WO2010009387A1

WO2010009387A1 - Methods for controlling protein loading variability in reverse phase protein microarrays

Info

Publication number: WO2010009387A1
Application number: PCT/US2009/050981
Authority: WO
Inventors: Emanuel Petricoin Iii; Lance Liotta; Gabriela Lavezzari
Original assignee: Theranostics Health, Llc
Priority date: 2008-07-18
Filing date: 2009-07-17
Publication date: 2010-01-21

Abstract

Methods for controlling for loading or printing variability between samples in protein microarray assays are provided, as well as protein microarrays and kits for use in employing the methods. These methods provide an advantage over conventional microarray techniques by a providing a more accurate determination of analyte quantity in a sample.

Description

METHODS FOR CONTROLLING PROTEIN LOADING VARIABILITY IN REVERSE PHASE PROTEIN MICRO ARRAYS

BACKGROUND

[0001] An important aspect to diagnosing or treating a disease or condition using an in vitro protein assay is to ensure that accurate measurements of protein analytes or biomarkers in a sample are made using the assay.

[0002] Protein microarray assays are robust and tremendously sensitive methods that can be used to quantitate protein analytes from small quantities of biological material. However, despite the robustness of these methods, a major source of variance of this method is often the inability to effectively account for and control for loading or printing variability between samples. This is especially true for samples containing low total protein concentrations or for sample that are comprised of a lysate from a small number of cells, such as from a laser capture microdissected sample or from a FACS (fluorescent activated cell sorter) cell sample. A critical step in analyzing protein microarray results is to normalize the intensity values obtained for a specific protein analyte by a total protein determinant. Normalization is used to account for differences in analyte signal intensity due to simple differences in the protein concentration of each sample when multiple samples are tested, as well as differences in the amount of protein actually printed in each microarray dot when one sample is tested. Thus, use of a normalization step allows for the identification of differences in analyte intensity that are related to the underpinning biology, rather than to variances arising from microarray methodology.

[0003] The current state of the art is to determine the total protein value for a sample loaded on one slide, measure an analyte from the sample on a second, different, slide, and then use the total protein value determined from the first slide to normalize the analyte measurement. This method typically utilizes total protein assays such as ruthenium-based dyes (e.g., SYPRO Ruby Red stain (Molecular Probes®, from Invitrogen Corporation (Carlsbad, CA)) or Deep Purple™ total protein stain (GE Healthcare Bio-Sciences Corp. (Piscataway, NJ)), which employ chemical dyes that bind to all protein and provide a fluorescent signal, or colloidal gold or colloidal coomassie, which provide a colorimetric display. [0004] The current method suffers from several disadvantages. One major disadvantage is that the method assumes that the amount of sample printed on each slide (e.g., in each microarray dot), and in any given slide, is equivalent. If the amount of sample is not the same, then normalization will not be accurate. Even with current high-end printing technologies, exact amounts of sample are often not printed every time (for example, due to evaporation from the sample reservoir during printing of individual spots), so that use of current methods to quantitate the amount of analyte in a sample can produce significant variance.

[0005] Another disadvantage is that this method is potentially limited by the amount of sample available. Since one sample is needed for the assay and another to determine total protein, the method cannot be used for samples having low amounts of protein. For example, with lysate samples generated from laser capture microdissected cells, which are often used in reverse phase protein microarray assays, it is often impossible to measure protein directly using standard dye-based solution methods without using up most, if not all, of the sample to make a protein determination.

[0006] Improved accuracy in the measurement of protein analytes or biomarkers is particularly important when a concentration is being determined for an analyte that is a biomarker associated with a disease or condition. Accurate measurement of the biomarker may be critical for diagnosis and for use in guiding therapeutic decision making by health professionals.

[0007] Thus a need exists for improved methods of controlling for loading or printing variability between samples in protein microarray assays, to improve the accuracy of analyte measurements in these assays.

SUMMARY

[0008] Methods for controlling for loading or printing variability between samples in protein microarray assays are provided, as well as protein microarrays and kits for use in employing the methods. These methods provide an advantage over conventional microarray techniques by a providing a more accurate determination of analyte quantity in a sample.

[0009] In some aspects, a method is provided for correcting protein microarray assay results for variable sample loading, the method comprising: (a) determining an amount of analyte protein in a sample present in one or more dots in a microarray by determining an amount of the analyte protein in each of the one or more dots; (b) determining an amount of total protein in the sample present in the one or more dots in the microarray by determining an amount of total protein in each of the one or more dots; and (c) normalizing the amount of analyte protein determined in (a) using the amount of total protein therein determined in (b); wherein both the amount of analyte protein and the amount of total protein are determined within the exact same dots in the microarray in the microarray.

[0010] In other aspects, a method is provided for correcting an amount of analyte protein determined using a protein microarray assay for variable sample loading in the assay, the method comprising: (a) determining an amount of analyte protein present in a microarray dot; (b) determining an amount of total protein in the microarray dot; and (c) using the amount of total protein determined in (b) to normalize the amount of analyte protein determined in (a) for the microarray dot; wherein both the amount of analyte protein in (a) and the amount of total protein in (b) are determined within the same microarray dot.

[0011] In yet other aspects, a method is provided for normalizing results obtained in a protein microarray assay, the method comprising: (a) providing a biological sample on a substrate, wherein the sample is placed in one or more individual spots on the substrate to form a set of spots for the sample; (b) contacting each of the one or more spots in the set with an antibody specific for a target protein; (c) detecting the antibody in each of the one or more spots to determine an amount of the target protein present in each spot; (d) using the amount of the target protein present in each of the one or more spots to determine an amount of the target protein present in the sample; (e) determining an amount of total protein present in each of the one or more spots to determine an amount of total protein present in the sample; and (f) normalizing the amount of the target protein determined in (d) using the amount of total protein determined in (e); wherein both the amount of target protein and the amount of total protein are determined within the exact same spot in the set.

[0012] In some aspects, a method is provided for normalizing results obtained in a protein microarray assay, the method comprising: (a) providing a plurality of biological samples on a substrate, wherein each biological sample is placed in one or more individual spots on the substrate to form a set of spots for the sample, and the plurality of samples form a plurality of sets arranged in a microarray on the substrate; (b) for the sample in each set of spots, contacting each of the one or more spots in the set with an antibody specific for a target protein; (c) detecting the antibody in each of the one or more spots in the set to determine an amount of the target protein present in each spot of the set; (d) using the amount of the target protein present in each spot of the set to determine an amount of the target protein present in the sample; (e) determining an amount of total protein present in each of the one or more spots of the set to determine an amount of total protein present in the sample; and (f) normalizing the amount of the target protein in the sample determined in (d) using the amount of total protein for the sample determined in (e) for each set of spots.

