AU2008347152A1 - Discovery tool with integrated microfluidic biomarker optical detection array device and methods for use - Google Patents

Description

WO 2009/088408 PCT/US2008/012369 DISCOVERY TOOL WITH INTEGRATED MICROFLUIDIC BIOMARKER OPTICAL DETECTION ARRAY DEVICE AND METHODS FOR USE TECHNICAL FIELD [00011 Particular embodiments relate to scientific and medical research, the diagnosis of diseases such as cancer, cardiovascular disease, diabetes, renal disease, pulmonary diseases, infectious diseases of viral and microbial nature, as well as neurodegenerative, immunological, and metabolic diseases, etc. In particular, the detection of biomarkers and the measurement of protein and enzymatic activities, interactions, inhibition and activation with relevant scientific and medical applications are provided. BACKGROUND [00021 Recent, rapid increases in the scientific understanding of molecular physiology have been driven by, among many reasons, the completion of the sequence of the human genome and the advent of both highly sensitive and massively parallel systems for detection of biologically or medically interesting analytes. In particular, such detection systems for biological analytes of interest, or biomarkers, are of growing importance in scientific research and, increasingly, for patients in clinical settings. Analytical methods that employ spectroscopic detection systems are frequently used in the detection and quantification of biomarkers, often providing information about the interaction of biomarkers with various test molecules. Such assay methods may be employed initially during the identification, characterization, and development of molecular diagnostics, and may also be employed as molecular diagnostic tests used to assay biological samples. Thus, these assay methods may be employed to measure the health status of patients or to provide information that may support medical decisions. 100031 Raman spectroscopy is a spectroscopic technique that measures the inelastic scattering of monochromatic light (known as Raman Scattering) commonly used to interrogate molecular vibrational or rotational aspects of a sample. Typically, a laser in the range of visible, near infrared or near ultraviolet light is used to excite the sample/system. The energy of laser photons is then shifted up or down (known as the Raman effect or Raman shift), and this shift in energy (wavelength, frequency or wave number) provides information about molecular vibrational or rotational aspects of the system. The Raman WO 2009/088408 PCT/US2008/012369 effect occurs when light interacts With the electron cloud of the bonds of a molecule or a molecular complex with multiple molecules or atoms; the magnitude of deformation in the electron cloud caused by the incident light is a reflection of the polarizability of the molecule, which determines the intensity and frequency of the reflected energy and the characteristic, fingerprint-like Raman spectra. [0004] Surface Enhanced Raman Spectroscopy (SERS) is a highly sensitive method that can enhance the signal intensity of low-probability or weak Raman spectra emitted from a small sample. SERS, in fact has been demonstrated to detect the Raman spectra of single molecules. SERS systems for the detection of biologically or medically interesting analytes typically immobilize or fix the analyte, substrate, or complex of interest onto or adjacent to a solid, usually metal or metal alloy surface, or metal complexed with other non-metal materials with Raman enhancing, dampening or tuning capabilities. This is often referred to as a SERS-active structure. Interactions between the analyte, substrate, or complex of interest and the metal surface and the metal surface derivatives, result in an increase or a modulation in the intensity and specific profiles of the Raman-scattered radiation. Accordingly, different binding events and chemical reactions, such as phosphorylation and de-phosphorylation may be detected and compared based on the characteristic, fingerprint-like Raman spectra they create. [0005] The use of SERS in biological and medical applications has tremendous potential for directly measuring medically and scientifically interesting molecular interactions and protein and enzymatic activity. In particular, SERS may be employed to measure protein substrate binding events and reactions, such as those involving protein-protein, protein small molecule, small molecule-small molecule, nucleic acid-protein, and riboprotein nucleic acid interactions, for example. The sensitivity of such applications, perhaps enabling single-molecule detection, thus offers the potential to detect very low copy number proteins and components of lysates from rare cells. While recent advances have been made in high-throughput measurement of DNA (sequencing), RNA (gene