Global analysis of protein activities using proteome chips
First Claim
1. A positionally addressable array comprising a plurality of proteins, with each protein being at a different position on a solid support, wherein the plurality of proteins comprises at least one protein encoded by at least 50% of the known genes in a single species.
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Abstract
The present invention relates to proteome chips comprising arrays having a large proportion of all proteins expressed in a single species. The invention also relates to methods for making proteome chips. The invention also relates to methods for using proteome chips to systematically assay all protein interactions in a species in a high-throughput manner. The present invention also relates to methods for making and purifying eukaryotic proteins in a high-density array format. The invention also relates to methods for making protein arrays by attaching double-tagged fusion proteins to a solid support. The invention also relates to a method for identifying whether a signal is positive.
55 Citations
80 Claims
- 1. A positionally addressable array comprising a plurality of proteins, with each protein being at a different position on a solid support, wherein the plurality of proteins comprises at least one protein encoded by at least 50% of the known genes in a single species.
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3. A positionally addressable array comprising a plurality of proteins, with each protein being at a different position on a solid support, wherein the plurality of proteins comprises at least 50% of all proteins expressed in a single species, wherein protein isoforms and splice variants are counted as a single protein.
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4. A positionally addressable array comprising a plurality of proteins, with each protein being at a different position on a solid support, wherein the plurality of proteins comprises at least 1000 proteins expressed in a single species.
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5. A positionally addressable array comprising a plurality of proteins, with each protein being at a different position on a solid support, wherein the plurality of proteins in aggregate comprise proteins encoded by at least 1000 different known genes in a single species.
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11. A method for making a positionally addressable array comprising the step of attaching a plurality of proteins to a surface of a solid support, with each protein being at a different position on the solid support, wherein the plurality of proteins comprises at least one protein encoded by at least 50% of the known genes in a single species.
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12. A method for making a positionally addressable array comprising the step of attaching a plurality of proteins to a surface of a solid support, with each protein being at a different position on the solid support, wherein the plurality of proteins comprises at least 50% of all proteins expressed in a single species, wherein protein isoforms and splice variants are counted as a single protein.
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13. A method for making a positionally addressable array comprising the step of attaching a plurality of proteins to a surface of a solid support, with each protein being at a different position on the solid support, wherein the plurality of proteins comprises at least 1000 proteins expressed in a single species.
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14. A method for making a positionally addressable array comprising the step of attaching a plurality of proteins to a surface of a solid support, with each protein being at a different position on the solid support, wherein the plurality of proteins in aggregate comprise proteins encoded by at least 1000 different known genes in a single species.
- 15. A method for making a positionally addressable array comprising the step of attaching a plurality of fusion proteins to a surface of a solid support, with each fusion protein being at a different position on the solid support, wherein the fusion protein comprises a first tag, a second tag, and a protein sequence encoded by genomic nucleic acid of an organism.
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20. A method for making and isolating a plurality of purified protein samples, comprising the steps of:
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at each site of a plurality of sites of a multi-site array;
(a) growing a eukaryotic cell having a heterologous nucleotide sequence operatively linked to a regulatory sequence;
(b) contacting the regulatory sequence with an inducer that enhances expression of a protein encoded by the heterologous nucleotide sequence;
(c) lysing the cell to produce a cell lysate;
(d) contacting the cell lysate or protein-containing sample therefrom with a binding agent such that a complex between said protein and binding agent is formed; and
(e) isolating the protein from the complex;
wherein each step is conducted in a multi-array format. - View Dependent Claims (21, 22, 23, 24, 72, 79)
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25. A method for detecting a lipid-binding protein comprising the steps of:
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(a) contacting a probe comprising a lipid with a positionally addressable array comprising a plurality of proteins, with each protein being at a different position on a solid support; and
(b) detecting any protein-probe interaction, wherein detection of the interaction at a position on the solid support indicates the presence of a lipid-binding protein at said position. - View Dependent Claims (26, 27, 28, 44, 45, 46, 47, 48, 49)
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29. A method for detecting a binding protein comprising the steps of:
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(a) contacting a probe with a positionally addressable array comprising a plurality of proteins, with each protein being at a different position on a solid support, wherein the plurality of proteins comprises at least one protein encoded by at least 50% of the known genes in a single species; and
(b) detecting any protein-probe interaction. - View Dependent Claims (34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 50, 51)
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30. A method for detecting a binding protein comprising the steps of:
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(a) contacting a probe with a positionally addressable array comprising a plurality of proteins, with each protein being at a different position on a solid support, wherein the plurality of proteins comprises at least 50% of all proteins expressed in a single species, wherein protein isoforms and splice variants are counted as a single protein; and
(b) detecting any protein-probe interaction.