[0013] In other aspects, a method is provided for normalizing an amount of a cell signaling protein for variable sample loading in a protein microarray assay, the method comprising: (a) providing a plurality of biological samples on a substrate, wherein each biological sample is placed in one or more individual spots on the substrate to form a set of spots; (b) for each set of spots, contacting the one or more spots in the set with a first antibody specific for a cell signaling protein, wherein the first antibody is conjugated to a first detectable label; (c) detecting the first antibody in the one or more spots to determine an amount of the cell signaling protein in each spot in the set; (d) using the amount of the cell signaling protein in each of the one or more spots of the set to determine an amount of the target protein present in the sample; (e) contacting the one or more spots in the set with a second antibody specific for a second protein distinct from the cell signaling protein, wherein the second protein is present in each spot of the set in an amount substantially equal to the amount of protein present in the spot, and wherein the second antibody is conjugated to a second detectable label; (f) detecting the second antibody in the one or more spots in the set to determine an amount of the second protein in each spot; (g) using the amount of the second protein in each spot to determine an amount of total protein present in the sample; and (h) normalizing the amount of the cell signaling protein determined in (d) for the sample using the amount of the second protein determined in (g) for the same sample.

[0014] In other aspects, a method is provided for determining an amount of one or more cell signaling proteins in a biological sample, the method comprising: (a) providing a biological sample on a substrate, wherein the sample is placed in multiple spots on the substrate to form a microarray; (b) providing a plurality of first antibodies, wherein each first antibody is specific for a different cell signaling protein, wherein each first antibody is conjugated to a different first detectable label, and wherein each first antibody is placed on at least one spot in the microarray such that no two first antibodies are placed together in the same spot; (c) detecting the first antibodies to determine an amount of the cell signaling protein in each spot; (d) determining an amount of total protein in each spot; and (e) normalizing the amount of the cell signaling protein determined in (c) using the amount of total protein determined in (d).

[0015] In yet other aspects, a method is provided for detecting the presence of a disease in a subject, the method comprising: (a) providing a biological sample on a substrate, wherein the sample is placed in multiple spots on the substrate to form a microarray; (b) providing a plurality of first antibodies, wherein each first antibody is specific for a different cell signaling protein or for a different post-translationally-modified cell signaling protein, wherein each first antibody is conjugated to a different first detectable label, and wherein each first antibody is placed on at least one spot in the microarray such that no two first antibodies are placed together in the same spot; (c) detecting the first antibody in each spot to determine an amount of the cell signaling protein in the spot; (d) determining an amount of total protein in each spot; (e) normalizing the amount of the cell signaling protein in each spot using the amount of total protein in the spot; and (f) determining the presence of a disease in a subject based on the normalized amounts of the cell signaling proteins.

[0016] In further aspects, a method is provided for screening a plurality of candidate compounds for treating a disease caused by a defect in a cell signaling pathway, the method comprising: (a) providing a plurality of biological samples on a substrate in individual spots to form a microarray, wherein each sample is derived from the same cell type, and wherein each sample has been cultured with a different candidate compound; (b) contacting each spot with a first antibody, wherein the first antibody is specific for a cell signaling protein, and wherein the first antibody is conjugated to a first detectable label; (c) detecting the first antibody in each spot to determine a corresponding amount of cell signaling protein in the spot; (d) determining an amount of total protein in each spot; (e) normalizing the amount of the cell signaling protein in each spot using the corresponding amount of total protein in the spot; and (f) determining an effective candidate compound based on the normalized amounts of the cell signaling proteins determined in (e).

[0017] In yet further aspects, a kit for detecting a disease caused by a defect in a cell signaling pathway in a subject, the kit comprising: (a) a first antibody conjugated to a first detectable label, wherein the first antibody is specific for a protein the cell signaling pathway; and (b) a second antibody conjugated to a second detectable label, wherein the second antibody is specific for a housekeeping protein.

[0018] In other aspects, a protein microarray is provided comprising: (a) a plurality of biological samples on a substrate, wherein each biological sample is present in one or more individual spots on the substrate, and the individual spots form a microarray on the substrate; (b) a plurality of first antibodies disposed on one or more spots in the microarray, wherein each first antibody is specific for a target protein present in one or more spots in the microarray, and each first antibody is conjugated to a first detectable label, and (c) a plurality of second antibodies disposed on one or more spots in the microarray, wherein: each second antibody is specific for a second protein in one or more spots in the microarray, the second protein is distinct from the target protein, the second protein is present in one or more spots in an amount representative of total protein present in the spot, and each second antibody is conjugated to a second detectable label.

[0019] In other aspects, a method is also provided for screening a plurality of candidate compounds for treating a disease caused by a defect in a cell signaling pathway, the method comprising: (a) treating animal cells with a candidate compound; (b) obtaining a lysate of said treated animal cells and placing the lysate on one or more spots on a substrate to form a microarray; (c) contacting each spot with a first antibody, wherein the first antibody is specific for a cell signaling protein, and wherein the first antibody is conjugated to a first detectable label; (d) detecting the first antibody in each spot to determine a corresponding amount of cell signaling protein in the spot; (e) determining the amount of total protein in each spot; (f) normalizing the amount of the cell signaling protein in each spot using the corresponding amount of total protein value obtained in step (d); and (g) identifying an effective candidate compound.

[0020] Other objects, features and advantages will become apparent from the following detailed description. The detailed description and specific examples are given for illustration only since various changes and modifications within the spirit and scope of the invention will become apparent to those skilled in the art from this detailed description. Further, the examples demonstrate the principle of the invention and cannot be expected to specifically illustrate the application of this invention to all the examples where it will be obviously useful to those skilled in the prior art. BRIEF DESCRIPTION OF FIGURES

[0021] Figure 1 presents a series of bar graphs showing the results of experiments described in Example 1 below. "CV" refers to coefficient of variance.

DETAILED DESCRIPTION

[0022] Methods have been developed for controlling loading and printing variability in protein microarray assay results, which provide a more accurate determination of the amount of an analyte in a sample. The methods involve directly measuring the total protein of a sample in the exact same spot or dot of a microarray from which the analyte of interest is measured, thus eliminating the major source of analytical variability for the array analysis. In the methods, the amount of an analyte present in a microspot or dot of a microarray is determined, the amount of total protein in the same dot is determined, and then the amount of analyte is normalized using the amount of total protein determined for the dot. In contrast, conventional protein microarray assay methods measure the total protein of a sample in a separate reference sample distinct from the sample used to measure the analyte. Compared to conventional methods, the current method provides more accurate analyte measurements by eliminating variability due to differences in sample loading between the reference and samples that is often present in conventional microarray assays methods.

[0023] The new methods provide the experimentalist with the ability to measure both the analyte and the total protein loaded on that spot directly, and often at substantially the same time, providing a superior analytical method with reduced coefficient of variance. The methods can be used both for both research and clinical purposes. In particular, the methods can be used therapeutically, in methods of detecting or diagnosing diseases or conditions, such as cancer, as well as in methods of treating such diseases and conditions. The methods can also be used to screen compounds for treating a disease. See, for example, U.S. Pat. Appl. No. 10/798,799, "Combinatorial Therapy for Protein Signaling Diseases," filed March 10, 2004, as well as International Publication Nos. WO 2007/047754, WO 2007/106432, WO 2008/057305, WO 2008/054764, and WO 2008/054763. Each of these applications and publications is herein incorporated by reference in its entirety. Protein Microarrays and Assays

[0024] Although the present methods can be used in any number of assay systems in which a protein analyte is measured, the methods are most advantageously used in protein microarray assays.