expression technologies) and proteins (proteomics); to date, high-throughput measurement of protein activity, in particular enzyme activity, has remained technically out of reach. Such information is clearly valuable both medically and scientifically. For example, while the value is clear in knowing a patient's complete DNA sequence or the expression levels of all genes or proteins in a cell, understanding the activity of all proteins in a cell is actually 2 WO 2009/088408 PCT/US2008/012369 more informative and represents a higher order of biological information. This is because proteomic-level information is directly tied to function and cell phenotype. [0006] Microfluidic devices and systems of integrated microfluidics devices employ small capillaries or microchannels attached or integrated with a solid substrate to perform a variety of operations in a number of analytical, chemical and biochemical applications on a very small scale. For example, integrated microfluidic devices can first employ electrical fields to effectively separate nucleic acids, proteins or other macromolecules of interest and then use microscale detection systems for characterization and analysis of the separation products. Such microfluidic devices accomplish these operations using remarkably small reaction volumes that can be at least several orders of magnitude smaller than conventional methods. The small size of these systems allows for increased reaction rates that use less reagent volume and that take up far less laboratory, clinical, or industrial space. Microfluidic systems thus offer the potential for attractive efficiency gains, and consequently, substantial economic advantages. [0007] Microfluidic devices are particularly well-suited to conduct analytical methods that employ spectroscopic detection systems. A variety of spectroscopic techniques can be employed in conjunction with microfluidic devices, including light scattering spectroscopy, such as Raman spectroscopy. In research or industrial settings, microfluidic devices are typically employed in biochemical or cell-based assays that use spectroscopic detection systems to quantify labeled or unlabeled molecules of interest. For example, such an assay measures the expression of green fluorescent protein in mammalian cells following treatment by a candidate small molecule or biologic drug of interest. Another example is the use of the quantitative polymer chain reaction technique (PCR) in microfluidics devices for gene amplification and analysis with intercalating fluorescence dye as the spectroscopic indicator. Other examples include, but are not limited to, enzymatic and biochemical reactions in general, chemical reactions, phase transition detections, etc. 10008] Microfluidic devices typically employ networks of integrated microscale channels and reservoirs in which materials are transported, mixed, separated and detected, with various detectors and sensors embedded or externally arranged for quantification, as well as actuators and other accessories for manipulations of the fluidic samples. The development of sophisticated material transport systems has permitted the development of systems that are readily automatable and highly reproducible. Such operations are potentially automatable and can be incorporated into high-throughput systems with 3 WO 2009/088408 PCT/US2008/012369 tremendous advantages for numerous industrial and research applications. Microfluidic devices often use plastics as the substrate. While polymeric materials offer advantages of easy fabrication, low cost and availability, they tend to be fluorescent. For example, when irradiating a sample with excitation light, light scatter may result in a significant background signal, particularly when the excitation pathway and emission pathway are the same. Other materials, such as glass, silicon, metal, and metal oxides may be used as well. [0009] Analysis of biomarkers is fast becoming the preferred method for early detection of disease, patient stratification and monitoring efficacy of treatment. Rapid and highly sensitive detection of changes in a biomarker is often technically impossible, or may require a cumbersome procedure involving multiple processing steps, necessitating large sample volumes and a prolonged diagnosis/prognosis timeline. The sample from a patient is often of a limited volume and not amenable to processing or to procedures requiring multiple steps that extend the processing time. The devices and methods of the current application provide considerable advantages that work to mitigate these problems, such that SERS spectral detection of biological and chemical samples may be performed in a real-time, microfluidic environment. SUMMARY [0010] In one embodiment, the invention involves the integration of SERS substrates into microfluidics