being reacted with a different solid support and therefore a different reporter) and the resin bound components of the sample that do not react with the reactive group can be optionally washed away.
  • the labeled analyte or analytes can then be removed from each solid support by treating the support under conditions that cleave the cleavable linker S′ and thereby release the reporter-linker-analyte complex from the support.
  • Each support can be similarly treated under conditions that cleave the cleavable linker to thereby obtain two or more different samples, each sample comprising one or more labeled analytes wherein the labeled analytes associated with a particular sample can be identified and/or quantified by the unique reporter linked thereto.
  • the collected samples can then be mixed to form a sample mixture, as previously described.
  • each different labeling reagent of the set used in the previously described method can be attached to a solid support.
  • the support comprising a labeling reagent can be prepared by any of several methods (see the Example section below).
  • the amino, hydroxyl or thiol group of an isobaric mass tag can be reacted with the cleavable linker of a suitable support.
  • the cleavable linker can be a “sterically hindered cleavable linker”. Cleavage of the cleavable linker will release the labeled analyte from the support.
  • Non-limiting examples of sterically hindered solid supports include: Trityl chloride resin (trityl-Cl, Novabiochem, P/N 01-64-0074), 2-Chlorotrityl chloride resin (Novabiochem, P/N 01-64-0021), DHPP (Bachem, P/N Q-1755), MBHA (Applied Biosystems P/N 400377), 4-methyltrityl chloride resin (Novabiochem, P/N 01-64-0075), 4-methoxytrityl chloride resin (Novabiochem, P/N 01-64-0076), Hydroxy-(2-chorophnyl)methyl-PS (Novabiochem, P/N 01-64-0345), Rink Acid Resin (Novabiochem P/Ns 01-64-0380, 01-64-0202), NovaSyn TGT alcohol resin (Novabiochem, P/N 01-64-0074).
  • Trityl chloride resin trityl-Cl, Novabiochem, P/N
  • methods of the invention can further comprise digesting each sample with at least one enzyme to partially, or fully, degrade components of the sample prior to performing the labeling of the analytes of the sample as more fully described above in the section entitled: “Sample Processing”.
  • the enzyme can be a protease (to degrade proteins and peptides) or a nuclease (to degrade oligonucleotides). The enzymes may also be used together to thereby degrade sample components.
  • the enzyme can be a proteolytic enzyme such as trypsin, papain, pepsin, ArgC, LysC, V8 protease, AspN, pronase, chymotrypsin or carboxypeptidase C.
  • methods can further comprise separating the sample mixture prior to performing the first mass analysis as more fully described above in the section entitled: “Separations”. In this manner the first mass analysis can be performed on only a fraction of the sample mixture.
  • the separation can be performed by any separations method, including by chromatography or by electrophoresis.
  • LC/MS liquid chromatography/mass spectrometry
  • any chromatographic separation process suitable to separate the analytes of interest can be used. Non-limiting examples of suitable chromatographic and electrophoretic separations processes have been described herein.
  • the methods of the invention can comprise both an enzyme treatment to degrade sample components and a separations step.
  • the amount of reporter can be determined by peak intensity in the mass spectrum. In some embodiments, the amount of reporter can be determined by analysis of the peak height or peak width of the reporter (signature ion) signal obtained using the mass spectrometer. Because each sample can be labeled with a different labeling reagent and each labeling reagent can comprise a unique reporter that can be correlated with a particular sample, determination of the different reporters in the second mass analysis identifies the sample from which the ions of the selected analyte originated. Where multiple reporters are found (e.g.
  • the relative amount of each reporter can be determined with respect to the other reporters. Because the relative amount of each reporter determined correlates with the relative amount of an analyte in the sample mixture, the relative amount (often expressed as concentration and/or quantity) of the analyte in each sample combined to form the sample mixture can be determined. As appropriate, a correction of peak intensity associated with the reporters can be performed for naturally occurring, or artificially created, isotopic abundance, as previously discussed in the section entitled: “Relative and Absolute Quantitation of Analytes”. More specifically, where the volume and/or quantity of each sample that is combined to the sample mixture is known, the relative amount (often expressed as concentration and/or quantity) of the analyte in each sample can be calculated based upon the relative amount of each reporter determined.
  • This analysis can be repeated one or more times on selected ions of a different mass to charge ratio to thereby obtain the relative amount of one or more additional analytes in each sample combined to form the sample mixture.
  • a correction of peak intensity associated with the reporters can be performed for naturally occurring, or artificially created, isotopic abundance.
  • the amount of the unique reporter associated with the calibration standard can be used to determine the absolute amount (often expressed as a concentration and/or quantity) of the analyte in each of the samples combined to form the sample mixture. This is possible because the amount of analyte associated with the reporter for the calibration standard is known and the relative amounts of all other reporters can be determined for the labeled analyte associated with the selected ions.
  • This analysis can be repeated one or more times on selected ions of a different mass to charge ratio to thereby obtain the absolute amount of one or more additional analytes in each sample combined to form the sample mixture.
  • a correction of peak intensity associated with the reporters can be performed for naturally occurring, or artificially created, isotopic abundance.
  • the methods can be practiced with digestion and/or separation steps.
  • the steps of the methods, with or without the digestion and/or separation steps can be repeated one or more times to thereby identify and/or quantify one or more other analytes in a sample or one or more analytes in each of the two or more samples (including samples labeled with support bound labeling reagents).