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31. A method for detecting a binding protein comprising the steps of:
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(a) contacting a probe with a positionally addressable array comprising a plurality of proteins, with each protein being at a different position on a solid support, wherein the plurality of proteins comprises at least 1000 proteins expressed in a single species; and
(b) detecting any protein-probe interaction.
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32. A method for detecting a binding protein comprising the steps of:
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(a) contacting a probe with a positionally addressable array comprising a plurality of proteins, with each protein being at a different position on a solid support, wherein the plurality of proteins in aggregate comprise proteins encoded by at least 1000 different known genes in a single species; and
(b) detecting any protein-probe interaction.
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33. A method for detecting a binding protein comprising the steps of:
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(a) contacting a probe with a positionally addressable array comprising a plurality of fusion proteins, with each fusion protein being at a different position on a solid support, wherein the fusion protein comprises a first tag, a second tag, and a protein sequence encoded by genomic nucleic acid of an organism; and
(b) detecting any protein-probe interaction.
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52. A method of labeling a protein for use in a binding assay, comprising the steps of:
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(a) contacting separate aliquots of said protein with a biotin-transferring compound under conditions and for a period of time to produce said proteins that are biotinylated to differing degrees among the different aliquots; and
(b) combining together said different aliquots to produce a sample of differentially biotinylated protein. - View Dependent Claims (53, 54)
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55. A method for determining whether a protein preferentially binds phosphatidylinositol as compared with phosphatidylcholine, comprising the steps of:
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(a) contacting a probe comprising phosphatidylinositol with a positionally addressable array comprising a plurality of proteins, with each protein being at a different position on a solid support;
(b) detecting protein-probe interaction, wherein said interaction at a position on the solid support indicates the presence of a phosphatidylinositol-binding protein;
(c) contacting a probe comprising phosphatidylcholine with a positionally addressable array comprising a plurality of proteins, said proteins comprising at least some of the same proteins as in step (a), with each protein being at a different position on a solid support;
(d) detecting protein-probe interaction, wherein the interaction at a position on the solid support indicates the presence of a phosphatidylcholine-binding protein; and
(e) comparing, for each of a plurality of proteins, the results of steps (b) and (d).
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56. A method for determining if a phospholipid regulates a metabolic pathway or signal transduction pathway in a cell, or if said metabolic or signal transduction pathway occurs on membrane surfaces, comprising the steps of:
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(a) contacting a probe comprising phospholipid with a positionally addressable array comprising a plurality of proteins, with each protein being at a different position on a solid support, wherein the plurality of proteins comprise one or more proteins that form at least part of said pathway; and
(b) detecting interaction of said probe with a protein in said pathway;
wherein said interaction indicates that said probe regulates said metabolic pathway or signal transduction pathway, or that said pathway occurs on membrane surfaces.
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57. A method for making a non-naturally occurring protein that binds calmodulin comprising making a non-naturally occurring protein comprising the following sequence:
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I/L-Q-X—
X—
K—
K/X-G-B
(SEQ ID NO;
1)wherein X is any amino acid and B is a basic amino acid.