systems. The SERS substrates include various nanoscale structures such as nanopillars, nanorings, nanotriangles, nanobowties, nanospheres, nanorods, and/or nanospirrals. [00111 In one embodiment, the invention provides a method for determining the activity of a target biomolecule using a surface enhanced Raman spectroscopy (SERS) system. The method comprises introducing a fluid sample into a microfluidic optical chamber wherein the optical chamber comprises a Raman active surface with a plurality of substrates extending therefrom. Passage of the fluid sample through the microfluidic optical chamber allows for specific binding and/or interaction between a biomolecule in the fluid sample and a plurality of said substrates. The enzymes or proteins in the fluidic sample exert an effect on the surface-immobilized biomolecule, either by cleavage or addition of chemical groups. These alteration effects can be detected by reading the Raman signal on the surface with SERS. 4 WO 2009/088408 PCT/US2008/012369 [0012] In one embodiment, the invention has minimal to no requirement for washing of the fluid sample. The change to the surface-bound biomolecules can be measured without significant interference from the molecules in the fluidic sample. [0013] In some embodiments, a laser is directed at the fluid sample in the microfluidic optical chamber, wherein the interaction of the laser with the fluid sample produces a SERS signal that is specific for the interaction between the biomolecule and the substrate. [0014] In some embodiments, the presence, quantity and/or activity of a biomolecule may be detected by recording a change in the Raman scattering spectrum of the biomolecule upon binding to the plurality of substrates. [0015] In one embodiment, cells are lysed and the lysates are applied to target molecules on a SERS surface, without purification of enzymes from the lysates. The absence of the enzyme purification steps allows for direct and quick measurement of enzyme activity, and reduction of result variation due to sample manipulation. [0016] In one embodiment, the labeling of target proteins with additional labels is not required. [0017] In a further embodiment, a set of protease substrate peptides are immobilized on the surface in a microarray format, or in a linear row, or in a folded channel such as a serpentined channel, for example. [0018] In another embodiment, Raman label molecules, metal ions, and/or nanocomposite are conjugated to the enzyme substrate to enhance the Raman signal. Organic solvents may also be added in the sample to enhance the Raman signal. [0019] In one embodiment, a set of kinase substrate peptides are immobilized on the surface in a microarray format, or in a linear row, or a folded channel such as a serpentined channel, for example. [00201 In one embodiment, the sample volume is 10 microliters or less, and in a preferred embodiment, the sample volume is less than 1 microliter. The concentration range required for detection may be I micromolar or less. [0021] In one embodiment, the reaction dynamics and kinetics measurements may be detected in real-time, rather than in end-point fashion, as labeling methods in the art require. Multiple data points may be obtained from the reaction at a data rate of between about 1 millisecond to 1 minute per measurement, and at a time duration from between about 1 minute to 24 hours. 5 WO 2009/088408 PCT/US2008/012369 [0022] In a further embodiment, a washing step is not required in the real time measurement as the SERS detection is a near field optical detection method, and thus only molecular reaction events at the SERS substrate surface can be detected. Reactions taking place at roughly100 nanometers distant from the surface will not contribute significantly to the signal. In this embodiment, the removal of noise generated from background compounds is realized by the natural or facilitated diffusion of the background compounds from the SERS substrate surface. [0023] In another embodiment, multi-channel measurement can be performed by employing a multichannel microfluidic system. These measurements can be completed simultaneously without interfering with each other. [00241 In one embodiment, a high speed optical scanning system can be used for scanning multiple channels in a timely manner. In a particular embodiment, the high speed optical system involves using a motorized galvo mirror to scan multiple samples. [0025] In one embodiment, the microfluidic operation is fully automated including sample loading, sample mixing, reagent exchange, sample heating and temperature control, etc. The fluidic actuation methods include, but are not limited to, mechanical pumping, optical pumping, and thermal pumping. [00261 In one embodiment, the liquid flow can be controlled during the optical measurement to facilitate reagent mixing, to increase diffusion of lytic reaction end products from the surface, and to prevent molecule precipitation, and so forth. [0027] In a further embodiment, a polarized laser may be used as the excitation source, and molecular chirality may be measured with increased signal-to-noise ratio. BRIEF DESCRIPTION OF THE DRAWINGS [00281 Particular embodiments are best understood from the following detailed description when read in conjunction with the accompanying drawings. It is emphasized that, according to common practice, the various features of the drawings may not necessarily be to-scale. In some cases, the dimensions of various features may be arbitrarily expanded or reduced for clarity. 