  • the quantitation can be relative to the other labeled analytes, or it can be absolute.
  • Such an analysis method can be particularly useful for proteomic analysis of multiplex samples of a complex nature, especially where a preliminary separation of the labeled analytes (e.g. liquid chromatography or electrophoretic separation) precedes the first mass analysis.
  • the analytes can be peptides in a sample or sample mixture.
  • Analysis of the peptides in a sample, or sample mixture can be used to determine the amount (often expressed as a concentration and/or quantity) of identifiable proteins in the sample or sample mixture wherein proteins in one or more samples can be degraded prior to the first mass analysis.
  • the information from different samples can be compared for the purpose of making determinations, such as for the comparison of the effect on the amount of the protein in cells that are incubated with differing concentrations of a substance that may affect cell growth.
  • Other, non-limiting examples may include comparison of the expressed protein components of diseased and healthy tissue or cell cultures.
  • This may encompass comparison of expressed protein levels in cells, tissues or biological fluids following infection with an infective agent such as a bacteria or virus or other disease states such as cancer.
  • an infective agent such as a bacteria or virus or other disease states such as cancer.
  • changes in protein concentration over time (time-course) studies may be undertaken to examine the effect of drug treatment on the expressed protein component of cells or tissues.
  • the information from different samples taken over time may be used to detect and monitor the concentration of specific proteins in tissues, organs or biological fluids as a result of disease (e.g. cancer) or infection.
  • the analyte can be a nucleic acid fragment in a sample or sample mixture.
  • the information on the nucleic acid fragments can be used to determine the amount (often expressed as a concentration and/or quantity) of identifiable nucleic acid molecules in the sample or sample mixture wherein the sample was degraded prior to the first mass analysis. Moreover, the information from the different samples can be compared for the purpose of making determinations as described above.
  • this invention pertains to mixtures (e.g. sample mixtures).
  • the mixtures can comprise at least two differentially labeled analytes, wherein each of the two-labeled analytes can originate from a different sample and comprise the formula: RP—X-LK—Y-Analyte.
  • RP—X-LK—Y-Analyte For each different label, some of the labeled analytes of the mixture can be the same and some of the labeled analytes can be different.
  • the atoms, moieties or bonds, X, Y, RP and LK have been previously described and their characteristics disclosed.
  • the mixture can be formed by mixing all, or a part, of the product of two or more labeling reactions wherein each labeling reaction uses a different labeling reagent of the general formula: RP—X-LK—Y—RG, wherein atoms, moieties or bonds X, Y, RP, LK RG have been previously described and their characteristics disclosed.
  • the labeling reagents can be isotopically coded isomeric or isobaric labeling reagents.
  • the unique reporter of each different labeling reagent can indicate from which labeling reaction each of the two or more labeled analytes is derived.
  • the labeling reagents can be isomeric or isobaric.
  • two or more of the labeled analytes of a mixture can be isomeric or isobaric.
  • the mixture can be the sample mixture as disclosed in any of the above-described methods. Characteristics of the labeling reagents and labeled analytes associated with those methods have been previously discussed.
  • the analytes of the mixture can be peptides.
  • the analytes of the mixture can be proteins.
  • the analytes of the mixture can be peptides and proteins.
  • the analytes of the mixture can be nucleic acid molecules.
  • the analytes of the mixture can be carbohydrates.
  • the analytes of the mixture can be lipids.
  • the analytes of the mixture can be steroids.
  • the analytes of the mixture can be small molecules of less than 1500 daltons.
  • the analytes of the mixture comprise two or more analyte types.
  • the analyte types can, for example, be selected from peptides, proteins, oligonucleotides, carbohydrates, lipids, steroids and/or small molecules of less than 1500 daltons.
  • a mixture of the invention comprises at least two labeled analytes, wherein at least one of the labeled analytes originates from a different sample from the other labeled analytes, combined to form the mixture.
  • the analyte can be a protein, a peptide, a nucleotide, a carbohydrate, a lipid, a steroid or a small molecule of less than 1500 daltons.
  • the labeled analytes can be represented by any of Structural Formulas I w , I # , I-S′ to VI-S′ or I to VI, typically one of I to VI, wherein RG represents the reaction product of a nucleophilic group or electrophilic group and the analyte, e.g., the labeled analytes can be represented by one of the following formulas: or a salt form and/or hydrate form thereof, wherein the variables are as defined above.
  • RP/LK typically at least one of RP/LK (or RP 1 /LK 1 , RP 2 /LK 2 , RP 3 /LK 3 , RP 4 /LK 4 , RP 5 /LK 5 or RP 6 /LK 6 ) can be isotopically enriched with one or more heavy atom isotopes; and the group RP—X-LK— (or RP 1 —X-LK 1 —, RP 2 —X-LK 2 —, RP 3 —X-LK 3 —, RP 4 —X-LK 4 —, RP 5 —X-LK 5 —, or RP 6 —X-LK 6 —) of each labeled analyte can have the same mass.
  • each of RP and LK comprise at least two heavy atom isotopes. In some embodiments, each of RP and LK comprise at least three heavy atom isotopes.
  • the method of the invention comprises reacting two or more samples, each sample comprising one or more reactive analytes, with a set of labeling reagents or “isobaric mass tags” to thereby produce two or more differentially labeled samples each comprising one or more labeled analytes, and mixing two or more of the differentially labeled samples, or a portion thereof, and optionally one or more calibration standards to thereby produce a sample mixture.
