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WO2004111646A1 - Nouveaux reactifs pour spectrometrie de masse - Google Patents

Nouveaux reactifs pour spectrometrie de masse Download PDF

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Publication number
WO2004111646A1
WO2004111646A1 PCT/SE2004/000860 SE2004000860W WO2004111646A1 WO 2004111646 A1 WO2004111646 A1 WO 2004111646A1 SE 2004000860 W SE2004000860 W SE 2004000860W WO 2004111646 A1 WO2004111646 A1 WO 2004111646A1
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WO
WIPO (PCT)
Prior art keywords
group
reagent
groups
reagent molecule
bridge
Prior art date
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Ceased
Application number
PCT/SE2004/000860
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English (en)
Inventor
Peter James
Jean-Luc Maloisel
Ulrika Carlsson
Philippe Busson
Åsa WÅHLANDER
Ronnie Palmgren
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Cytiva Sweden AB
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Amersham Bioscience AB
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Amersham Bioscience AB filed Critical Amersham Bioscience AB
Publication of WO2004111646A1 publication Critical patent/WO2004111646A1/fr
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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Classifications

    • 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
    • C07K—PEPTIDES
    • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
    • C07K1/13—Labelling of peptides

Definitions

  • the present invention relates to novel mass spectrometry reagents suitable to be used in a fully automated system for, for example, protein expression analysis.
  • the reagents may be used for differential isotopic labelling of whole cell digests and these may be subsequently analysed by multi-dimensional chromatography and/or electrophoresis coupled to mass spectrometry and optionally database searching.
  • proteomics The study of the protein version of the genome, 'proteomics', must deal with 40,000 or more genes which can be arranged to give some 800,000 proteins (corresponding to some 10 7 tryptic peptides), which in turn can be modified with over 300 different chemicals. Not only that, proteomics must also define which proteins are being produced in a certain type of cell at a specific time, how they are modified, where they are in the cell and with whom they are in contact and finally and most difficult, what is the function of the protein.
  • WOOO/11208 discloses a method in which a protein is derivatised with an isotopically labelled molecule.
  • the labelled protein is captured, digested, released and analysed by mass spectrometry.
  • WOO 1/74842 Proteome Systems teaches a method in which the desired protein sample is subjected to 2D-electrophoresis separation (2D-SDS), specific residues protected be- fore digestion, and derivatisation with a labelled reagent and analysed by mass spectrometry.
  • 2D-electrophoresis separation 2D-SDS
  • these methods have shown to be limited due to the use of 2D- electrophoresis, which does not allow a separation, which leads to a visualisation of all proteins.
  • Many proteins are incompatible with this method, either being too small or too large, too acidic or alkaline, or just too insoluble.
  • Membrane proteins which is one of the most important group of proteins, both physiologically and pharmaceutically, are completely underrepresented due to their tendency to aggregate and precipitate during the various steps in 2D electrophoresis. Therefore, these proteins tend to be excluded from labelling and thus also from the analysis.
  • the method disclosed in WO 01/74842 cannot be used in MS in parent ion-scanning mode, since the reagent described therein is not capable of generating any signature ions.
  • WO 01/86306 (Purdue Research) relates to a method for protein identification in complex mixtures that utilises affinity selection of constituent proteolytic peptide fragments unique to a protein analyte.
  • These "signature peptides" which are low abundance amino acids such as Cys or Met, act as analytical surrogates for chemical capture of re- agents. Mass spectrometric analysis of the proteolysed mixture permits identification of a protein in a complex sample without purifying the protein or obtaining its composite signature, since the use of "signature peptides" will reduce the complexity of the analysis.
  • such "signature peptides” should not be confused with the signature ions required in MS in parent ion mode, which is not possible with the method disclosed in WO 01/86306.
  • Aebersold et al (American Genomic/Proteomic Technology (Aug. 2001), Vol. 1(1), p. 22-27) discloses isotope-coded affinity tag reagents for quantitative proteomics. However, this method requires the reduction in peptide complexity to be achieved by affinity purification and not by MS in parent ion-scanning mode. Likewise, Goodlet et al (Rapid Communications in MS, 2001, 15, 1214-1221) discloses a chemical tagging of proteins specific to Asp and GIu. The reagents are MS/MS stable, and cannot generate the specific fragment signature ions required in MS in parent ion-scanning mode.
  • WO 02/48717 relates to an acid-labile isotope-coded extractant and its use in quantitative mass spectrometric analysis of protein mixtures.
  • the reagents used in such method must be thiol specific and MS/MS stable. Thus, this method can not generate any signature fragment ions, and is consequently not useful in MS in parent ion-scanning mode.
