WO2008010846A2 - Procédés d'évaluation d'interactions protéine-protéine au moyen de points quantiques et de spectrométrie icp-ms - Google Patents

Procédés d'évaluation d'interactions protéine-protéine au moyen de points quantiques et de spectrométrie icp-ms Download PDF

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WO2008010846A2
WO2008010846A2 PCT/US2007/000583 US2007000583W WO2008010846A2 WO 2008010846 A2 WO2008010846 A2 WO 2008010846A2 US 2007000583 W US2007000583 W US 2007000583W WO 2008010846 A2 WO2008010846 A2 WO 2008010846A2
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protein
quantum dot
labeled
sample
analyte
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PCT/US2007/000583
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WO2008010846A3 (fr
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Viorica Lopez-Avila
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Agilent Technologies, Inc
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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6845—Methods of identifying protein-protein interactions in protein mixtures
    • B—PERFORMING OPERATIONS; TRANSPORTING
    • B82—NANOTECHNOLOGY
    • B82Y—SPECIFIC USES OR APPLICATIONS OF NANOSTRUCTURES; MEASUREMENT OR ANALYSIS OF NANOSTRUCTURES; MANUFACTURE OR TREATMENT OF NANOSTRUCTURES
    • B82Y15/00—Nanotechnology for interacting, sensing or actuating, e.g. quantum dots as markers in protein assays or molecular motors
    • 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
    • G01N33/58—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances
    • G01N33/588—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving labelled substances with semiconductor nanocrystal label, e.g. quantum dots
    • 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

Definitions

  • Methods for detecting protein-protein interactions have many uses. For example, certain protein-protein interaction detection methods may be employed to identify proteins that bind to a protein of known identity. Other protein-protein interaction detection methods may be employed to determine if a protein of known identity is present in a sample. Many protein-protein interaction detection methods are employed in diagnostic and research applications.
  • This disclosure provides a method and a system for detecting protein-protein interactions.
  • a method for sample analysis comprises contacting a test sample with a quantum dot-labeled protein, and evaluating binding of an analyte protein in the test sample to the quantum dot-labeled protein using inductively coupled plasma mass spectrometry (ICPMS).
  • ICPMS inductively coupled plasma mass spectrometry
  • Fig. 1 shows an HPLC chromatogram of a QDots 655 streptavidin conjugate eluted from an SEC column.
  • Fig. 2 shows an ICPMS scans at 66, 82, 111 and 208 Da.
  • Fig. 3 shows an HPLC/UV chromatogram of an antitransferrin antibody.
  • Fig. 4 shows an FIPLC-I CPMS chromatograpm of the anti-transferrin Ab (Fe scan).
  • Fig. 5 shows an HPLC/UV chromatogram of human transferrin.
  • Fig. 6 shows an HPLC-ICPMS chromatogram of human transferring (Fe scan).
  • Fig. 7 shows an HPLC/UV chromatogram of an anti-transferrin (Ab) and transferrin
  • Fig. 8 shows an HPLC-ICPMS chromatogram of an anti-transferrin (Ab) and transferrin (Ag) complex (Fe scan).
  • Fig. 9 shows an HPLC/UV chromatogram of an anti-TIMP2 and human transferrin mixture.
  • Fig. 10 shows an HPLC-ICPMS chromatogram of an anti-TIMP2 and transferring mixture (Fe scan).
  • Fig. 11 shows an HPLC/UV chromatogram of an anti-TIMP2 antibody.
  • Fig. 12 shows an HPLC-ICPMS chromatogram of an anti-TIMP2 antibody (Fe scan).
  • sample as used herein relates to a material or mixture of materials, typically, although not necessarily, in fluid form, e.g., aqueous, containing one or more components of interest.
  • Samples may be derived from a variety of sources such as from food stuffs, environmental materials, a biological sample such as tissue or fluid isolated from an individual, including but not limited to, for example, plasma, serum, spinal fluid, semen, lymph fluid, the external sections of the skin, respiratory, intestinal, and genitourinary tracts, tears, saliva, milk, blood cells, tumors, organs, and also samples of in vitro ceil culture constituents (including but not limited to conditioned medium resulting from the growth of cells in cell culture medium, putatively virally infected cells, recombinant cells, and cell components).