[0025] Protein microarray assays are well known in the art. See, for example, Liotta et al., Cancer Cell. 3(^:317-25 (2003), and Paweletz et al., Oncogene 20(16)Λ98\-9 (2001). See also Kingsmore, S.F., Nat. Rev. Drug Discov. 5(4) :310-320 (2006). A protein microarray is an assay format that utilizes a substrate for simultaneously testing multiple samples. The microarray format is not limited to particular embodiments, but can comprise any arrangement and substrate that serves to provide a plurality of individual samples for testing. For example, in some embodiments, the microarray comprises a flat substrate with rows and columns of individual spots, each spot comprising a sample, while in other embodiments, the microarray comprises a flat substrate with a plurality of depressions, for example, a 96-well plate, in which each depression contains one sample.

[0026] Substrates suitable for use in protein microarrays should have a (1) high binding capacity, (2) minimum effect on the protein structure and (3) low background. Examples of typical microarray substrates include nitrocellulose, derivitized glass slides, and three- dimensional substrates such as hydrogels. In some embodiments of the method, the substrate is a nitrocellulose coated glass slide. Examples of nitrocellulose-coated glass slides include FAST slides (Schleicher & Schuell BioSciences, Keene, NH), which have protein binding capacities of 75-150 ug/cm² in a volume of 0.3-2nL/spot. Nitrocellulose-coated glass slides are particularly useful, as a variety of detection methods can be used with this substrate, including chromogenic, fluorometric and luminescent detection methods.

[0027] The number of samples deposited onto a microarray substrate can vary. For example, the size of the substrate can often determine how many samples are located on the substrate. In some embodiments, the protein microarray comprises about 100 spots, while in other embodiments, it comprises about 500 spots, about 1,000 spots, about 5000 spots, or about 10,000 spots. In yet other embodiments, the microarray comprises about 1 to about 10,000 spots, about 100 to about 10,000 spots, or about 500 to about 10,000 spots. In some embodiments, the microarray comprises less than about 100,000 spots, or less than about 10,000 spots. Typically, the microarray comprises about 500 to about 10,000 spots.

[0028] The sample volume deposited on each spot, or used to form each spot, on the microarray can also vary. The volume can depend on the diameter of the pin (contact printing), the inherent qualities of the pin hydrophobicity, as well as the method of supplying the sample. In some embodiments, for example, the amount of sample deposited or printed can range from less than about 1 picoliter to about 100 nanoliters, about 10 picoliters to about 10 nanoliters, or about 100 picoliters to about 1 nanoliter. In other embodiments, the amount of sample deposited or printed can range from about 1 picoliter to about 1 nanoliter, about 500 nanoliters to about 1 nanoliter, about 100 nanoliters to about 1 nanoliter, or about 75 nanoliters to about 1 nanoliter.

[0029] Samples can be placed or loaded onto the substrate using any one of a number of mechanisms known in the art. See Schena, "Microarray biochip technology." Eaton Pub., Natick MA, 2000, incorporated herein by reference in its entirety. For example, in some embodiments, the samples are printed onto the microarray using a printer. The printing technique can be contact or non-contact printing. Contact printing involves direct contact between a print head and the substrate. Examples of embodiments used for contact printing include a solid pin, quill, and pin and ring assemblies. Non-contact printing involves dispensing a minute volume of sample above the substrate. Examples of embodiments used for non-contact printing include piezoelectric, acoustic wave, or syringe solenoid devices. In yet other embodiments, the printing is automated.

[0030] For example, in some embodiments, contact printing is performed by a printer having a plurality of pins. The spacing between the pins (pinhead spacing) can be, for example, 9.0 mm, a spacing that is compatible with 96-well, 384-well, or 1536 well plates for holding the sample volume. The pins are dipped into the wells within the plates to allow sample to be deposited onto the pins. The pins are then transferred to a flat substrate, e.g. glass slide, such that the pins make contact with the substrate and spots of samples are correspondingly transferred onto the slide at the points where the pins make contact thereon. For example, printing of the samples using a pinhead spacing of 9.0 mm can be performed on a 25 x 75 mm glass slide. Spots produced using the pins can be greater than the diameter of the pins because the sample can spread outward over the substrate surface. For example, the spots can be about 1.5 times the diameter of each pin. [0031] Protein microarray formats can fall into two major classes, the Forward Phase Array (FPA) and the Reverse Phase Array (RPA), depending on whether the analyte is capture from solution phase or bound to solid substrate.

[0032] Forward Phase Arrays immobilize a bait molecule, such as a antibody designed to capture a specific analyte within a mixture of test sample proteins. In FPAs, the capture molecule specific for the analyte is immobilized on a substrate. The capture molecule is then exposed to the sample, binding the analyte in the sample and immobilizing the analyte onto the substrate. The bound analyte can then be detected using a detectable label. The label can bind to the analyte directly, or can be attached to a secondary "sandwich" antibody that is specific for the analyte. The capture molecule can be any molecule that has specificity for an analyte. Examples of capture molecules include, but are not limited to, peptides, proteins (including, for example, antibodies or fragments thereof, and affybodies), oligomers, nucleic acids (, e.g. DNA and RNA), including aptamers, and PNA.

[0033] For example, in some embodiments, the capture molecule is an antibody or fragment thereof specific for the analyte. In other embodiments, the capture molecule is an affybody. "Affybody" or "Affϊbody®" refers to a small robust protein, derived from a bacterial receptor structure, which may be engineered to bind to virtually any molecular moiety. Affibodies are commercially available through Affϊbody, Teknikringen 30, floor 6, Box 700 04, Stockholm SE-10044, Sweden, and are the subject of U.S. Pat. No. 5,831,012.

[0034] In some embodiments, a series of capture molecules are arranged in an array format to provide a microarray of individual spots with capture molecules in each spot. Multiple spots can comprise the same type of capture molecule or can comprises different types of capture molecules. In some embodiments, the microarray comprises different types of capture molecules at each spot, thereby allowing multiple types of analytes to be simultaneously immobilized from one sample.

[0035] Reverse Phase Arrays immobilize the test sample analytes on the solid substrate. In RPAs, the sample is placed directly on the substrate, allowing analyte in the sample to bind directly to the substrate. A detection molecule specific for the analyte is then exposed to the substrate, allowing an analyte-detection molecule complex to form. The detection molecule can comprise a detectable label to indicate the presence of the analyte. Alternatively, a secondary molecule specific for the detection molecule and comprising a detectable label can be provided, allowing for an analyte-detection molecule-labeled secondary molecule complex to form.