10029] FIGS. lA-IF show an example fabrication process for a silicon based surface enhanced Raman scattering (SERS) substrate device in accordance with embodiments of the present invention. 6 WO 2009/088408 PCT/US2008/012369 [0030] FIGS. 2A-2F show process diagrams of printing various molecular probes on a SERS chip in accordance with embodiments of the present invention. [0031] FIGS. 3A-3B show an example assembly process with a completed assembly of an example microfluidic molecular diagnostic device in accordance with embodiments of the present invention. [00321 FIGS. 4A-4B show an example of use of microfabrication masks for making two channel devices in accordance with embodiments of the present invention. [0033] FIGS. 5A-5B show principles of protease and/or nuclease biomarker detections in an example microfluidic SERS chip in accordance with embodiments of the present invention. [0034] FIGS. 6A-6B show principles of a phosphorylation event. Alterations in biomarkers are detected in an example microfluidic SERS chip in accordance with embodiments of the present invention. [0035] FIGS. 7A-7B show example views of an integrated well plate and silicon microfluidic device structure in accordance with embodiments of the present invention. [0036] FIG. 8 shows an example configuration of a fluorescence detection system for a microfluidic protease/nuclease biomarker diagnostic device in accordance with embodiments of the present invention. [0037] FIG. 9 shows an example configuration of a Raman detection system for the microfluidic protease/nuclease biomarker diagnostic device in accordance with embodiments of the present invention. [00381 FIG. 10 shows an example configuration of a high throughput Raman detection system for a microfluidic protease/nuclease biomarker diagnostic device in accordance with embodiments of the present invention. [0039] FIG. 11 shows an example Raman signal enhancement of peptide probes in kinase biomarker detections in accordance with embodiments of the present invention. [00401 FIG. 12 shows a flow diagram of an example method of fabricating a structure for a microfluidic optical device in accordance with embodiments of the present invention. [0041] FIG. 13 shows a flow diagram of an example method of making a device for discovery of characteristics of a fluid sample in accordance with embodiments of the present invention. 10042] FIG. 14 shows a flow diagram of an example method of using a discovery device for fluid sample analysis in accordance with embodiments of the present invention. 7 WO 2009/088408 PCT/US2008/012369 [0043] FIG. 15. shows a galvo mirror drawing. The motorized galvo mirror allows for the quick scan of multiple substrate coordinates. DETAILED DESCRIPTION [00441 Before the methods and devices of embodiments of the present invention are described, it is to be understood that the invention is not limited to any particular embodiment described, as such may, of course, vary. It is also to be understood that the terminology used herein is with the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention which will be limited only by the appended claims. [0045] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limits of that range is also specifically disclosed. Each smaller range between any stated value or intervening value in a stated range and any other stated or intervening value in that stated range is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included or excluded in the range, and each range where either, neither or both limits are included in the smaller ranges is also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention. [0046] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited. The present disclosure is controlling to the extent there is a contradiction between the present disclosure and a publication incorporated by reference. [0047] It must be noted that as used herein and in the appended claims, the singular forms "a", "and", and "the" include plural referents unless the context clearly dictates otherwise. Thus, for example, reference to "a peptide" includes a plurality of such