  • bond Y can link the linker to the analyte; at least one of RP (respectively represented by RP 1 , RP 2 , RP 3 , RP 4 , RP 5 , and RP 6 in the various formula) and LK (respectively represented by LK 1 , LK 2 , LK 3 , LK 4 , LK 5 and LK 6 in the various formula), e.g.
  • the labeling reagents can be useful for the multiplex analysis of one or more analytes in the same sample, or in two or more different samples.
  • At least one heavy atom isotope can be incorporated into a carbonyl or thiocarbonyl group of the labeling reagent and at least one other heavy atom isotope cam be incorporated into the reporter group of the labeling reagent.
  • the labeling reagents can comprise a carbonyl or thiocarbonyl linker.
  • Labeling reagents comprising a carbonyl or thiocarbonyl linker can be used in active ester form for the labeling of analytes.
  • an alcohol group forms a leaving group (LG), e.g., in some embodiments, the leaving groups depicted in FIG. 9 .
  • the active ester can be an N-hydroxysuccinimidyl ester.
  • Protocol I A respective amine (1-400 ⁇ mol) was dissolved in aqueous sodium bicarbonate (0.2 M) and acetonitrile (v/v 2:1 or 1:1). Typically, the concentration of the amine was in the range of between about 0.01 to about 0.1 M. N-Hydroxysuccinimidyl iodoacetate in acetonitrile (about 0.4 M, around 10 fold excess relative to the free amine) was added while vortexing the reaction mixture. The mixture was shaken at room temperature for about 10 min. to about 30 min. The product was purified with HPLC, and confirmed with mass spectrometry (MS).
  • MS mass spectrometry
  • Protocol II Iodoacetic anhydride (0.74 g, 2.1 mmol) in CH 2 Cl 2 (3 mL) was added to a stirred solution of a respective amine (1.9 mmol) with N, N-diisopropylethylamine (DIEA, 1.9 mmol) at room temperature. The reaction solution was further stirred at room temperature for 1.5-3 hour, then partitioned between methylene chloride and water. The organic layer was dried with anhydrous Na 2 SO 4 , concentrated in vacuo, and purified with silica gel flash chromatography. The product was characterized with NMR and/or MS.
  • DIEA N, N-diisopropylethylamine
  • FIG. 11 illustrates the synthesis of Mass Tag (2).
  • 4-Amino-benzylamine Aldrich, 1 mmol
  • N-succinimidyl iodoacetate Pierce, 1 mmol
  • DIEA N,N-diisopropyl ethylamine
  • FIG. 12 illustrates the synthesis of Mass Tag (3).
  • DMF hexamethyleneimine
  • More DMF (2 ml) was added.
  • the mixture was shaken at room temperature for 2 hours to form Boc-Asp(But)-HMI.
  • Boc-Asp(But)-HMI was purified with preparative HPLC, and characterized with MS ([M+H] + : 371.3, calculated; 371.1, found).
  • Boc-protected amine group of Boc-Asp(But)-HMI was deprotected by exposure to a solution of methylene chloride (0.1 ml) and trifluoroacetic acid (TFA, 0.1 ml) at room temperature for 20 minutes. The solvents were evaporated in vacuo at 40° C. to dryness. The free amine was then acylated with Protocol I to furnish Mass Tag (3) ([M+H] + : 383.0, calculated; 383.0, found).
  • FIG. 13 illustrates Mass Tags (4) and (5).
  • Mass Tag (4) was prepared by acylating the amine group of commercially available O-benzyl serine using Protocol I. ([M+H] + in MS: 364.0, calculated; 364.0, found).
  • Mass Tag (5) was prepared by acylating the amine group of commercially available S-(p-methyl benzyl)cysteine using Protocol I. ([M+H] + in MS: 394.0, calculated; 394.0, found).
  • FIG. 14 illustrates the syntheses of Mass Tags (6), (7) and (8).
  • Boc-protected amine group of BocNH—O(Bzl) was deprotected by exposure to 4-8 ml of 25% TFA in methylene chloride at room temperature for 30 minutes.
  • the deprotected compound, NH 2 —O(Bzl) was extracted with water twice, and then either purified with preparative HPLC or used directly in the acylation reaction after evaporation of solvents.
  • the Boc-protected amine group of BocNH—CH 2 CH 2 —O(Bzl) was deprotected by exposure to 4-8 ml of 25% TFA in methylene chloride at room temperature for 30 minutes.
  • the deprotected compound, NH 2 —CH 2 CH 2 —O(Bzl) was extracted with water twice, and then either purified with preparative HPLC or used directly in the acylation reaction after evaporation of solvents.
  • the Boc-protected amine group of BocNH—(CH 2 ) 5 —O(Bzl) was deprotected by exposure to 4-8 ml of 25% TFA in methylene chloride at room temperature for 30 minutes.
  • the deprotected compound, NH 2 —(CH 2 ) 5 —O(Bzl) was extracted with water twice, and then either purified with preparative HPLC or used directly in the acylation reaction after evaporation of solvents.
  • FmocGly (Applied Biosystems, 1 mmol), N,N,N′,N′-tetramethyl(succinimido)-uranium tetrafluoroborate (TSTU, Advanced ChemTech, 1 mmol) and N,N-diisopropylethylamine (DIEA, Aldrich, 2 mmol) were dissolved in N,N-dimethylformamide (DMF, Burdick & Jackson, 6 ml). The mixture was shaken at room temperature for half an hour. The solvent was evaporated to form FmocGly-OSu, which was used directly in the following steps.