  • the sample is a complex sample containing at least about 10 2 , 5xlO 2 , 10 3 , 5x10 3 , 10 4 , 5xlO 4 , 10 5 , 5xlO 5 , 10 6 , 5xlO 6 , 10 7 , 5xlO 7 , 10 8 , 10 9 , 10 10 , 10 n , 10 12 or more species of analyte.
  • analyte is used herein to refer to a known or unknown component of a sample, which will specifically bind to a protein, e.g., a capture agent if the analyte and the protein are members of a specific binding pair.
  • analytes are biopolymers, i.e., an oligomer or polymer such as an oligonucleotide, a peptide, a polypeptide, an antibody, or the like.
  • an "analyte” is referenced as a moiety in a mobile phase (e.g., fluid), to be detected by a protein, e.g., a capture agent, which, in some embodiments, is bound to a substrate, or in other embodiments, is in solution.
  • a protein e.g., a capture agent
  • either of the "analyte” or “protein” may be the one which is to be evaluated by the other (thus, either one could be an unknown mixture of analytes, e.g., polypeptides, to be evaluated by binding with the other).
  • a "biopolymer” is a polymer of one or more types of repeating units, regardless of the source. Biopolymers may be found in biological systems and particularly include polypeptides and polynucleotides, as well as such compounds containing amino acids, nucleotides, or analogs thereof.
  • the term “polynucleotide” refers to a polymer of nucleotides, or analogs thereof, of any length, including oligonucleotides that range from 10-100 nucleotides in length and polynucleotides of greater than 100 nucleotides in length.
  • polypeptide refers to a polymer of amino acids of any length, including peptides that range from 6-50 amino acids in length and polypeptides that are greater than about 50 amino acids in length.
  • polypeptide includes polypeptides in which the conventional backbone has been replaced with non-naturally occurring or synthetic backbones, and peptides in which one or more of the conventional amino acids have been replaced with one or more non-naturally occurring or synthetic amino acids.
  • fusion protein or grammatical equivalents thereof references a protein composed of a plurality of polypeptide components, that while not attached in their native state, are joined by their respective amino and carboxyl termini through a peptide linkage to form a single continuous polypeptide. Fusion proteins may be a combination of two, three or even four or more different proteins.
  • polypeptide includes fusion proteins, including, but not limited to, fusion proteins with a heterologous amino acid sequence, fusions with heterologous and homologous leader sequences, with or without N-terrninal methionine residues; immunologically tagged proteins; fusion proteins with detectable fusion partners, e.g., fusion proteins including as a fusion partner a fluorescent protein, ⁇ -galactosidase, luciferase, and the like.
  • Polypeptides may be of any length, e.g., greater than 2 amino acids, greater than 4 amino acids, greater than about 10 amino acids, greater than about 20 amino acids, greater than about 50 amino acids, greater than about 100 amino acids, greater than about 300 amino acids, usually up to about 500 or 1000 or more amino acids.
  • “Peptides” are greater than 2 amino acids, greater than 4 amino acids, greater than about 10 amino acids, greater than about 20 amino acids, usually up to about 50 amino acids. In some embodiments, peptides are between 5 and 30 amino acids in length.
  • capture agent refers to an agent that binds an analyte through an interaction that is sufficient to permit the agent to bind and concentrate the analyte from a homogeneous mixture of different analytes.
  • the binding interaction may be mediated by an affinity region of the capture agent.
  • Representative capture agents include polypeptides and polynucleotides, for example antibodies, peptides or fragments of single stranded or double stranded DNA may employed. Capture agents usually "specifically bind" one or more analytes.
  • capture agent refers to a molecule or a multi-molecular complex which can specifically bind an analyte, e.g., specifically bind an analyte for the capture agent, with a dissociation constant (K D ) of less than about 10 "6 M without binding to other targets.
  • K D dissociation constant
  • telomere binding refers to the ability of a capture agent to preferentially bind to a particular analyte that is present in a homogeneous mixture of different analytes. In certain embodiments, a specific binding interaction will discriminate between desirable and undesirable analytes in a sample, in some embodiments more than about 10 to 100-fold or more (e.g., more than about 1000- or 10,000-fold).