[0036] RPAs are highly sensitive and do not require a large amount of sample. The high sensitivity exhibited by RPAs is due in part to the detection molecule, which can be conjugated to a detectable label, and is also due in part to the fact that the signal from the label can be amplified independently from the immobilized analyte. Amplification chemistries that are available take advantage of methods developed for highly sensitive commercial clinical immunoassays (see, for example, King et al., J. Pathol. 183:231-241 (1997)). For example, RPA can utilize tyramide amplification, which generates high fluorescent signals on each spot. Additionally, if a nitrocellulose substrate is used, detectable labels for the analyte can be chosen which emit fluorescent signals in the near-IR range, avoiding conflicting signals from nitrocellulose autofluorescence. Using commercially available automated equipment, RPAs can also exhibit excellent "within run" and "between run" analytical precision. RPAs do not require direct labeling of the sample analyte, and do not utilize a two-site antibody sandwich. Therefore, there is no experimental variability introduced due to labeling yield, efficiency or epitope masking.

[0037] In RPA assays, the detection molecule and secondary molecule can be any molecule with specificity for the analyte and capture molecule, respectively. Examples of detection and secondary molecules include, but are not limited to, peptides, proteins (including, for example, antibodies or fragments thereof, and affybodies), oligomers, nucleic acids (, e.g. DNA and RNA), including aptamers, and PNA. In those embodiments in which both a detection molecule and a secondary molecule are present, the detection and secondary molecules can be the same type of molecule, e.g., a protein, or can be different types of molecules, e.g., the detection molecule can be DNA, and the secondary molecule can be an antibody. In some embodiments, both the detection molecule and the secondary molecule are antibodies or fragments thereof.

[0038] In some embodiments using either an RPA or FPA format, the detection or capture molecule, and, if present, the secondary molecule, are both antibodies or fragments thereof. As described above, the antibody or fragment thereof that functions as the capture or detection molecule is specific for the analyte being tested, or "target protein." The antibody or fragment thereof that functions as the secondary molecule, if present, is typically specific for the detection antibody. Antibodies suitable for use in the methods can be chosen readily by those skilled in the art. A variety of antibodies to biomarkers are commercially available. See, for example, U.S. Pat. Appl. No. 10/798,799, "Combinatorial Therapy for Protein Signaling Diseases," filed March 10, 2004, the entire contents of which is herein incorporated by reference. Antibodies can also be obtained from Cell Signaling, Inc. (Danvers, MA), and BD Biosciences (San Jose, CA).

[0039] In both FRAs and RPAs, the capture molecule, the detection antibody, and the secondary molecule, if present, can comprise a detectable label. For example, the capture molecule, the detection molecule, or the secondary molecule, if present, can be conjugated to a detectable label. The detectable label can be detected using any suitable method known in the art, including, but not limited to, a colorimetric, fluorometric, luminescent, electrical (e.g., electrochemical), or radioactive method of detection. For example, suitable detectable labels include, but are not limited to, fluorescent, radioactive, luminescent, electrical (e.g., electrochemical), and colorimetric labels. The detectable label can also be an enzyme-based label. Methods and techniques for detecting each type of label are well known in the art.

[0040] For fluorescent labels, the labels can have excitation and/or emission spectra in the infrared, near-infrared, visible, or ultra-violet wavelengths. A wide range of fluorescent probes are commercially available (see, e.g., Invitrogen Corporation (Carlsbad, CA), and LI- COR Biosciences (Lincoln, NE)). Examples of suitable fluorescent probes include, but are not limited to, phycoerythrin and other phycobilliproteins, including, for example, allophycocyanin; lanthanide dye based probes; and phthalocyanine dyes. Methods and reagents for coupling fluorescent probes to proteins, including antibodies, are well known in the art. See, for example, technical handbooks from Invitrogen Corporation (Carlsbad, CA) and Pierce (Thermo Fisher Scientific, Inc. (Rockford, IL))

[0041] Examples of suitable radioactive labels include, but are not limited to, C-14, P-32 and S-35.

[0042] Examples of suitable luminescent labels include, but are not limited to, quantum dots, 1,2-dioxetanes and luminol.

[0043] Examples of suitable colorimetric labels, include, but are not limited to, diaminobenzidine (3,3 '-diaminobenzi dine, or 3,3'-diaminobenzidine tetrahydrochloride). [0044] Examples of suitable electrical labels mclude, but are not limited to, enzyme-based labels such as those used in electrochemical enzyme-based immunoassays (such as, for example, electrochemical-based ELISA assays). See, for example, Wilson et al , Anal Chem 75:6476-6483 (2006), Wilson et al , Anal Chem 75:2507-2513 (2006); and Brooks et al , Appl Environ Microbiol J6-3278-3284 (1990); and U S Pat No 5,137,827 The contents of each of these documents are incorporated by reference herein in their entireties

[0045] In some embodiments, the signal from the label can be amplified. Amplification is helpful for achieving sensitivity adequate for analysis of relatively low abundance proteins Amplification of the label signal can be achieved by enzymatic cleavage of colorimetric, luminescent or fluorescent substrates, by utilizing avidin/biotm signal amplification systems known in the art, or by taking advantage of the polymerase chain reaction by coupling nucleic acids to protein for detection. For example, amplification chemistries can take advantage of methods developed for highly sensitive commercial clinical immunoassays See, for example, King et al , J Pathol 183 237-241 (1997) Coupling the capture molecule with highly sensitive tyramide-based avidin/biotin signal amplification systems can also yield detection sensitivities down to fewer than 1,000-5,000 molecules/spot. In a particular embodiment, a biopsy of 10,000 cells can yield 100 RPA microarrays, and each array can be probed with a different antibody.

[0046] In some embodiments of the method, a RPA protein microarray assay is employed Examples of RPA protein microarray assays suitable for use with the present methods are described in U S Pat Appl No 10/798,799, filed March 10, 2004, referenced above

[0047] In the present methods, the analyte to be measured, or "target protein," can be any protein of interest The protein of interest is typically a "biomarker," or biological marker protein that correlates with a specific physiological outcome or state in an individual. For example, in some embodiments, the target protein is a target for a therapeutic drug.

[0048] In some embodiments, the target protein is a cell signaling protein. A "cell signaling protein" is a protein that is part of a cellular signaling pathway, or signal transduction pathway, in an individual

[0049] The terms "signaling pathway" and "signal transduction pathway" refer to an identifiable signal cascade through which a signal flows A signaling pathway can be a series of cellular proteins, such as a cell surface receptor, an intracellular signaling protein or both, along which a signal flows in response to a stimulus. The stimulus can be an extracellular or intracellular stimulus that interacts with a cell at a cell surface receptor. Upon such a stimulus, a series of coordinated intracellular processes can occur, whereby a living cell transduces the external or internal signal into one or more cellular responses.