  • TSTU N,N,N′,N′-tetramethyl(succinimido)-uranium tetrafluoroborate
  • DIEA N,N-diisopropylethylamine
  • FmocGly-Ser(Bzl)-Gly (1 mg) was exposed to a solution of 0.2 ml of 20% piperidine in DMF for 10 minutes to remove the Fmoc-protecting group. After evaporation of solvents in vacuo at 40° C., the deprotected amine was acylated using Protocol I to furnish Mass Tag (11) ([M+H] + : 478.0, calculated; 478.0, found).
  • BocNH—O(Bzl) (see Section V.A. and FIG. 14 for preparation) (0.2 mmol) was exposed to a solution of 5 ml of 25% TFA in methylene chloride for 30 minutes to remove the Boc-protecting group to form NH 2 —O(Bzl).
  • NH 2 —O(Bzl) was extracted with water, purified with preparative HPLC, and characterized with MS ([M+H] + : 124.1, calculated; 124.2, found).
  • FmocGly-Gly (0.02 mmol), TSTU (0.02 mmol), and DIEA (0.03 mmol) were dissolved in DMF (0.2 ml). The mixture was shaken at room temperature for 40 minutes, and then transferred to a solution of NH 2 —O(Bzl) (2 mg) in DMF (0.1 ml) and 0.2 M aqueous sodium bicarbonate (0.1 ml) while vortexing. The mixture was shaken at room temperature for 20 minutes. The product, FmocGly-Gly-NH—O(Bzl), was purified with HPLC, and characterized with MS ([M+H] + : 406.0, calculated; 405.8, found).
  • FIG. 17 illustrates Mass Tags (14) and (15).
  • FIG. 18 illustrates a general protocol for syntheses of Mass Tags (16), (17), (18), (19) and (20).
  • Mass Tag (16) ([M+H] + in MS: 335.0, calculated; 335.0, found). Mass Tag (17) ([M+H] + in MS: 377.0, calculated; 377.0, found). Mass Tag (18) ([M+H] + in MS: 407.1, calculated; 407.2, found). Mass Tag (19) ([M+H] + in MS: 451.1, calculated; 451.0, found). Mass Tag (20) ([M+H] + in MS: 479.1, calculated; 479.2, found).
  • FIG. 19 illustrates the syntheses of Mass Tags (21), (22), (23) and (24).
  • FmocOrn(Z)-Gly (2 mg) was exposed to a solution of 0.1 mL of 20% piperidine in DMF for 10 minutes to remove the Fmoc-protecting group. After evaporation of all the solvents, the deprotected amine was acylated using Protocol I to furnish Mass Tag (21) [M+H] + : 492.1, calculated; 492.0, found).
  • FmocOrn(Z)-Ala (2 mg) was exposed to a solution of 0.1 mL of 20% piperidine in DMF for 10 minutes to remove the Fmoc-protecting group. After evaporation of all the solvents, the deprotected amine was acylated using Protocol I to furnish Mass Tag (22) ([M+H] + : 506.1, calculated; 505.8, found).
  • FmocOrn(Z)-Gly (0.1 mmol), TSTU (0.2 mmol), and DIEA (0.3 mmol) were dissolved in DMF (1 ml). The mixture was shaken at room temperature for 1 hour, and then transferred to a solution of sodium bicarbonate (1.5 mmol) and L-alanine (1 mmol) in water (1 ml). The mixture was shaken at room temperature for 30 minutes.
  • the product, FmocOrn(Z)-Gly-Ala was purified with preparative HPLC, and characterized with MS ([M+H] + : 617.2, calculated; 617.2, found).
  • FmocOrn(Z)-Ala-Gly (2 mg) was exposed to a solution of 0.1 ml of 20% piperidine in DMF for 10 minutes. After evaporation of all the solvents, the deprotected amine was acylated using Protocol I to furnish Mass Tag (24) ([M+H] + : 563.1, calculated; 563.2, found).
  • Glu 1 ]-Fibrinopeptide B human [Glu-Fib, SEQ ID No.: 25 GVNDNEEGFFSAR), CAS#: 103213-49-6]: The peptide was assembled on trityl chloride resin (P/N: Novabiochem, 01-64-0074) using standard Fmoc-peptide synthesis protocol (Novabiochem caltalog, 2004-2005).
  • Fmoc-Glu-Fib-Trityl-chloride resin was treated with 20% (v/v) piperidine in DMF (2 mL ⁇ 1 min, filtered, then 2 mL ⁇ 5 min), filtered and washed (NMP).
  • 4-(Fmoc-amino)benzoic acid (P/N: Bachem, B-3260; 10 eqv to Glu-Fib amount on the resin) was activated with HATU (P/N: Applied Biosystems 4317033, 9.5 eqv) and N,N-Diisopropylethylamine (30 eqv) in NMP ( ⁇ 1 mL), added to the resin and mixed for 30 min.
  • MS/MS analyses of Mass Tag labeled Glu-Fib peptide (38) were performed on MALDI and electrospray platforms. Data indicate that the Mass Tag is a good candidate for MALDI platform but the intensity of the signature ion peak was very weak in electrospray platforms (data not shown). Differentially 13 C, 15 N and/or 2 H labeled aminobenzoic acids are not commercially available and would need to be synthesized.