  • the affinity between a capture agent and analyte when they are specifically bound in a capture agent/analyte complex is characterized by a KD (dissociation constant) of less than 10 "6 M, less than ICT 7 M, less than 10 "8 M, less than 10 '9 M 5 or less than about 10 '10 M.
  • protein/analyte complex is a complex that results from the specific binding of a protein, e.g., a capture agent, with an analyte, i.e., a "binding partner pair".
  • a capture agent and an analyte for the capture agent specifically bind to each other under "conditions suitable for specific binding", where such conditions are those conditions (in terms of salt concentration, pH, detergent, protein concentration, temperature, etc.) which allow for binding to occur between capture agents and analytes to bind in solution.
  • conditions suitable for specific binding typically permit capture agents and target pairs that have a dissociation constant (K D ) of less than about 10 '6 M to bind to each other, but not with other capture agents or targets.
  • binding partners refer to pairs of molecules that can be found in a protein/analyte complex, i.e., exhibit specific binding with each other.
  • surface-bound protein refers to a protein that is immobilized on a surface of a solid substrate, where the substrate can have a variety of configurations, e.g., a sheet, bead, or other structure, such as a plate with wells.
  • the collections of capture agents employed herein are present on a surface of the same support, e.g., in the form of an array.
  • pre-determined refers to an element whose identity is known prior to its use.
  • a "pre-determined protein” is a protein whose identity is known prior to any binding to a capture agent.
  • An element may be known by name, sequence, molecular weight, its function, or any other attribute or identifier.
  • protein of interest i.e., a known protein that is of interest, is used synonymously with the term "pre-determined protein”.
  • antibody protein and “immunoglobulin” are used interchangeably herein to refer to a capture agent that has at least an epitope binding domain of an antibody. These terms are well understood by those in the field, and refer to a protein containing one or more polypeptides that specifically binds an antigen.
  • One form of antibody constitutes the basic structural unit of an antibody. This form is a tetramer and consists of two identical pairs of antibody chains, each pair having one light and one heavy chain. In each pair, the light and heavy chain variable regions are together responsible for binding to an antigen, and the constant regions are responsible for the antibody effector functions.
  • the recognized immunoglobulin polypeptides include the kappa and lambda light chains and the alpha, gamma (IgGi, IgG 2 , IgG3, IgG 4 ), delta, epsilon and mu heavy chains or equivalents in other species.
  • Full-length immunoglobulin "light chains" (of about 25 kDa or about 214 amino acids) comprise a variable region of about 110 amino acids at the NH 2 -terrninus and a kappa or lambda constant region at the COOH-terminus.
  • Full-length immunoglobulin "heavy chains” (of about 50 kDa or about 446 amino acids), similarly comprise a variable region (of about 116 amino acids) and one of the aforementioned heavy chain constant regions, e.g., gamma (of about 330 amino acids).
  • antibodies and “immunoglobulin” include antibodies or immunoglobulins of any isotype, fragments of antibodies which retain specific binding to antigen, including, but not limited to, Fab, Fv, scFv, and Fd fragments, chimeric antibodies, humanized antibodies, single-chain antibodies, and fusion proteins comprising an antigen- binding portion of an antibody and a non-antibody protein.
  • the antibodies may be detectably labeled, e.g., with a radioisotope, an enzyme which generates a detectable product, a fluorescent protein, and the like.
  • the antibodies may be further conjugated to other moieties, such as members of specific binding pairs, e.g., biotin (member of biotin- avidin specific binding pair), and the like.
  • the antibodies may also be bound to a solid support, including, but not limited to, polystyrene plates or beads, and the like. Also encompassed by the terms are Fab', Fv, F(ab')2, and or other antibody fragments that retain specific binding to antigen.
  • Antibodies may exist in a variety of other forms including, for example, Fv, Fab, and (Fab')2, as well as bi-functional (i.e. bi-specific) hybrid antibodies (e.g., Lanzavecchia et al., Eur. J. Immunol. 17, 105 (1987)) and in single chains (e.g., Huston et al., Proc. Natl. Acad. Sci. U.S.A., 85, 5879-5883 (1988) and Bird et al., Science, 242, 423-426 (1988), which are incorporated herein by reference).
  • Monoclonal antibodies and "phage display” antibodies are well known in the art and encompassed by the term "antibodies”.