[0050] Signal transduction may, for example, involve an enzymatic reaction. In this type of a reaction, enzymes include protein kinases, GPTAses, ATPases, protein phosphatases, and phosholipases. The cellular responses that are controlled by signaling pathways include gene transcription, secretion, proliferation, mechanical activity, metabolic activity and cell death. A signaling pathway can carry a signal from the plasma membrane to the nucleus in response to an extracellular stimulus, or can carry a signal from one part of a cell to another, for example, where two or more intracellular signaling pathways interact. Thus, a signal generated in a particular signaling pathway by an extracellular stimulus can flow into other signaling pathways by an extracellular stimulus can flow into other signaling pathways within the cell. In some instances, the signal is mediated by a series of post-translational modifications of the proteins in the pathway such as in protein kinase signaling cascades where, for example, one kinase phosphorylates another kinase that in turn phosphorylates another kinase. In other instances, the signal is mediated by protein translocations or protein- protein interactions. For example, post-translational modification of one protein in a signaling pathway can lead to translocation of the protein to another part of the cell, where the translocated protein then post-translationally modifies yet another protein to pass the signal along the pathway. Alternatively, a post-translational modification of a particular protein can promote its association with another protein to form a dimer, and then the dimmer post-translationally modifies yet another protein to pass the signal along the pathway. Examples of signaling pathways include the integrin pathway, the focal adhesion signaling pathway, the Akt signaling pathway, the IL-6R pathway, growth factor pathways, chemokine receptor signal pathways, cell-cycle signaling pathways, stress signal pathways, apotosis signaling pathways, Tau/beta signaling pathways, pro-inflammatory pathways, differentiation signaling pathways, T-cell receptor pathways, death-receptor signaling pathways, survival signaling pathways, MAPK signaling pathways, p38 MARK signaling pathways, G-coupled Receptor signaling pathways, SAPK/JNK signaling pathways, insulin receptor signaling pathways, Wnt signaling pathways, B-cell antigen signaling pathways, cKit signaling pathways, and Jak/Stat signaling pathways. These and other signaling pathways may be intermediately interconnected to each other by interactions that lead to cross-talk between the pathways to form a signaling network. Interconnections between pathways may be mediated by, for example, protein-protein interactions, post-translational modifications, protein cleavage, translocations to an organelle or compartment, ion channel activation, soluble mediators, protein-nucleic acid interactions, protein-lipid interactions, or protein- carbohydrate interactions can pass signals from one signaling pathway to another.

Methods

[0051] The methods described herein provide accurate quantitation of an analyte in a sample in a protein microarray assay by controlling or correcting for loading and printing variability in the assay. In the methods, the amount of an analyte present in a spot or dot of a microarray is determined, the amount of total protein in the same spot is determined, and then the amount of analyte is normalized using the amount of total protein determined for the spot.

[0052] In the methods, protein microarrays can comprise biological samples from one patient or from a plurality of patients. In some embodiments, biological samples from several patients (e.g., a "plurality of patients") are placed on a substrate to form a microarray, such that each individual microarray spot or dot contains a sample from a different patient.

[0053] In other embodiments, a biological sample of one patient is placed on a substrate such that the sample is placed in two or more spots or dots to form a set of spots on the substrate, each spot in the set containing an aliquot of the same sample. In these embodiments, the sample is placed on the substrate in multiple spots, e.g., duplicate or triplicate spots, so that more accurate measurements of a protein in the sample can be made.

[0054] In yet other embodiments, a plurality of biological samples (either from one patient or from different patients) can be placed on the substrate as a plurality of sets of spots to form a microarray on the substrate, with each set of spots representing a different sample. In these embodiments, for example, each sample would be present in a set of three (triplicate) spots.

[0055] The terms "patient, "subject," and "individual," are used herein interchangeably to refer to a mammal, including a human. Examples of mammals include, but are not limited to, humans; primates; farm animals such as horses, sheep, or cattle; laboratory animals such as mice, rats, rabbits, and guinea pigs; and domestic animals such as dogs, cats and the like.

[0056] The term "set," as used herein in reference to microarray spots or dots, is meant to refer to one or more spots or dots in a microarray. Thus, a "set" of spots can contain only one spot on a microarray substrate. Alternatively, a "set" can contain two or more spots on a microarray substrate. The terms "spot" and "dots," as used herein in reference to a microarray, are used interchangeably and are meant to refer to an aliquot of sample deposited on a microarray.

[0057] In some embodiments of the method, a RPA protein microarray assay (RPMA) is employed. See, for example, U.S. Pat. Appl. No. 10/798,799, filed March 10, 2004, referenced above.

[0058] The biological sample(s) used to form the microarray contain at least one target protein and are generally in the form of a solution. Typically, the biological sample contains more than one target protein. Suitable biological samples include, but are not limited to, cellular lysates obtained from one or more tissue samples from one individual, e.g., a patient, or from different individuals; or obtained from one or more cultured cell lines. Cellular lysates can be obtained using methods known in the art.

[0059] For example, in some embodiments, the biological sample is a cellular lysate obtained from a tissue sample. In some embodiments, the cellular lysate is made using a substantially homogeneous population of cells from the tissue sample. A substantially homogeneous population of cells can be obtained from a tissue sample (or from a biological sample) using methods known in the art such as, for example, microdissection techniques, magnetic bead sorting, and fluorescent activated cell sorting (FACS). Suitable microdissection techniques include, but are not limited to, laser capture microdissection. See, for example, U.S. Pat. No. 6,969,614, which is incorporated by reference herein in its entirety. The techniques of magnetic bead sorting and fluorescent activated cell sorting (FACS) are known in the art. Thus, for example, in some embodiments, the biological sample is a cellular lysate obtained from a tissue sample obtained using laser capture microdissection. In other embodiments, the biological sample is a cell lysate of a sample of cells obtained from an aspirated tissue sample (e.g., a biopsy) that is subsequently subjected to FACS to obtain a substantially homogenous population of cells.

[0060] Suitable biological samples can also include one or more body fluids, such as, for example, serum, urine, vitreous fluid, tears, saliva, nipple fluid, semen, bronchiolar washings, lymph, plasma, peritoneal washings, ascitic fluid and sweat. Thus, in some embodiments, the biological sample is selected from the group consisting of serum, urine, vitreous fluid, tears, saliva, nipple fluid, semen, bronchiolar washings, lymph, plasma, peritoneal washings, ascitic fluid and sweat.

[0061] The amount of analyte or target protein is determined for one or more spots or dots in the microarray.

[0062] The term "amount," as used herein in reference to a protein in a sample, refers to the quantity or level of the protein in the sample. For example, in some embodiments, the amount of the protein can be expressed as the quantity of protein in the sample in terms of total mass of the protein in the sample (e.g., micrograms), as the mass per unit volume of the sample, as the number of moles of the protein (e.g., micromoles), or moles protein per unit volume of sample. The amount of protein can also be expressed in terms of the signal produced by the label used to detect the protein (e.g., a fluorescent label) that is proportional to the quantity of the protein present. Thus, in some embodiments in which the protein in a sample is detected using a fluorescent label, the amount of protein can be expressed as the intensity of the fluorescent signal (e.g., in units of fluorescence intensity) that is measured for the protein.

[0063] The term "determine" or "determining," as used herein in reference to the amount of a protein in a sample, refers to measuring (directly or indirectly) the amount of the protein (e.g., to measuring the signal from a label used to detect the protein, that is proportional to the quantity of protein), as well as to calculating the quantity of the protein (e.g., calculating the quantity of protein using a signal from a label used to detect the protein to arrive at the mass or number of moles of the protein). The terms "determine" and "measure" are also used herein interchangeably.