  • Carr et al have described methods for following phosphate loss from phos- phopeptides (Selective detection and sequencing of phosphopeptides at the femtomole level by mass spectrometry, Anal. Biochem. 239(2): 180-92, 1996).
  • the method relies on the generation of a natural signature ion -79 m/z that is due to the loss of phosphate.
  • the occurrence of phosphate can also be followed by the loss of phosphate as a neutral molecule using the neutral loss-scanning mode.
  • an object of the invention is to provide a novel MS reagents solving the posed problems.
  • the inventors have developed a method, which meets the demands of the proteomics research society.
  • This method is for labelling of a protein or a polypeptide mixture, which has been extracted from a set of cells, with an isotopic labelled reagent molecule, and analysing it with MS parent ion-scanning.
  • two different sets of cells, representing two different states are ana- lysed by the method, whereby each set of cells is labelled with different reagent molecules, allowing for a subtractive parent ion or neutral loss scanning.
  • the present invention relates to improved reagent molecules, and in a second aspect the invention relates to a kit, comprising the reagent molecules.
  • the invention relates to use of said reagents for analytical and diagnostic pur- poses.
  • the reagent molecules are designed to be used for identification and quantification of proteins/peptides by MS and specifically via analysis of parent ions.
  • the invention relates to a Reagent Molecule of the following formula:
  • LG2 is Linker Group 2
  • RG is Reactive Group and CG, LGl, BG, LG2, and RG are covalently connected.
  • a Reagent Molecule is meant a molecule having the ability to covalently react to a specific site in a biomolecule, such as a protein, thereby, if labelled, being used to detect the bound protein in an analysis.
  • specific site in a protein is meant a chemical group, which can react covalently with a Reagent Molecule.
  • the specific site can be an amino, a hy- droxyl, a thiol, a vinyl or an allyl group.
  • labelled is meant that a Reagent Molecule can fully or partly contain a label/labels, which is possible to detect, by subsequent analysis, such as mass spectrometry.
  • the label is selected from the group that consists of 1H/ 2 D,
  • Charged Group is meant a part in the Reagent Molecule, which is positively charged under desired conditions.
  • the Charged Group is se- lected from the group that consists of positively charged aromatic amines, positively charged alkyl amines, positively charged tertiary amines, positively charged quaternary amines, heterocycle containing one or several positively charged amines, and phosphorous based compounds.
  • the Charged Group is selected from the group that consists of positively charged solid supports and polymeric structures.
  • the Charged Group contains one or several labelled groups.
  • Linker Group 1 is meant a part in the Reagent Molecule which is covalently bonded to the Charged Group and to the Bridge Group.
  • a general schematic structure of a Linker Group 1 is shown below:
  • Rl -R4 are selected from the group that consists of H, halogens, such as F, Cl, Br, I, alkyl groups, such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, aryl group, such as phenyl, diphenyl, naphthyl.
  • n 0 to 20
  • m 0 to 20
  • X is selected from the elements or atoms that consist of C, O.
  • the Linker Group 1 contains one or several labelled groups.
  • Linker Group 2 is meant a part in the Reagent Molecule which is covalently bonded to the Bridge Group and to the Reactive Group.
  • a general schematic structure of a Linker Group 2 is shown below:
  • R5-R8 are selected from the group that consists of H, halogens, such as F, Cl, Br, I, alkyl groups such as methyl, ethyl, propyl, butyl, pentyl, hexyl, heptyl, octyl, nonyl, aryl group, such as phenyl, diphenyl, naphthyl.
  • n 0 to 20
  • m 0 to 20
  • X is selected from the elements or atoms that consist of C, O.
  • All the groups are covalently bonded by single bonds, double bonds or triple bonds.
  • the Linker Group 2 contains one or several labelled groups.
  • Bridge Group is meant a part in the Reagent Molecule, which, after cleavage, allows detection of a unique labelled mass marker in a subsequent analysis, such as mass spectrometry.
  • a general schematic structure of a Bridge Group is shown below:
  • Bridge 1 and Bridge 2 are covalently connected.
  • Reactive Group is meant a part of the Reagent Molecule having the ability to react to a specific site on a desired protein as defined earlier, forming a covalent bond.
  • the Reactive Group is selected from the group that consists of ep- oxy-activated groups, allyl-activated groups, cyanobromide-activated groups, thiol groups, activated esters, carboxylic acids and acid chlorides.
  • activated esters are N-hydroxysuccinimide-, />-nitrophenyl-, pentachlorophenyl-, and pentafluoro- phenyl esters.
  • the coupling of the Reagent Molecule to the specific site can be performed in different ways.
  • the Reagent Molecule is coupled directly to the specific site.
  • the coupling of the Reagent Molecule to the specific site is performed in two or several steps.
  • the part Linker Group 2- Reactive Group is first reacted to the specific site and in a second step the part Charged Group-Linker Group 1 is reacted to form the Reagent Molecule coupled to the specific site (see experimental).
  • Reagent Molecules are:
  • the invention relates to a kit comprising one or more of the above reagent molecules.