  • mixture refers to a combination of elements, e.g., proteins or analytes, that are interspersed and not in any particular order.
  • a mixture is homogeneous and not spatially separable into its different constituents.
  • examples of mixtures of elements include a number of different elements that are dissolved in the same aqueous solution, or a number of different elements attached to a solid support at random or in no particular order in which the different elements are not spatially distinct. In other words, a mixture is not addressable.
  • an array of capture agents as is commonly known in the art, is not a mixture of capture agents because the species of capture agents are spatially distinct and the array is addressable.
  • Isolated or purified generally refers to isolation of a substance (compound, polynucleotide, protein, polypeptide, polypeptide composition) such that the substance comprises a significant percent (e.g., greater than 2%, greater than 5%, greater than 10%, greater than 20%, greater than 50%, or more, usually up to about 90%- 100%) of the sample in which it resides.
  • a substantially purified component comprises at least 50%, 80%-85%, or 90-95% of the sample.
  • Techniques for purifying polynucleotides and polypeptides of interest are well-known in the art and include, for example, ion-exchange chromatography, affinity chromatography and sedimentation according to density.
  • a substance is purified when it exists in a sample in an amount, relative to other components of the sample, that is not found naturally.
  • the term "evaluating” includes any form of measurement, and includes determining if an element is present or not.
  • the terms “determining”, “measuring”, “evaluating”, “assessing” and “assaying” are used interchangeably and may include quantitative and/or qualitative determinations. Evaluating may be relative or absolute. "Evaluating the presence of includes determining the amount of something present, and/or determining whether it is present or absent.
  • the term "using" has its conventional meaning, and, as such, means employing, e.g., putting into service, a method or composition to attain an end.
  • a program is used to create a file
  • a program is executed to make a file, the file usually being the output of the program.
  • a computer file it is usually accessed, read, and the information stored in the file employed to attain an end.
  • a unique identifier e.g., a barcode
  • the unique identifier is usually read to identify, for example, an object or file associated with the unique identifier.
  • atom refers to uncharged atoms as well as charged atoms.
  • Charged atoms may be referred to as "ions”. Ions may be positively or negatively charged.
  • a "metalloprotein” is a protein that comprises a metal atom cofactor.
  • the metal atom cofactor may be a non-complexed metal atom (such as Zn 2+ in the case of a zinc- finger protein), or a complexed metal atom (e.g., heme, which is a complex between an Fe 2+ and protoporphyrin IX).
  • the metal atom cofactor may be coordinated, i.e., held in place by amino-acid side chains.
  • the metal atom of a metal atom cofactor of a metalloprotein may be an ion of a transition metal, e.g., iron (which is present in heme groups of cytochrome c oxidase and hemoglobin, for example), zinc (as in carbonic anhydrase, for example), magnesium (as in certain kinases, for example), or molybdenum (as in nitrate reductase, for example).
  • a metalloprotein without its metal atom cofactor is called an apoprotein, while a metalloprotein combined with its metal atom cofactor is called a holoprotein.
  • a “quantum dot” is a metal-containing particle that luminesces, i.e., emits light of a particular wavelength upon stimulation.
  • the metal-containing particle of a quantum dot confines the motion of conduction band electrons, valence band holes, or excitons (pairs of conduction band electrons and valence band holes) in all three spatial directions.
  • the confinement can be due to electrostatic potentials (generated by external electrodes, doping, strain, impurities), due to the presence of an interface between different semiconductor materials (e.g., in the case of self-assembled quantum dots), due to the presence of the semiconductor surface (e.g., in the case of a semiconductor nanocrystal), or due to any combination of the above.
  • Quantum dots are described in Lin et al ⁇ Methods for labeling quantum dots to biomolecules. J Nanosci Nanotechnol. 2004 4:641-5), Hotz et al (Applications of quantum dots in biology: an overview. Methods MoI Biol. 2005 303:1-17), Chan et al (Luminescent quantum dots for multiplexed biological detection and imaging. Curr Opin Biotechnol. 2002 13 :40-6), Medintz et al (Quantum dot bioconjugates for imaging, labelling and sensing Nat Mater. 2005 4:435-46) and Goldman et al (Fluoroimmunoassays using antibody-conjugated quantum dots. Methods MoI Biol. 2005;303:19-34), for example.