  • MS/MS analyses of Mass Tag labeled Glu-Fib peptide (39) were performed on MALDI and electrospray platforms. Data indicate that the Mass Tag is a good candidate for MALDI platform but the intensity of the signature ion peak was very weak in electrospray platforms (data not shown). Differentially 13 C, 15 N and/or 2 H labeled aminobenzoic acids are not commercially available and would need to be synthesized.
  • Fmoc-Glu-Fib-Trityl-chloride resin was treated with 20% (v/v) piperidine in DMF (2 mL ⁇ 1 min, filtered, then 2 mL ⁇ 5 min), filtered and washed (NMP).
  • Fmoc-4-carboxymethyl-piperazine P/N: Chem-Impex, 04960
  • 10 eqv to Glu-Fib amount on the resin was activated with HATU (P/N: Applied Biosystems 4317033, 9.5 eqv) and N,N-Diisopropylethylamine (30 eqv) in NMP ( ⁇ 1 mL), added to the resin and mixed for 30 min.
  • MS/MS analyses of Mass Tag labeled Glu-Fib peptide (40) were performed on MALDI and electrospray platforms. Data indicate that the Mass Tag is a good candidate for MALDI and electrospray platforms. Signature ion intensity and fragmentation pattern of the peptide were similar to current iTRAQTM reagent N-Methyl piperazine acetic acid (data not shown).
  • thymine acetic acid propyl ester (41a) can be synthesized according to: Alahiane, A.; Taourirte, M.; Rochdi, A.; Redwane, N.; Sebti, S.; Engels, J. W.; Lazrek, H.
  • Fmoc-Glu-Fib-Trityl-chloride resin was treated with 20% (v/v) piperidine in DMF (2 mL ⁇ 1 min, filtered, then 2 mL ⁇ 5 min), filtered and washed (NMP).
  • Compound (41c) (10 eqv to Glu-Fib amount on the resin) was activated with HATU (P/N: Applied Biosystems 4317033, 9.5 eqv) and N,N-Diisopropylethylamine (30 eqv) in NMP ( ⁇ 1 mL), added to the resin and mixed for 30 min. Resin was then filtered, washed with NMP, and Fmoc group was cleaved.
  • MS/MS analyses of the Mass Tag labeled Glu-Fib peptide (41) were performed on MALDI and electrospray platforms. Data indicates that the Mass Tag is good candidate for MALDI and electrospray platform. Signature ion intensity was strong and desired peptide fragmentation pattern was observed (data not shown).
  • Fmoc-Glu-Fib-Trityl-chloride resin was treated with 20% (v/v) piperidine in DMF (2 mL ⁇ 1 min, filtered, then 2 mL ⁇ 5 min), filtered and washed (NMP).
  • Compound (42f) (10 eqv to Glu-Fib amount on the resin) was activated with HATU (P/N: Applied Biosystems 4317033, 9.5 eqv) and N,N-Diisopropylethylamine (30 eqv) in NMP ( ⁇ 1 mL), added to the resin and mixed for 30 min. Resin was then filtered, washed with NMP, and Fmoc group was cleaved.
  • MS/MS analyses of the Mass Tag labeled Glu-Fib peptide (42) were performed on MALDI and electrospray platforms. Data indicates that the Mass Tag is good candidate for MALDI and electrospray platform. Signature ion intensity was strong and desired peptide fragmentation pattern was observed (data not shown).
  • Fmoc-OSu P/N: Advance ChemTech RC8015, 0.239 g, 0.71 mmol in acetone (2 mL)
  • MS/MS analyses of the Mass Tag labeled Glu-Fib peptide (43) were performed on MALDI and electrospray platforms. Data indicates that the Mass Tag is good candidate for MALDI and electrospray platform. Signature ion intensity was strong and desired peptide fragmentation pattern was observed (data not shown).
  • Compound (46f) Compound (46e) (0.200 g, 0.304 mmol) was treated with TFA-CH 2 Cl 2 (9:1, 10 mL) for 30 min at RT and then TFA-CH 2 Cl 2 removed under reduced pressure. The yellow oil so obtained was co-evaporated with THF until the oil was colorless (re-tritylation of thiol group). The oil was then dissolved in DMF (5 mL) and basified with N,N-Diisopropylethylamine (P/N: Applied Biosystems 400136, pH 9-10, moist pH paper).
  • N,N-Diisopropylethylamine (0.206 mL, 1.19 mmol) was added to a solution of N-Me-piperazine acetic acid.2TFA (0.152 g, 0.395 mmol) and HATU (Applied Biosystems, 4317033, 0.138 g, 0.364 mmol) in DMF (2 mL); mixed for 1 min and added to the above solution of Boc deprotected (46c). After 1 h the reaction mixture was acidified with HCl (1 M), diluted with brine (150 mL) and extracted with CH 2 Cl 2 (150 mL).
  • MS/MS analyses of the Mass Tag labeled Glu-Fib peptide (46) were performed on a MALDI platform. Data indicate that the Mass Tag is a good candidates for MALDI platform (data not shown). In MS analysis (electrospray) the amount of +3 charged species was much lower than in the case of only thymine nucleobase containing tags. This compound gave only ‘y’ ion series upon peptide fragmentation when analyzed on MALDI platform (data not shown)—thus substantially reducing the complexity of the MS/MS spectra for analysis.