peptides and 8 WO 2009/088408 PCT/US2008/012369 reference to "the method" includes reference to one or more methods and equivalents thereof known to those skilled in the art, and so forth. [0048] The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates which may need to be independently confirmed. DEFINITIONS [0049] The terms "nucleic acid" and "polynucleotide" are used interchangeably herein to refer to deoxyribonucleotides or ribonucleotides, and polymers thereof, in either single- or double-stranded form. The terms generally encompass nucleic acids containing known nucleotide analogs or modified backbone residues or linkages, which are synthetic, naturally occurring, and non-naturally occurring, which have similar binding properties as the reference nucleic acid, and which are metabolized in a manner similar to the reference nucleotides. Examples of such analogs include, without limitation, phosphorothioates, phosphoramidates, methyl phosphonates, chiral-methyl phosphonates, 2-0-methyl ribonucleotides, peptide-nucleic acids (PNAs). [00501 The term "amino acid" refers to naturally occurring and synthetic amino acids, as well as amino acid analogs and amino acid mimetics that function in a manner similar to the naturally occurring amino acids. Naturally occurring amino acids are those encoded by the genetic code, as well as those amino acids that are later modified, e.g., hydroxyproline, y-carboxyglutamate, and 0-phosphoserine. Amino acid analogs refers to compounds that have the same basic chemical structure as a naturally occurring amino acid, e.g., an a carbon that is bound to a hydrogen, a carboxyl group, an amino group, and an R group, e.g., homoserine, norleucine, methionine sulfoxide, methionine methyl sulfonium. Such analogs have modified R groups (e.g., norleucine) or modified peptide backbones, but retain the same basic chemical structure as a naturally occurring amino acid. Amino acid mimetics refers to chemical compounds that have a structure that is different from the general chemical structure of an amino acid, but that functions in a manner similar to a naturally occurring amino acid. [0051] "Biological sample" as used herein is a sample of biological tissue or chemical fluid that is suspected of containing an analyte of interest. Samples include, for example, body 9 WO 2009/088408 PCT/US2008/012369 fluids such as whole blood, serum, plasma, cerebrospinal fluid, urine, lymph fluids, and various external secretions of the respiratory, intestinal and genitourinary tracts such as tears, saliva, semen, milk, and the like; and other biological fluids such as cell culture suspensions, cell extracts, cell culture supernatants. Samples may also include tissue biopsies, e.g., from the lung, liver, brain, eye, tongue, colon, kidney, muscle, heart, breast, skin, pancreas, uterus, cervix, prostate, salivary gland, and the like. Samples may also be microbiopsies, small samples or even single cells extracted from patients and subsequently processed, for example, using laser capture microdisecction. A sample may be suspended or dissolved in, e.g., buffers, extractants, solvents, and the like. A sample can be from any naturally occurring organism or a recombinant organism including, e.g., viruses, prokaryotes or eukaryotes, and mammals (e.g., rodents, felines, canines, and primates). The organism may be a nondiseased organism, an organism suspected of being diseased, or a diseased organism. A mammalian subject from whom a sample is taken may have, be suspected of having, or have a disease such as, for example, cancer, autoimmune disease, or cardiovascular disease, pulmonary disease, gastrointestinal disease, musculoskeletal, disorders, central nervous system disorders, infectious disease (e.g., viral, fungal, or bacterial infection). The term biological sample also refers to research samples which have been deliberately created for the study of biological processes or discovery or screening of drug candidates. Such examples include, but are not limited to, aqueous samples that have been doped with bacteria, viruses, DNA, polypeptides, natural or recombinant proteins, metal ions, or drug candidates and their mixtures. [00521 The terms "peptide" and "peptidic compound" are used interchangeably herein to refer to a polymeric form of amino acids of from about 10 to about 50 amino acids (may consist of at least 10 and not more than 50 amino acids), which can comprise coded and non-coded amino acids, chemically or biochemically modified or derivatized amino acids, L- or D- amino acids, peptides having modified peptide backbones, and peptides comprising amino acid analogs. The amino acid may be limited to only amino acids naturally occurring in humans. The peptidic compounds may be polymers of: (a) naturally occurring amino acid residues; (b) non-naturally