  • a "quantum dot-labeled protein” is a protein that is linked with, e.g., covalently or non-covalently bound to, a quantum dot.
  • ICPMS Inductively coupled plasma mass spectrometry
  • ICPMS is a type of mass spectrometry that employs inductively coupled plasma to ionize components of a sample to produce ions, and then performs an elemental analysis of the ions.
  • ICPMS is capable of simultaneous analysis of a number of different metal ions that are present at very low concentration, e.g., less than one parts per a trillion.
  • the principles of ICPMS are further described in Vela et al, Elemental speciation with plasma mass spectrometry. (Analytical Chemistry 1993 65: 585A-597A).
  • a method for sample analysis comprises contacting a test sample with a quantum dot-labeled protein, and evaluating binding of an analyte protein in the test sample to the quantum dot-labeled protein using inductively coupled plasma mass spectrometry (ICPMS).
  • ICPMS inductively coupled plasma mass spectrometry
  • ICPMS is used to perform elemental analysis to evaluate the metal atoms contained in the quantum dot of a quantum dot-labeled protein (e.g., the types of metal atoms present and, in certain cases, the abundance of those ions).
  • the presence of a quantum dot-labeled protein may be evaluated by detecting the metal atoms contained in the quantum dot, rather than by detecting light emitted by the quantum dot.
  • the presence of the quantum dot-labeled protein may be evaluated by detecting the metal atoms contained in the quantum dot, as well as by detecting light emitted by the quantum dot.
  • the quantum dot-labeled protein and the analyte protein may be, independently, any type of protein, e.g., a capture agent such as an antibody, a metalloprotein, a non-metalloprotein, a kinase, a receptor, a phosphatase, a peptide hormone, a transcription factor, or a structural protein, or any fragment thereof.
  • a capture agent such as an antibody, a metalloprotein, a non-metalloprotein, a kinase, a receptor, a phosphatase, a peptide hormone, a transcription factor, or a structural protein, or any fragment thereof.
  • the quantum dot-labeled protein and the analyte protein may be, independently, of known identity, unknown identity, known amino acid sequence, or unknown amino acid sequence, for example.
  • the quantum dot-labeled protein and/or the analyte protein may be known to specifically bind other proteins or to each other. In other embodiments, the binding specificity of the quantum dot-labeled protein and/or the analyte protein may be unknown.
  • the quantum dot-labeled protein and the analyte protein may be different proteins, or the same proteins (excepting the quantum dot label).
  • the quantum dot-labeled protein and/or the analyte protein may be a metalloprotein, which, in certain embodiments, may contain a coordinated Fe, Cu, B, Mo, Mn, Cu, La, Cd, Hg, Pb, Tl, Zn, Ca, or W atom or ionic form thereof.
  • metalloprotein which, in certain embodiments, may contain a coordinated Fe, Cu, B, Mo, Mn, Cu, La, Cd, Hg, Pb, Tl, Zn, Ca, or W atom or ionic form thereof.
  • Such proteins are well described in the art (see, e.g., Castagnetto et al, MDB: the Metalloprotein Database and Browser at The Scripps Research Institute. Nucleic Acids Res. 2002, 30: 379-382), • and the amino acid sequence of many metalloproteins have been deposited in NCBI' s GenBank Database.
  • Examplary metalloproteins include cytochrome (having a Fe- containing cofactor), adrenodoxin (having a Fe-containing cofactor), plastocyanin (having a Cu-containing cofactor), LHC (having a Fe-containing cofactor), nitrile hydratase (having a Fe-containing cofactor), DMSO reductase (having a molybdenum or tungsten cofactor), nitrogenase (having a Mo and Fe-containing cofactor), superoxide dismutase (containing Cu 5 Zn, Fe and/or Mn cofactor), hydroxylamine oxireductase (having a Fe-containing cofactor), peroxidase (having a Mn-containing cofactor), ferrochelatase (having a Fe- containing cofactor), aconitate hydrolase (having a Fe-containing cofactor), copper- transporting ATPase (having a Cu-containing cofactor),
  • the quantum dot-labeled protein is labeled with a quantum dot, which, as discussed above, is a semiconductor nanoparticle composed of a crystalline inorganic metal- containing material that is luminescent (i.e., capable of emitting electromagnetic radiation upon excitation).