  • Fmoc-Glu-Fib-Trityl-chloride resin was treated with 20% (v/v) piperidine in DMF (2 mL ⁇ 1 min, filtered, then 2 mL ⁇ 5 min), filtered and washed (NMP).
  • Fmoc-cysteic-acid (10 eqv to Glu-Fib amount on the resin) was individually activated with HATU (P/N: Applied Biosystems 4317033, 9.5 eqv) and N, N-Diisopropylethylamine (30 eqv) in NMP ( ⁇ 1 mL), added to the resin and mixed for 30 min.
  • MS/MS analyses of the Mass Tag labeled Glu-Fib peptide (47) were performed on a MALDI platform. Data indicate that the Mass Tag is a good candidate for MALDI platform (data not shown). In MS analysis (electrospray) the amount of +3 charged species was much lower than in the case of only thymine nucleobase containing tags. This compound gave only ‘y’ ion series upon peptide fragmentation when analyzed on MALDI platform (data not shown)—thus substantially reducing the complexity of the MS/MS spectra for analysis.
  • Fmoc-Glu-Fib-Trityl-chloride resin was treated with 20% (v/v) piperidine in DMF (2 mL ⁇ 1 min, filtered, then 2 mL ⁇ 5 min), filtered and washed (NMP).
  • Compound (49b) (10 eqv to Glu-Fib amount on the resin) was activated with HATU (P/N: Applied Biosystems 4317033, 9.5 eqv) and N,N-Diisopropylethylamine (30 eqv, 60 eqv for compound 21) in NMP ( ⁇ 1 mL), added to the resin and mixed for 30 min. Resin was then filtered, washed with NMP followed by CH 3 CN.
  • MS/MS analyses of the Mass Tag labeled Glu-Fib peptide (49) was performed on MALDI and electrospray platforms. Data indicate that propionic acid derivative (49) could be a suitable candidate (data not shown). However a close analysis of the MS/MS pattern reveals presence of some peptide fragments in which part of the tag was still attached.
  • FmocSer(Bzl)-Gly 50 mg was exposed to 20% piperidine in DMF (5 ml) for 10 minutes to remove the Fmoc-protecting group. After evaporation of solvents, the product, Ser(Bzl)-Gly, was purified with preparative HPLC, and characterized with MS ([M+H] + : 253.1, calculated; 253.0, found).
  • FmocGlycine-2,2-d 2 (ISOTEC, 0.14 mmol), TSTU (0.21 mmol), and DIEA (0.28 mmol) were dissolved in DMF (1 ml). The mixture was shaken at room temperature for 45 minutes, and then transferred to a solution of Ser(Bzl)-Gly (0.14 mmol) in 0.2 M aqueous sodium bicarbonate (2 ml). More DMF (1 ml) was added. The mixture was shaken at room temperature for 20 minutes. The compound, FmocGly(d 2 )-Ser(Bzl)-Gly, was purified with preparative HPLC, and characterized with MS ([M+H] + : 534.2, calculated; 534.2, found).
  • FmocGly(d 2 )-Ser(Bzl)-Gly was exposed to 20% piperidine in DMF (5 ml) for 15 minutes to remove the Fmoc-protecting group. After evaporation of solvents, the compound, Gly(d 2 )-Ser(Bzl)-Gly, was purified with preparative HPLC, and characterized with MS ([M+H] + : 312.1, calculated; 312.0, found).
  • BocSer(Bzl-d 2 ) (0.4 mmol), TSTU (0.6 mmol), and DIEA (0.8 mmol) were dissolved in DMF (2 ml). The mixture was shaken at room temperature for 1 hour, and then transferred dropwise to a solution of glycine (2 mmol) in 3 mL of 1M aqueous sodium bicarbonate. The mixture was shaken at room temperature for 30 minutes.
  • the product, BocSer(Bzl-d 2 )-Gly was purified with preparative HPLC, and characterized with MS ([M+H] + : 355.3, calculated; 355.2, found).
  • BocSer(Bzl-d 2 )-Gly 64 mg was exposed to a solution of trifluoroacetic acid (TFA, Applied Biosystems, 1 ml) and methylene chloride (2 ml) at room temperature for 30 minutes to remove the Boc-protecting group. The mixture was extracted with water twice (1.5 ml each). The extracts were combined, and purified with preparative HPLC. The product, Ser(Bzl-d 2 )-Gly, was characterized with MS ([M+H] + : 255.1, calculated; 255.2, found).
  • FmocGly (0.3 mmol), TSTU (0.3 mmol), and DIEA (0.45 mmol) were dissolved in DMF (2 ml). The mixture was shaken at room temperature for 1 hour, and then transferred dropwise to a solution of Ser(Bzl-d 2 )-Gly in 0.2 M in aqueous sodium bicarbonate (2 ml). More DMF (1 ml) was added. The mixture was shaken at room temperature for 20 minutes. The product, FmocGly-Ser(Bzl-d 2 )-Gly, was purified with preparative HPLC, and characterized with MS ([M+H] + : 534.3, calculated; 534.4, found).
  • FIG. 22 illustrates the synthesis of Mass Tag (27).