[0064] In some embodiments, the amount of target protein is determined by contacting the spot with a capture or detection molecule specific for the analyte or target protein.. Examples of suitable capture or detection molecules are described above and include, but are not limited to, peptides, proteins (including, for example, antibodies or fragments thereof, and affybodies), oligomers, nucleic acids (, e.g. DNA and RNA), including aptamers, and PNA. For example, in some embodiments, the capture or detection molecule is an antibody or a fragment thereof specific for the target protein.

[0065] The capture or detection molecule can also be conjugated to a detectable label as described above. For example, in some embodiments, the capture or detection molecule is an antibody or fragment thereof specific for the target protein, and is conjugated to a fluorescent label.

[0066] The amount of total protein is also determined for the same one or more spots or dots in the microarray that were analyzed for the target protein. In some embodiments, this is accomplished by contacting the spot with a capture or detection molecule that is a dye, such as fluorescent dye, that binds to proteins in general, based on charge, and functions as a detectable label. An example of this type of dye suitable for use in the method is the fluorescent dye SYPRO Ruby Red (Molecular Probes®, from Invitrogen Corporation (Carlsbad, CA))).

[0067] In other embodiments, the amount of total protein is determined using a capture or detection molecule specific for a second protein that is representative of the total protein present in the spot. This second protein is distinct from the target protein or analyte. Suitable examples of this second protein include one or more "housekeeping proteins," such that the amount of the housekeeping protein on the microarray spot or dot is essentially invariant between any given sample. Representative housekeeping proteins suitable for use as the second protein in the method include, but are not limited to, β-actin and GAPDH.

[0068] The capture or detection molecule specific for the second protein can be, for example, a peptide, protein (including, for example, an antibody or a fragment thereof, or an affybody), an oligomer, nucleic acid (, e.g. DNA and RNA), including an aptamer, and PNA. The capture or detection molecule can also be conjugated to a detectable label, as described above.

[0069] For example, in some embodiments, the capture or detection molecule used to detect the second protein is an antibody or fragment thereof that is conjugated to a detectable label, such as a fluorescent label, as described above ("second detectable label"). This second detectable label is typically distinct from the detectable label used to detect the target protein ("first detectable label').

[0070] The first detectable label used to detect the target protein, and the second detectable label used to determine total protein, can be the same type of label, or they can be different types of labels. For example, in some embodiments, the second detectable label is the same type of label used to detect the target protein, but is still distinct from it. For example, in some embodiments, a first fluorescent label is used to label the target protein and a second fluorescent label is used to label the protein (e.g., housekeeping protein) used for determining total protein. The first and second fluorescent labels have different excitation and emission spectra in order for the user to distinguish between analyte and total protein. For example, the fluorescent label conjugated to the first antibody (used to detect the target protein) and the fluorescent label conjugated to the second antibody (used to detect the housekeeping protein) can be two near-IR coupled dyes. Near-IR coupled dyes can be commercially obtained (see, for example, LI-COR Biosciences UK Ltd, Cambridge, United Kingdom).

[0071] In other embodiments, the first detectable label used to detect the target protein, and the second detectable label used to determine total protein, are different types of labels. For example, in some embodiments, one of the detectable labels is a fluorescent label, while the second label is a chemiluminescent label.

[0072] The target protein and total protein in each spot can be determined substantially simultaneously, or they can be determined at different times, e.g., sequentially. For example, in some embodiments, the first detectable label that is used to detect the target protein, and the second detectable label that is used to detect total protein, in each spot are different fluorescent labels that are detected sequentially, e.g., read using the same fluorometric device in sequential order, using two different emission filters.

[0073] In the present methods, each spot is analyzed for both the target protein and for total protein. The amount of analyte obtained from each spot is then normalized using the amount of total protein determined for the same spot.

[0074] In general, normalizing a sample data point is accomplished by dividing the signal from the detectable label used to measure the target protein in a spot by the signal from the detectable label used to measure total protein in the same spot. For example, in some embodiments, the target protein is detected using a fluorescent label, such as a LI-COR near- IR dye, and the total protein is detected using a fluorescent label such as SYPRO Ruby Red, as described above. In these embodiments, for a microarray spot, the signal for target protein in the spot is normalized by dividing the fluorescence intensity value obtained at least 2 standard deviations above background for the LI-COR near-IR dye in the spot, with the fluorescence intensity value obtained at least 2 standard deviations above background for the SYPRO Ruby Red-stained image for the same spot. [0075] In some embodiments, as described above, a sample is analyzed using multiple spots, e.g., in duplicate or triplicate (i.e., the sample is placed on the microarray substrate as two (duplicate) or three (triplicate) separate spots), so that multiple measurements are obtained for the one sample. When multiple spots are used to measure the amount of target protein (and total protein) in a sample, the mean of the amounts of the target protein determined for the multiple spots is determined, and then divided by the mean of the amounts of total protein determined for the same multiple spots. For example, in those embodiments in which duplicate measurements are made, the sample is spotted onto the substrate in the microarray as two separate spots. The amount of target protein in each spot is measured as described above to give two values for the amount of target protein in the sample (i.e., one value for the target protein in each spot). Similarly, two values for the amount of total protein in the sample are obtained (i. e. , one value for total protein in each spot). In such embodiments, the target protein values for the sample are normalized by (i) first calculating a mean (arithmetic mean, or average) value for the amount of target protein in the duplicate spots (e.g., by adding the two target protein values from the two separate spots, and dividing by 2, the number of spots); (ii) calculating a mean value for the amount of total protein in the same two spots; and them (iii) dividing the mean value for the target protein by the mean value for total protein of the two spots.

[0076] The present methods can be used in any application in which a protein microarray assay is used, to provide more accurate determinations of the amount of an analyte in a sample. The methods can be used both for both research and clinical purposes. For example, the methods can be used therapeutically, in a method for detecting or diagnosing a disease or condition, such as cancer, as well as in a method for treating such disease and condition. See, for example, U.S. Pat. Appl. No. 10/798,799, "Combinatorial Therapy for Protein Signaling Diseases," filed March 10, 2004, as well as International Publication Nos. WO 2007/047754, WO 2007/106432, WO 2008/057305, WO 2008/054764, and WO 2008/054763. Each of these applications and publications is herein incorporated by reference in its entirety. In some embodiments, the methods are useful for detecting or diagnosing a disease or condition caused by a defect in a cell signaling pathway.

[0077] The methods can also be used to screen compounds for treating a disease. For example, the methods can be used in a method for screening a plurality of candidate compounds, to identify a compound or combination of compounds effective to treat a disease or condition. In some embodiments, the method is used in a method of screening candidate compounds to identify a compound or combination of compounds effective to treat a disease or condition caused by a defect in a cell signaling pathway.

Kits

[0078] Kits can also be prepared comprising materials for performing a protein microarray assay employing the present methods. In some embodiments, the kit comprises a substrate, a labeled antibody specific for an analyte or target protein and a labeled antibody specific for a housekeeping protein. In other embodiments, the kit comprises a substrate, multiple antibodies for different analytes, each antibody labeled with a different detectable label, and a labeled antibody specific for a housekeeping protein. In yet other embodiments, the kit comprises an antibody specific for a protein that is representative of total protein.