  • a quantum dot may contain an inner core of one or more first semiconductor materials that is contained within an overcoating or "shell" of a second inorganic material.
  • the inner core and the shell of a quantum dot generally contain different metals.
  • a quantum dot may be a CdSe-containing quantum dot.
  • a quantum dot may be a CdSe/ZnS core-shell quantum dot.
  • the quantum dot may be an inorganic crystallite that has a diameter in the range of about 1 nm to about 1000 ran or less, e.g, about 2 nm to about 50 nm or 2 run to about 20 nm.
  • a quantum dot may have a diameter of about 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 nm.
  • Quantum dots are also characterized by their uniform nanometer size, and their ability to emit electromagnetic radiation upon excitation (i.e., the ability to luminesce). As noted above, many quantum dots contain a "core" of one or more first semiconductor materials, which may be surrounded by a "shell" of a second semiconductor material.
  • a core surrounded by a shell is referred to as a "core/shell” quantum dot.
  • the surrounding "shell” material may have a bandgap energy that is larger than the bandgap energy of the core material and may be chosen to have an atomic spacing close to that of the "core” substrate.
  • the core and/or the shell can independently be a semiconductor material including, but not limited to, those of the group II-VI (ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, HgS, HgSe, HgTe, MgS, MgSe, MgTe, CaS, CaSe, CaTe 1 SrS, SrSe, SrTe, BaS, BaSe, BaTe, and the like) and group III-V (GaN, GaP, GaAs, GaSb, InN, InP, InAs, InSb, and the like) and group IV (Ge, Si, and the like) materials, Pb 5 PbS, PbSe, and an alloy or a mixture thereof.
  • the quantum dot may include CdS and ZnS.
  • Suitable quantum dots can be prepared as described in Murray et al, (J. Am. Chem.
  • Quantum dots can be made more luminescent using overcoating procedures as described in Danek et al (Chem. Mat. 1966 8:173-180), Hines et al (J. Phys. Chem. 1996 100:468-471), Peng et al ( J. Am. Chem. Soc. 1997 119:7019-7029), or Daboussi et al (J. Phys. Chem.-B. 1997 101:9463-9475), for example.
  • Quantum dots may be linked to a protein by a variety of techniques that may include modifying the surface of the quantum dot so that it becomes reactive, and then reacting the surface with an isolated protein or a compound that can bind to an isolated protein.
  • a quantum dot may be may conjugated to biotin, and the biotinylated quantum dot may be bound to a streptavidin-containing protein.
  • a quantum dot may be may conjugated to streptavidin, and the streptavidin-conjugated quantum dot may be bound to a biotinylated protein.
  • Methods of labeling proteins with quantum dots are reviewed in, for example, Lin et al (Methods for labeling quantum dots to biomolecules.
  • quantum dots having reactive carboxylate or hydroxyl groups on their surface can be made using the methods of, e.g., Bruchez et al, (Science 1998 281 :2013- 2016), Ghan et al (Science 1998 281 :2016-2018), Golvin et al (J. Am. Chem. Soc. 1992 114:5221-5230), Katari et al (J. Phys. Chem. 1994 98:4109-4117) or Steigerwald et al (J. Am. Chem. Soc. 1987 110:3046), and then coupled to other materials containing reactive amine group.
  • either the quantum dot-labeled protein or the analyte protein may be a metalloprotein.
  • the metal atom of the metalloprotein may be different than the metal atom of the quantum dot used.
  • a protein is produced recombinantly, e.g., using methods described in Ausubel, et al, (Short Protocols in Molecular Biology, 3rd ed., Wiley & Sons, 1995) and Sambrook, et al, (Molecular Cloning: A Laboratory Manual, Third Edition, (2001) Cold Spring Harbor, N. Y.) and linked to a quantum dot using known methods, to produce a quantum dot-labeled protein.
  • the quantum dot-labeled protein may be contacted with a test sample containing an analyte protein under conditions suitable for specific binding between the quantum dot-labeled protein and the analyte protein in the test sample.