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58. A method for determining the presence or absence of a posttranslational modification in a protein comprising the steps of:
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(a) contacting a probe that binds to said posttranslational modification with a positionally addressable array comprising a plurality of proteins, with each protein being at a different position on a solid support; and
(b) detecting any interaction of said probe with a protein;
wherein said interaction at a position on the solid support indicates that the protein at said position has said posttranslational modification. - View Dependent Claims (59)
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60. A method for preparing a culture of yeast cells, comprising the steps of:
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(a) growing a plurality of yeast cells in a growth medium until the OD600 is between 0.3 and 1.0, wherein said plurality of yeast cells comprises a heterologous nucleotide sequence operatively linked to a regulatory sequence, (b) contacting said cell with an inducer that enhances expression of a protein encoded by said heterologous nucleotide sequence;
(c) separating said cells from said medium;
(d) contacting said cells with cold water;
(e) separating said cells from said cold water;
(f) contacting said cells with cold lysis buffer;
(g) separating said cells from said lysis buffer; and
(h) freezing said cells semi-dry for storage.
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61. A method for purifying a protein from a cell, comprising the steps of:
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(a) for each of a plurality of cell samples, lysing cells in each of said sample to produce a cell lysate, wherein said cell comprises a fusion protein having an affinity tag, and wherein said lysing step is performed using a paint shaker;
(b) separating each said lysate into a soluble fraction and a non-soluble fraction;
(c) transferring each said soluble fraction into a different site of a multi-site array, wherein said transferring step is performed using a wide-open tip;
(d) contacting each said soluble fraction with a binding agent such that a complex between said fusion protein and binding agent is formed;
(e) isolating each said fusion protein from the complex; and
(f) storing each said fusion protein in a buffer of high viscosity.
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62. A method for identifying whether a signal is positive, comprising the steps of:
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(a) determining foreground and background signals for each spot locally and determining net signals from the difference between said foreground and background signals;
(b) determining the lower quartile, median, and upper quartile values of a first and second net signal distribution;
(c) subtracting a first median value from said first net signal distribution, and subtracting a second median value from said second net signal distribution to obtain a first and second subtracted value, respectively;
(d) dividing said first subtracted value by the difference between said upper and lower quartile values of said first signal distribution, and dividing said second scaled value by the difference between said upper and lower quartile values to obtain a first and second scaled value, respectively;
(e) determining a local median value of a scaled signal distribution of a neighborhood region, wherein said neighborhood region comprises a plurality of sites in the area; and
(f) subtracting the local median value from the scaled signal to obtain a scaled excess value. - View Dependent Claims (63, 64, 65, 74)
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66. A method for identifying positive signals among signals measured with a plurality of different arrays, comprising the steps of:
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(a) transforming signals measured with different arrays to generate transformed signals;
(b) correcting each said transformed signal by a method comprising subtracting from said transformed signal a local median signal to generate a corrected transformed signal, wherein said local median signal is the median of signals in a neighborhood region, said neighborhood region comprising one or more sites around site of said transformed signal; and
(c) comparing said each said corrected transformed signal to a threshold value, and identifying said corrected transformed signal as positive if said corrected transformed signal is greater than said threshold value;
wherein said array comprises a positionally addressable array comprising a plurality of proteins, with each protein being at a different position on a solid support. - View Dependent Claims (67, 68, 69, 70, 71)
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73. A method for making a positionally addressable array comprising the step of attaching a plurality of fusion proteins to a surface of a solid support, with each fusion protein being at a different position on the solid support, wherein the fusion protein comprises a first tag, a second tag, and a protein sequence encoded by genomic nucleic acid of an organism.
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75. A method for determining the presence or absence of an enzymatic activity in a protein comprising the steps of:
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(a) contacting a probe that is a substrate for said enzymatic activity with a positionally addressable array comprising a plurality of proteins, with each protein being at a different position on a solid support; and
(b) detecting any catalysis of said substrate at a position on the solid support;
wherein said catalysis at a position on the solid support indicates that the protein at said position has said enzymatic activity.
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76. A method for determining the presence or absence of an enzyme substrate in a protein comprising the steps of:
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(a) contacting a probe that is an enzyme for said enzyme substrate with a positionally addressable array comprising a plurality of proteins, with each protein being at a different position on a solid support; and
(b) detecting any catalysis of said substrate at a position on the solid support;
wherein said catalysis at a position on the solid support indicates that the protein comprises said enzyme substrate.
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Specification