[0079] The kit can comprise solutions and/or reagents necessary to perform an assay using a protein microarray.

[0080] The kit can be used for research purposes or for therapeutic or clinical purposes. For example, in some embodiments, the kit is used for detecting a disease or condition in a subject, for example, a disease or condition caused by a defect in a cell signaling pathway.

[0081] The following examples are provided to illustrate the invention and do not limit the scope thereof. One skilled in the art will appreciate that although specific reagents and conditions are outlined in the following examples, modifications can be made which are meant to be encompassed by the spirit and scope of the invention.

EXAMPLES Example 1

[0082] Three separate antibodies were used in a series of experiments employing reverse- phase microarray assays, to demonstrate the utility of the disclosed method for determining loading control for reverse phase protein microarrays.

[0083] For each experiment, a tissue sample lysate containing activated phosphoproteins or cleaved proteins was spotted 50 separate times on the same slide, as 50 separate microarray spots on the same slide, and across 50 separate slides, as separate microarray spots on 50 different slides, for within-slide (intra) and between-slides (inter) reproducibility precision assessments. Both sets of slides were then probed with one of the following three antibodies to detect the target protein in each spot: caspase-3 -cleaved D175, pErk 1-2 T202 Y204, and pp70 S6 Kinase T389. Total protein was determined in each spot using Sypro Ruby Red stain. A duplicate "control" experiment was performed in which the target protein was measured for each spot as described above, but the total protein for the sample was measured in one or more spots on a separate, external slide. The results for each experiment and it control are shown in the bar graphs in Figure 1.

[0084] The Figure 1 bar graphs for each set of experiments show a greatly improved (i. e. , lower) coefficient of variance ("CV") for both inter and intra-assay precision by normalizing the protein loading within the same spot using Sypro staining followed by analyte detection (left column, in white, for each set of bar graphs) compared to data obtained by normalizing the analyte signal to a total protein measurement obtained from a externally stained, separate slide ("control experiment", represented by the right column, in black, for each bar graph). The average decrease in CV by the new method was 20% and 30% for intra-slide and inter- side precision, respectively for the three analytes measured.

[0085] All publications, patents, and patent applications cited in this specification are herein incorporated by reference in their entirety to the same extent as if each individual document was specifically and individually indicated to be incorporated by reference in its entirety. While the invention has been described with reference to a particularly preferred embodiment, it will be appreciated that modifications can be made without departing from the spirit of the invention. Such modifications are intended to fall within the scope of the appended claims.

Claims

1. A method of correcting protein microarray assay results for variable sample loading, the method comprising:

(a) determining an amount of analyte protein in a sample present in one or more dots in a microarray by determining an amount of the analyte protein in each of the one or more dots;

(b) determining an amount of total protein in the sample present in the one or more dots in the microarray by determining an amount of total protein in each of the one or more dots; and

(c) normalizing the amount of analyte protein determined in (a) using the amount of total protein therein determined in (b); wherein both the amount of analyte protein and the amount of total protein are determined within the exact same dots in the microarray.

2. A method for correcting an amount of analyte protein determined using a protein microarray assay for variable sample loading in the assay, the method comprising:

(a) determining an amount of analyte protein present in a microarray dot;

(b) determining an amount of total protein in the microarray dot; and

(c) using the amount of total protein determined in (b) to normalize the amount of analyte protein determined in (a) for the microarray dot; wherein both the amount of analyte protein in (a) and the amount of total protein in (b) are determined within the same microarray dot.

3. A method of normalizing results obtained in a protein microarray assay, the method comprising:

(a) providing a biological sample on a substrate, wherein the sample is placed in one or more individual spots on the substrate to form a set of spots for the sample;

(b) contacting each of the one or more spots in the set with an antibody specific for a target protein;

(c) detecting the antibody in each of the one or more spots to determine an amount of the target protein present in each spot;

(d) using the amount of the target protein present in each of the one or more spots to determine an amount of the target protein present in the sample; (e) determining an amount of total protein present in each of the one or more spots to determine an amount of total protein present in the sample; and

(f) normalizing the amount of the target protein determined in (d) using the amount of total protein determined in (e); wherein both the amount of target protein and the amount of total protein are determined within the exact same spot in the set.

4. The method of claim 3, wherein the amount of total protein is determined by contacting each spot with a dye that binds to proteins in general, based on charge, and that functions as a detectable label.

5. The method of claim 3, wherein the dye is a fluorescent dye selected from the group consisting of Sypro Ruby Red or Deep Purple.

6. The method of claim 3, wherein the amount of total protein is determined by contacting each spot with an antibody capable of being used to measure the total protein in the spot.

7. The method of claim 3, wherein the antibody specific for the target protein is conjugated to a detectable label.

8. The method of claim 6, wherein the antibody capable of being used to measure the total protein is conjugated to a detectable label.

9. The method of claim 3, wherein the antibody is detected in (c) using a colorimetric, fluorometric, luminescent, electrical, or radioactive method of detection.

10. The method of claim 9, wherein the antibody is detected in (c) using a fluorometric method of detection.

11. The method of claim 7, wherein the detectable label is a fluorescent label.

12. The method of claim 8, wherein the detectable label is a fluorescent label.

13. The method of claim 6, wherein the antibody capable of being used to measure the total protein is an antibody to a cellular housekeeping protein.

14. The method of claim 13, wherein the cellular housekeeping protein is actin or GAPDH.

15. The method of claim 3, wherein the biological sample is a cellular lysate obtained from a sample of tissue.

16. The method of claim 15, wherein the sample of tissue is obtained by microdissection.

17. The method of claim 16, wherein the microdissection method is laser capture microdissection.

18. The method of claim 3, wherein the biological sample is obtained by magnetic bead sorting.

19. The method of claim 3, wherein the biological sample is obtained by Fluorescent Activated Cell Sorting (FACS).

20. The method of claim 3, wherein (a) comprises printing the biological sample onto the substrate.

21. The method of claim 3, wherein the target protein is a protein in a cell signaling pathway.

22. The method of claim 3, wherein the target protein is a target for a therapeutic drug.

23. The method of claim 3 , wherein the protein microarray assay is a reverse phase protein microarray assay.

24. The method of claim 3, wherein the biological sample is selected from the group consisting of serum, urine, vitreous fluid, tears, saliva, nipple fluid, semen, bronchiolar washings, lymph, plasma, peritoneal washings, ascitic fluid and sweat.

25. A method of normalizing results obtained in a protein microarray assay, the method comprising:

(a) providing a plurality of biological samples on a substrate, wherein each biological sample is placed in one or more individual spots on the substrate to form a set of spots for the sample, and the plurality of samples form a plurality of sets arranged in a microarray on the substrate;

(b) for the sample in each set of spots, contacting each of the one or more spots in the set with an antibody specific for a target protein;

(c) detecting the antibody in each of the one or more spots in the set to determine an amount of the target protein present in each spot of the set;

(d) using the amount of the target protein present in each spot of the set to determine an amount of the target protein present in the sample;

(e) determining an amount of total protein present in each of the one or more spots of the set to determine an amount of total protein present in the sample; and

(f) normalizing the amount of the target protein in the sample determined in (d) using the amount of total protein for the sample determined in (e) for each set of spots.