  • Conditions suitable for specific binding of protein to other proteins are well known in the art (see, e.g., Harlow and Lane (Antibodies: A Laboratory Manual Cold Spring Harbor Laboratory, Cold Spring Harbor, N. Y. (1989)) and include incubation in a defined buffer, . e.g., PBS (phosphate buffered saline) or TBS (Tris buffered saline) at a defined temperature (e.g., room temperature, 30 0 C or 37 0 C) for a defined length of time, e.g., in the range of 30 mins to 2 hr, 6r in the range of 4 hr to overnight, or longer.
  • a defined buffer e.g., PBS (phosphate buffered saline) or TBS (Tris buffered saline)
  • a defined temperature e.g., room temperature, 30 0 C or 37 0 C
  • a defined length of time e.g., in the range of 30 mins to 2
  • complexes containing the quantum dot-labeled protein and the analyte protein may be separated from unbound components of the sample.
  • this separation step may be done chromatographically.
  • quantum dot-labeled protein/analyte protein complexes may be chromatographically separated from other components of the sample by size, hydrophobicity, hydrophilicity, ionic strength or affinity.
  • either the quantum dot-labeled protein or the analyte protein may be immobilized on a substrate, e.g., a bead or a porous matrix, and the quantum dot-labeled protein/analyte protein complexes may be separated from unbound components of the sample by washing the insoluble substrate with a buffer, e.g., a wash buffer. Such separation methods have been routinely employed to investigate other protein-protein interactions.
  • the components of a quantum dot-labeled protein/analyte protein complex may be investigated by evaluating the metals (i.e., determining which metal atoms are present in the complex and/or determining the abundance of those metal atoms) of the quantum dot-labeled protein and/or the analyte protein by atomic spectroscopy, e.g., ICPMS.
  • the quantum dot-labeled protein and analyte protein of the complex are ionized together and analyzed simultaneously.
  • the quantum dot-labeled protein and analyte protein of the complex may be ionized and analyzed sequentially.
  • only the quantum dot-labeled protein or only the analyte protein of the complex is ionized and analyzed.
  • results obtained from such methods indicate the presence, absence and, in certain cases, the abundance of the quantum dot-labeled protein and/or the analyte protein).
  • results obtained from the instant methods may be compared to the results obtained using a suitable control, e.g., results obtained from an otherwise identical method except no quantum dot-labeled protein is employed, no analyte protein is employed, no sample is employed, a different quantum dot-labeled protein is employed, or a different analyte protein is employed, for example.
  • results obtained from the instant method may be compared to the results obtained from a control method in order to evaluate the complex. For example, in one embodiment, the difference in the metals that are present in the test sample and the control sample indicates whether binding between the quantum dot-labeled protein and the analyte protein has occurred.
  • a quantum dot-labeled protein may be contacted with a sample containing an analyte protein under suitable binding conditions to produce a contacted sample.
  • the components of the contacted sample may be chromatographically separated, e.g., by HPLC, and the separated components are analyzed using ICPMS. Binding between the quantum dot-labeled protein and the analyte protein may be identified by a difference in the binding characteristics of the quantum dot-labeled protein to a chromatography column.
  • binding between the quantum dot-labeled protein and the analyte protein may be identified by a difference in retention time of the quantum dot-labeled protein, as compared to the same quantum dot-labeled protein that has not contacted the sample.
  • chromatography methods may be employed in certain embodiments of the subject methods including, but not limited to, ion exchange chromatography, such as anion or cation exchange chromatography, affinity chromatography, reversed-phase chromatography, adsorption chromatography, gel filtration chromatography, hydrophobic chromatography, hydroxyapatite chromatography, phosphocellulose chromatography, and lectin chromatography (see, e.g., Strategies for Protein Purification and Characterization: A Laboratory Course Manual, Marshak et al. ed., Cold Spring Harbor Laboratory Press (1996)). Chromatography can be carried out using a liquid phase chromatography such as HPLC or FPLC.
  • a quantum dot-labeled protein may be contacted with an analyte protein that is attached to a substrate, e.g., a bead, under suitable binding conditions to produce a contacted support.
  • a substrate e.g., a bead
  • the material that is bound to the analyte protein can be separated from the analyte protein and analyzed using ICPMS. Binding between the quantum dot-labeled protein and the analyte protein may be identified by detecting the metal atoms of the quantum dot.
  • a quantum dot-labeled capture agent e.g., a quantum dot- labeled antibody
  • a sample containing a metalloprotein under suitable binding conditions to produce a contacted sample.