  • the kit comprises two or more reagent molecules of the same kind but labelled with different labels, wherein the one reagent molecule is labelled with a specific isotope and the other reagent molecule with another isotope of the same atom, and the third reagent molecule, if present, with yet a further isotope etc.
  • the label is selected from the group comprising 1H/ 2 D, 12 C/ 13 C/ 14 C, 14 N/ 15 N, 16 O/ 17 O/ 18 O,
  • the invention relates to use of a reagent molecule or kit above to label biomolecules for analysis or identification of biomolecules, such as protein/peptides, and/or their reactions with other compounds in vivo or in vitro.
  • biomolecules such as protein/peptides
  • the use is the analysis of protein expression profiles, especially comparison of normal versus different disease states.
  • the reagents of the invention are also intended for diagnosis of different disease states.
  • the present invention utilises reagent molecules that can be produced in two or more forms which confer the ability to distinguish the different forms of reagents and the peptides to which they are linked by mass, but which importantly do not affect the ionisation efficiency of the peptides to which they are linked when subject to mass spectrometry.
  • the sample is treated with a reagent molecule available in different forms that can be distinguished on the basis of mass.
  • the present invention can be used in a wide variety of applications, such as for example to identify peptides presented by a major histocompatibility complex (MHC) molecule.
  • MHC major histocompatibility complex
  • the reagents according to the present invention can be used e.g. in diagnosis of diseases.
  • the use of two or more labelling reagents with different labels allows a determination of relative amounts of proteins in two or more different samples.
  • the labelling techniques of the present invention may be used to compare protein expression in two different cells.
  • the two different cells may for example be cells of the same type but under different conditions (or states), or they may be cells of a different type (under the same or different conditions).
  • a first cell may be treated with an agonist and a second cell untreated, and the expression of one or more proteins in each cell compared.
  • the present invention may also be used within drug discovery to find new potential drugs affecting biomolecules.
  • the two conditions could also be cells resting versus cells induced or treated in some manner. Often, differential expression in cells under different conditions can provide useful information on the activity in the cells.
  • the protein-fragment mixture is analysed at a first frequency, thereby generating a first set of cells, and then at a second frequency, thereby generating a second set of cells, followed by an inversion of the intensity values of the second frequency and adding them to the first, whereby a difference spectrum is generated.
  • the second frequency is usually higher man the first, and the analysis is a scanning, such as a parent ion-scanning or a neutral loss scanning.
  • Example 1 Reaction of the Reagent Molecule and the specific site in two steps
  • the specific site of the protein (amine group) was modified in two steps to bind the Reagent Molecule, after protection of the Lys side chains and digestion of the protein.
  • Iodoacetic anhydride was reacted with the free amine group on the peptide to give an iodo functional peptide.
  • This iodo functional peptide was in turn reacted with several different thiol-containing reagents to give the Reagent Molecule tagged peptide.
  • the thiol containing reagents are shown in Figure 1.
  • Figure 1 Thiol containing reagents coupled to iodo functional peptide.
  • Succinic anhydride (9mg/ mL) was dissolved in potassium phosphate buffer (200 mM, pH 8.5) containing urea (2M) and pH was rapidly adjusted to 8.5 with NaOH.
  • a solution of pure protein (1-5 mg/ mL) in ddH 2 O was added (mol prote i n / mol sl UC- c i n i c anhyd ri de '• 1/ 1000-2000).
  • succinic anhydride in phosphate buffer was added, and reaction left to proceed for 1 more hour. The reaction was performed at room temperature and the pH continuously checked and adjusted to 8.5.
  • a buffer exchange to potassium phosphate 200 mM, pH 8.6 was performed.
  • the protease GIu-C was added to the solution (protein/ protease : 1/ 20 (w/ w)) and digestion left to proceed at 37 0 C, usually overnight.
  • Iodoacetic anhydride was dissolved in THF (200 mM) and added to the peptide solution 2 times during 1 hour, with 30 min intervals (molp ro tei n / mol iodoacetic a nh ydride : 1/ 200). The reaction has been successfully performed on both ice and at room temperature.
  • Example 2 Preparation of Linker Group 2-Reactive Group type compounds These compounds were used in the reaction between Linker Group 2-Reactive Group and the specific site of the protein (amine group) to obtain an iodo functional protein.
  • the crude product was dissolved in ethyl acetate and the extra precipitate of DCC urea was filtered off on a glass filter (P3). The solvent was then evaporated on a rotatory evaporator. The crude product was then recrys- tallized from a mixture of hexane/ethyl acetate. The product precipitated as a white powder, which was filtered off on a glass filter (P3) and dried under vacuum. 0.90 g of N- hydroxysuccinimide-4-iodobutyric ester was obtained as white crystals. Yield: 62%.
  • Example 3 Fragmentation results of the thiol containing reagents coupled to iodo functional peptide.
  • the tagged peptide was prepared by coupling of the Reagent Molecule synthesized ac- cording to Example 1 to the specific site of the peptide (amine group)