26. The method of claim 25, wherein the biological samples are cellular lysates obtained from one or more samples of tissue.

27. The method of claim 26, wherein the biological samples are cellular lysates obtained from one sample of tissue.

28. The method of claim 26, wherein the biological samples are cellular lysates obtained from different samples of tissue.

29. The method of claim 25, wherein the amount of total protein is determined by contacting each spot with a dye that binds to proteins in general, based on charge, and that functions as a detectable label.

30. The method of claim 29, wherein the dye is a fluorescent dye selected from the group consisting of Sypro Ruby Red or Deep Purple.

31. The method of claim 25, wherein the amount of total protein is determined by contacting each spot with an antibody capable of being used to measure the total protein in the spot.

32. The method of claim 25, wherein the antibody specific for the target protein is conjugated to a detectable label.

33. The method of claim 31 , wherein the antibody capable of being used to measure the total protein is conjugated to a detectable label.

34. The method of claim 25, wherein the antibody is detected in (c) using a colorimetric, fluorometric, luminescent, electrical, or radioactive method of detection.

35. The method of claim 34, wherein the antibody is detected in (c) using a fluorometric method of detection.

36. The method of claim 32, wherein the detectable label is a fluorescent label.

37. The method of claim 33, wherein the detectable label is a fluorescent label.

38. The method of claim 31 , wherein the antibody capable of being used to measure the total protein is an antibody to a cellular housekeeping protein.

39. The method of claim 38, wherein the cellular housekeeping protein is actin or GAPDH.

40. The method of claim 25, wherein the biological sample is a cellular lysate obtained from a sample of tissue.

41. The method of claim 26, wherein the one or more samples of tissue are obtained by laser capture microdissection.

42. The method of claim 25, wherein the target protein is a protein in a cell signaling pathway.

43. The method of claim 25, wherein the protein microarray assay is a reverse phase protein microarray assay.

44. A method for normalizing an amount of a cell signaling protein for variable sample loading in a protein microarray assay, the method comprising:

(a) providing a plurality of biological samples on a substrate, wherein each biological sample is placed in one or more individual spots on the substrate to form a set of spots; (b) for each set of spots, contacting the one or more spots in the set with a first antibody specific for a cell signaling protein, wherein the first antibody is conjugated to a first detectable label;

(c) detecting the first antibody in the one or more spots to determine an amount of the cell signaling protein in each spot in the set;

(d) using the amount of the cell signaling protein in each of the one or more spots of the set to determine an amount of the target protein present in the sample;

(e) contacting the one or more spots in the set with a second antibody specific for a second protein distinct from the cell signaling protein, wherein the second protein is present in each spot of the set in an amount substantially equal to the amount of protein present in the spot, and wherein the second antibody is conjugated to a second detectable label;

(f) detecting the second antibody in the one or more spots in the set to determine an amount of the second protein in each spot;

(g) using the amount of the second protein in each spot to determine an amount of total protein present in the sample; and

(h) normalizing the amount of the cell signaling protein determined in (d) for the sample using the amount of the second protein determined in (g) for the same sample.

45. A method for determining an amount of one or more cell signaling proteins in a biological sample, the method comprising:

(a) providing a biological sample on a substrate, wherein the sample is placed in multiple spots on the substrate to form a microarray;

(b) providing a plurality of first antibodies, wherein each first antibody is specific for a different cell signaling protein, wherein each first antibody is conjugated to a different first detectable label, and wherein each first antibody is placed on at least one spot in the microarray such that no two first antibodies are placed together in the same spot;

(c) detecting the first antibodies to determine an amount of the cell signaling protein in each spot;

(d) determining an amount of total protein in each spot; and

(e) normalizing the amount of the cell signaling protein determined in (c) using the amount of total protein determined in (d).

46. A method for detecting the presence of a disease in a subject, the method comprising: (a) providing a biological sample on a substrate, wherein the sample is placed in multiple spots on the substrate to form a microarray;

(b) providing a plurality of first antibodies, wherein each first antibody is specific for a different cell signaling protein or for a different post-translationally-modified cell signaling protein, wherein each first antibody is conjugated to a different first detectable label, and wherein each first antibody is placed on at least one spot in the microarray such that no two first antibodies are placed together in the same spot;

(c) detecting the first antibody in each spot to determine an amount of the cell signaling protein in the spot;

(d) determining an amount of total protein in each spot;

(e) normalizing the amount of the cell signaling protein in each spot using the amount of total protein in the spot; and

(f) determining the presence of a disease in a subject based on the normalized amounts of the cell signaling proteins.

47. A method for screening a plurality of candidate compounds for treating a disease caused by a defect in a cell signaling pathway, the method comprising:

(a) providing a plurality of biological samples on a substrate in individual spots to form a microarray, wherein each sample is derived from the same cell type, and wherein each sample has been cultured with a different candidate compound;

(b) contacting each spot with a first antibody, wherein the first antibody is specific for a cell signaling protein, and wherein the first antibody is conjugated to a first detectable label;

(c) detecting the first antibody in each spot to determine a corresponding amount of cell signaling protein in the spot;

(d) determining an amount of total protein in each spot;

(e) normalizing the amount of the cell signaling protein in each spot using the corresponding amount of total protein in the spot; and

(f) determining an effective candidate compound based on the normalized amounts of the cell signaling proteins determined in (e).

48. A kit for detecting a disease caused by a defect in a cell signaling pathway in a subject, the kit comprising: (a) a first antibody conjugated to a first detectable label, wherein the first antibody is specific for a protein the cell signaling pathway; and

(b) a second antibody conjugated to a second detectable label, wherein the second antibody is specific for a housekeeping protein.

49. A protein microarray comprising:

(a) a plurality of biological samples on a substrate, wherein each biological sample is present in one or more individual spots on the substrate, and the individual spots form a microarray on the substrate;

(b) a plurality of first antibodies disposed on one or more spots in the microarray, wherein each first antibody is specific for a target protein present in one or more spots in the microarray, and each first antibody is conjugated to a first detectable label, and

(c) a plurality of second antibodies disposed on one or more spots in the microarray, wherein: each second antibody is specific for a second protein in one or more spots in the microarray, the second protein is distinct from the target protein, the second protein is present in one or more spots in an amount representative of total protein present in the spot, and each second antibody is conjugated to a second detectable label.

50. A method for screening a plurality of candidate compounds for treating a disease caused by a defect in a cell signaling pathway, the method comprising:

(a) treating animal cells with a candidate compound;

(b) obtaining a lysate of said treated animal cells and placing the lysate on one or more spots on a substrate to form a microarray;

(c) contacting each spot with a first antibody, wherein the first antibody is specific for a cell signaling protein, and wherein the first antibody is conjugated to a first detectable label;

(d) detecting the first antibody in each spot to determine a corresponding amount of cell signaling protein in the spot; (e) determining the amount of total protein in each spot;

(f) normalizing the amount of the cell signaling protein in each spot using the corresponding amount of total protein value obtained in step (d); and

(g) identifying an effective candidate compound.