  • FmocGly( 13 C 2 , 15 N) (ISOTEC, 0.33 mmol), TSTU (0.66 mmol) and DIEA (0.66 mmol) were dissolved in DMF (2 ml). The mixture was shaken at room temperature for 40 minutes, and then transferred dropwise to a solution of L-Serine(Bzl) (NovaBiochem, 2 mmol) in DIEA (4 mmol), DMSO (8 ml) and water (2 ml) while vortexing. The mixture was shaken at room temperature for 20 minutes. After filtration, the filtrate, which contained the product, was purified with preparative HPLC. The product, FmocGly( 13 C 2 , 15 N)-Ser(Bzl), was characterized with MS ([M+H] + : 478.2, calculated; 478.2, found).
  • FIG. 23 illustrates the synthesis of Mass Tag (28).
  • Boc-L-Ser (NovaBiochem, 5.82 mmol) was dissolved in DMF (6 ml), and cooled with an ice-water bath. Sodium hydride (17.46 mmol) was added while vortexing. The mixture was shaken at room temperature for 15 minutes. After no more gas was released, benzyl ( ⁇ - 13 C) bromide (ISOTEC, 2.91 mmol) was added while vortexing. The mixture was shaken at room temperature for 4 hours, and then purified with preparative HPLC. The product, BocSer(Bzl- ⁇ - 13 C), was characterized with MS ([M+H] + : 297.1, calculated; 297.2, found).
  • BocSer(Bzl- ⁇ - 13 C) (300 mg) was deprotected with 10 ml of 30% TFA in methylene chloride for 30 minutes, and then extracted with water twice (3 mL each). The aqueous layers were combined, and purified with preparative HPLC. The product, Ser(Bzl- ⁇ - 13 C), was characterized with MS ([M+H] + : 197.1, calculated; 197.0, found).
  • FmocGly(2- 13 C, 15 N) (ISOTEC, 1 mmol), TSTU (2 mmol) and DIEA (2 mmol) were dissolved in DMF (3 ml). The mixture was shaken at room temperature for 1 hour, and then transferred to Ser(Bzl- ⁇ - 13 C) in 3 ml of 0.2 M aqueous sodium bicarbonate solution while vortexing. The mixture was shaken at room temperature for 20 minutes, and purified with preparative HPLC. The product, FmocGly(2- 13 C, 15 N)-Ser(Bzl- ⁇ - 13 C), was characterized with MS ([M+H] + : 478.2, calculated; 478.2, found).
  • FmocGly(2- 13 C, 15 N)-Ser(Bzl- ⁇ - 13 C) (0.021 mmol), TSTU (0.042 mmol) and DIEA (0.042 mmol) were dissolved in DMF (0.5 ml). The mixture was shaken at room temperature for 1 hour, and transferred to Glycine( 13 C 2 , 15 N) (ISOTEC, 0.1 mmol) in 0.5 ml of 0.2 M aqueous sodium bicarbonate solution. The mixture was shaken at room temperature for 20 minutes, and purified with preparative HPLC.
  • FIG. 24 illustrates the synthesis of FmocGly-Ser(Bzl- 13 C 6 ) (29)
  • the compound was prepared with the same procedures as those for preparing FmocGly(2- 13 C, 15 N)-Ser(Bzl- ⁇ - 13 C) ([M+H] + in MS: 481.2, calculated; 481.2, found) (see: Syntheses of Isobaric Mass Tags Isotopically Coded with heavy atom isotopes, ⁇ IIB).
  • FIG. 25 illustrates the syntheses of resin bound isobaric isotopically coded Mass Tags (30), (31) and (32).
  • the resin was washed with 5 ml of 20% piperidine in DMF once, and then fully deprotected with 5 ml of 20% piperidine at room temperature for 10 minutes. After filtration, the resin was washed twice with DMF, DCM, methanol, DCM and DMF.
  • FmocGly( 13 C 2 , 15 N) (ISOTEC, 0.1 mmol), TSTU (0.1 mmol) and DIEA (0.15 mmol) were dissolved in DMF (1 ml). The mixture was shaken at room temperature for 20 minutes, and then transferred to the resin suspended in approximately 1 ml of DMF. The mixture was shaken at room temperature for 2 hours. After filtration, the resin was washed twice with DMF, DCM, methanol, DCM and DMF.
  • the resin was washed with 5 ml of 20% piperidine in DMF once, and then fully deprotected with 5 ml of 20% piperidine at room temperature for 10 minutes. After filtration, the resin was washed twice with DMF, DCM, methanol, DCM and DMF.
  • the resin was washed with 5 ml of 20% piperidine in DMF once, and then fully deprotected with 5 ml of 20% piperidine at room temperature for 10 minutes. After filtration, the resin was washed twice with DMF, DCM, methanol, DCM and DMF.
  • FIG. 27A illustrates a known synthetic procedure for the synthesis of 6-methyl uracil in greater than 90% yield.
  • the general procedure outlined in FIG. 26 can be used to convert the 6-methyl uracil to the isomer (36b) analogous to Compound (36a), and similarly to compounds (37a) and (37b) containing reactive groups RG.
  • Compounds (36a) and (37b) are embodiments of compounds of the general formula RP—X-LK—Y—RG wherein the nucleobase is a component of the linker (LK) and the N-methyl piperazine is a component of the reporter (RP).
  • FIGS. 28A-28B illustrate numerous isotopically enriched versions of 6-methyl uracil that can be prepared using these commercially available isotopically substituted starting materials and the procedure illustrated in FIG. 27A .