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  • Engineering & Computer Science (AREA)
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  • Biochemistry (AREA)
  • General Health & Medical Sciences (AREA)
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  • Proteomics, Peptides & Aminoacids (AREA)
  • Biomedical Technology (AREA)
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  • Bioinformatics & Cheminformatics (AREA)
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  • Bioinformatics & Computational Biology (AREA)
  • Medicinal Chemistry (AREA)
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Abstract

La présente invention se rapporte à de nouveaux réactifs pour spectrométrie de masse (MS), qui sont représentés par la formule suivante : CG-LG1-BG-LG2-RG. Dans ladite formule, CG signifie groupe chargé, LG1 signifie groupe lieur 1, BG signifie groupe pont, LG2 signifie groupe lieur 2, RG signifie groupe réactif, et CG, LG1, BG, LG2 et RG sont liés par covalence. De préférence, la molécule de réactif contient tout ou partie d'un ou plusieurs marqueurs, que l'on peut ensuite détecter par le biais d'une analyse telle que la spectrométrie de masse. L'invention est destinée à servir à l'identification et à la quantification de protéines/peptides par MS, en particulier par l'intermédiaire de l'analyse d'ions parents. L'utilisation de deux molécules de réactif comportant un marquage différent permet, par exemple, la quantification relative de protéines/peptides dans un échantillon. Dans un second aspect, l'invention concerne un kit comprenant lesdites molécules de réactif. L'invention a également trait à l'utilisation des molécules de réactif pour l'analyse de biomolécules.
PCT/SE2004/000860 2003-06-19 2004-06-03 Nouveaux reactifs pour spectrometrie de masse Ceased WO2004111646A1 (fr)

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GB0314209A GB0314209D0 (en) 2003-06-19 2003-06-19 Novel MS reagents

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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008008859A3 (fr) * 2006-07-11 2008-03-06 Univ Utah Res Found Macromolécules modifiées par des groupes électrophiles et procédés de fabrication et d'utilisation de celles-ci
CN106053834A (zh) * 2016-06-22 2016-10-26 复旦大学附属中山医院 一种基于16o/18o标记的完整糖肽相对定量方法
CN108780072A (zh) * 2016-01-22 2018-11-09 普度研究基金会 带电质量标记系统

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WO2004019000A2 (fr) * 2002-08-20 2004-03-04 The Institute For Systems Biology Reactifs chimiques et procedes de detection de quantification de proteines presentes dans des melanges complexes
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Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008008859A3 (fr) * 2006-07-11 2008-03-06 Univ Utah Res Found Macromolécules modifiées par des groupes électrophiles et procédés de fabrication et d'utilisation de celles-ci
CN108780072A (zh) * 2016-01-22 2018-11-09 普度研究基金会 带电质量标记系统
CN106053834A (zh) * 2016-06-22 2016-10-26 复旦大学附属中山医院 一种基于16o/18o标记的完整糖肽相对定量方法

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