  • the non-binding components of the contacted sample may be separated from the quantum dot-labeled antibody, e.g., by washing or chromatography, to produce a separated sample, and the separated sample is analyzed using ICPMS.
  • a complex between the quantum dot-labeled protein and the metalloprotein can be identified by detecting the metal cofactor of the metalloprotein, and the metal atoms of the quantum dot.
  • the metal atom of the metalloprotein may be different to the metal atoms of the quantum dot.
  • results obtained from the instant methods may be qualitative (e.g., may indicate that two proteins bind) and/or quantitiative (e.g., may indicate the level of binding between two proteins).
  • the system may contain quantum dots, such as a CdSe/ZnS core/shell quantum dot discussed above, reagents for labeling a protein with the quantum dot, and a atomic spectroscope, e.g., an inductively coupled plasma mass spectrometer system.
  • the mass spectrometer system may comprise a quadrupole or electrostatic or time-of-flight mass analyzer for performing elemental analysis on an ionized sample.
  • the mass spectrometer system may be configured to simultaneously evaluate the presence of several different metal ions.
  • the liquid flow from the HPLC column was converted into aerosol droplets by a Micromist nebulizer with a dual pass spray chamber.
  • the elemental scan was performed on an Agilent 7500ce ICPMS system with a quadrupole mass analyzer and an Octapole Reaction System for matrix- based interference removal.
  • ICP conditions outer gas (Ar) flowrate 15 L/min; carrier gas (Ar) flowrate 0.8 L/min; forward power 1.55kW, sampling depth 8 mm; He was used as reaction gas at 3.5 mL/min.
  • Antibody-protein interaction to demonstrate that a protein-protein interaction can be observed by HPLC-ICPMS, an Fe-containing protein (transferrin) and its corresponding antibody were each analyzed individually and also analyzed after being incubated together for a defined time interval.
  • Figure 3 shows the HPLC chromatograms of the anti-transferrin antibody solution (retention times of 9.307 and 9.309 min at 215 and 280 nm, respectively) and
  • Figure 4 shows the corresponding Fe scan (m/z 56) in which no peak is seen at the retention window of the antibody reaching the ICPMS system (about 620 sec) but there is a small peak corresponding to inorganic iron (at about 840 sec).
  • Figures 5 and 6 are the corresponding figures for transferrin (HPLC retention time of 10.149 to 10.151 min) its corresponding Fe scan (peak at 620 sec corresponds to transferrin and peak at 840 sec to inorganic Fe).
  • transferrin solution concentration 5.6 rng/rnL
  • anti-transferrin antibody solution concentration 1 mg/mL
  • the large inorganic Fe peak was present in the transferrin solution and in this case is used as marker to verify that the compound retention times are not shifting.
  • a different antibody i.e., anti- TIMP2
  • anti-TIMP2 was incubated with transferring the complex did not form as illustrated in Figures 9 and 10 which correspond to the UV chromatogram and the Fe scan of the anti- TIMP2/transferrin composite solution after 24 hr incubation.
  • the anti-TIMP2 solution was analyzed separately by HPLC-ICPMS and the corresponding UV chromatograms and Fe — scan are shown in Figure 11 and 12, respectively.

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Abstract

L'invention concerne un procédé d'analyse d'échantillon et un système pour réaliser ce procédé. Dans certains modes de réalisation, le procédé consiste à mettre en contact un échantillon d'essai avec une protéine marquée par un point quantique, et à évaluer la liaison d'une protéine d'analyte dans l'échantillon d'essai et de la protéine marquée par un point quantique au moyen de la spectrométrie de masse couplée à un plasma inductif (ICP-MS).
PCT/US2007/000583 2006-01-10 2007-01-09 Procédés d'évaluation d'interactions protéine-protéine au moyen de points quantiques et de spectrométrie icp-ms WO2008010846A2 (fr)

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US20060019279A1 (en) * 2004-06-09 2006-01-26 Perkinelmer Las, Inc. Methods for target molecule detection using siderophores and related compositions

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US20060019279A1 (en) * 2004-06-09 2006-01-26 Perkinelmer Las, Inc. Methods for target molecule detection using siderophores and related compositions

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