  • the designations +1, +2, +3, +4, +5, +6 and +7 are used to denote versions of 6-methyl uracil comprising 1, 2, 3, 4, 5, 6 and 7 heavy atom isotopes, respectively.
  • versions of 6-methyl uracil can be prepared with any where from no heavy atom isotopes to those with up to 7 heavy atom isotopes, it is possible to prepare at least 8 different isobaric labeling reagents of the general formula (37b).
  • Some exemplary isotopically coded labeling reagents are illustrated in FIG. 28C .
  • 6-methyl uracil can be prepared according to: 1. Donleavy, J. J.; Kise, M. A. 6-Methyl Uracil, Organic Syntheses , Coll. Vol. 2, p. 422; Vol. 17, p. 63; 2. Jiang, Z.; Wang, Z.; Ma, D.; Zhou, Y. Improved synthesis of 6-methyluracil. Tongji Daxue Xuebao, Ziran Kexueban, 2003, 31(2), 250-252: 3. 6-Methyluracil. SAIJIYOU SHIGEYA; NISHINAKA TOSHIYOSHI (Yodogawa Pharmaceutical Co., Ltd., Japan). Jpn.
  • Mass tags (38) and (39) are a pair of mass tags that were tested extensively. Mass tags (38) and (39) have the following structural formulae: Mass tags (38) and (39) can be synthesized by employing appropriate isotopically substituted starting materials with any known amino acid syntheses, for example, appropriate isotopically substituted starting materials can be employed with the methods shown in FIG. 25 in combination with a cleavage step to release the mass tags from the solid resin support.
  • mass tags (38) and (39) have a mass of 479.05 Da and are expected to lose a benzyl group when subjected to dissociative energy levels. However, because of the placement of the deuterium substituents on each mass tag, mass tag (38) will have a signature ion having a mass of 91.05 Da and mass tag (39) will have a signature ion of 93.07 Da.
  • the cysteine amino acid residues of synthetic peptides SEQ ID No.: 1 and SEQ ID No.: 2 having the following formulae: IAVAAQNCYK SEQ ID No.:1 IIYGGSVTGATCK SEQ ID No.:2 were alkylated with a mass tag (38) and were purified by RP-HPLC.
  • the purified tagged-peptides were reconstituted in 0.1% TFA with a concentration at 1 ⁇ M.
  • a mass spectra was generated by infusion experiment on the QTRAPTM 2000 System using TurboIonSpray operation. Total 0.5 min (40 scans) were collected. As shown in FIGS.
  • tagged SEQ ID Nos.: 1 and 2 generated signature ions of 91 Da (see FIGS. 3A and 3B , respectively). Their intensities were peptide dependent and were typically at least about as intense as those of immonium ions. The sequence ions of the tagged peptides were comparable with those of corresponding peptides alkylated with iodoacetic acid in both presence and intensities.
  • SEQ ID No.: 1 and SEQ ID No.: 3 were alkylated with mass tag (38), purified by RP-HPLC, and diluted to 100 ⁇ L with 0.1% aqueous TFA. Each sample (1 ⁇ L) was mixed with the matrix (1 ⁇ L saturated solution), and each mixture (1 ⁇ L) was then loaded on a MALDI plate for analysis.
  • the parent ions for tagged SEQ ID No.: 1 and SEQ ID No.: 3 were m/z 1431.7 and 1880.8, respectively. Both peptides were stable, and loss of the signature ion by the parent ion in the MS stage was not observed (see FIGS. 4A and 4B ).
  • the MS/MS spectra for tagged SEQ ID No.: 1 at M/z 1431.7 and tagged SEQ ID No.: 3 at m/z 1880.8 were generated using CID gas pressure set at 1 ⁇ 10 ⁇ 5 Torr and a total of 2,000 shots were collected (see FIGS. 5A and 5B ).
  • the signature ions and the sequence ions are indicated on the figure.
  • the intensities of the signature ions in the MS/MS stage were peptide dependent and were typically less than the intensities obtained using QTRAPTM 2000. However, the intensities could be enhanced significantly when CID was increased (data not shown).
  • the sequence coverage was consistent with that obtained for corresponding peptides alkylated with iodoacetic acid.
  • FIGS. 6 A-C The HPLC chromatogram for the five peptides is as listed in FIGS. 6 A-C. These peptides are named as SEQ ID Nos.: 1-5, based on their retention time from the earliest to the longest IAVAAQNCYK SEQ ID No.:1 IIYGGSVTGATCK SEQ ID No.:2 DCGATWVVLGHSER SEQ ID No.:3 VPADTEVVCAPPTAYIDFAR SEQ ID No.:4 VAHALSEGLGVIACIGEK SEQ ID No.:5
  • the dynamic range was from 1/0.05 to 1/10, spanning more than 2 orders of magnitude. Since the 91 Da ion from mass tag (38) for the ratio 1/0.1 and the 93 Da ion from mass tag (39) for the ratio 1/10 were still above the background noise, the dynamic range may be to 3 orders of magnitude or more.
  • probes with dynamic ranges of 1-order of magnitude may be sufficient since typical relative protein expressions are less than 10-fold.
  • probes with a large dynamic range are desirable.
  • the mass tags of the invention have a dynamic range of greater than 2-orders of magnitude, they can be used for absolute quantification, as well as relative quantification of proteins. TABLE 1 Relative quantification of mass tagged proteins in a sample using QTRAP TM or 4700 Protein Analyzer.

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US8501498B2 (en) 2013-08-06
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