occurring amino acid residues, e.g., N substituted glycines, amino acid substitutes, etc.; or (c) both naturally occurring and non naturally occurring amino acid residues/substitutes. In other words, the subject peptidic compounds may be peptides or peptoids. Peptoid compounds and methods for their preparation are described in WO 91/19735, the disclosure of which is hereby incorporated 10 WO 2009/088408 PCT/US2008/012369 in its entirety by reference herein. A peptide compound of the invention may comprise or consist of 23 amino acids or from 18 to 28 amino acids or from 20 to 26 amino acids. The active amino acid sequence of the invention comprises or consists of three motifs which may be overlapping, which are: an integrin binding motif sequence, a glycosaminoglycan binding motif sequence, and a calcium-binding motif. [00531 By "protein" is meant a sequence of amino acids for which the chain length is sufficient to produce the higher levels of tertiary and/or quaternary structure. This is to distinguish from "peptides" or other small molecular weight drugs that do not have such structure. Typically, a protein will have a molecular weight of about 15-20 kD to about 20 kD. [00541 The terms "polypeptide," "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residue is an artificial chemical mimetic of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers and non-naturally occurring amino acid polymer. 100551 The term "substrate" when used in context of biochemistry, means a molecule upon which an enzyme acts. Enzymes catalyze chemical reactions involving substrates. A substrate binds to an enzyme's active site, and an enzyme-substrate complex is formed. The substrate is broken down into a product and is released from the active site. [0056] The term "substrate" when used in context of material science, is used to describe the base material or surface on which processing is conducted to produce new film or layers of material such as deposited coatings, attachment of nucleic acids, peptides, sugars, and fatty acids, etc. [00571 A "kinase" is an enzyme that catalyzes the transfer of a phosphate group (e.g., from ATP or GTP) to a target molecule such as a kinase substrate, leading to phosphorylation of the substrate. [00581 A "kinase substrate" refers to a molecule that can be partially or completely phosphorylated by a kinase. [0059] A "phosphatase" is an enzyme that catalyzes the removal of a phosphate group from a phosphatase substrate thereby resulting in the partial or complete dephosphorylation of that substrate. [0060] A "phosphatase substrate" refers to a molecule that can be partially or completely dephosphorylated by a phosphate. 11 WO 2009/088408 PCT/US2008/012369 [00611 The terms "treatment," "treating" and the like are used herein to refer to both therapeutic treatment and prophylactic or preventative measures. Those in need of treatment include those already with the disorder as well as those in which the disorder is to be prevented. In general, this encompasses obtaining a desired pharmacologic and/or physiologic effect, e.g., stimulation of angiogenesis. The effect may be prophylactic in terms of completely or partially preventing a disease or symptom thereof and/or may be therapeutic in terms of a partial or complete cure for a disease and/or adverse effect attributable to the disease. The terms as used herein cover any treatment of a disease in a mammal, particularly a human, and include: (a) preventing a disease or condition (e.g., preventing the loss of cartilage) from occurring in a subject who may be predisposed to the disease but has not yet been diagnosed as having it; (b) inhibiting the disease, e.g., arresting loss of cartilage; or (c) relieving the disease (e.g., enhancing the development of cartilage). [00621 The terms "subject," "individual," "patient," and "host" are used interchangeably herein and refer to any vertebrate, particularly any mammal and most particularly including human subjects, farm animals, and mammalian pets. The subject may be, but is not necessarily under the care of a health care professional such as a doctor. [00631 "Mammal" for purposes of treatment refers to any animal classified as a mammal, including humans, domestic and farm animals, and zoo, sports, or pet animals, such as dogs, horses, cats, cows, etc. Preferably, the mammal is human. [0064] A "disorder" is any condition that would benefit from treatment with the peptide. This includes chronic and acute disorders or diseases including those pathological conditions which predispose the mammal to the disorder in question. Non-limiting examples of disorders to be treated herein include skeletal loss or weakness and bone defects or breakage. [0065] "Surface Enhanced Raman Spectroscopy", or "Surface Enhanced Raman Scattering", often abbreviated SERS, is a surface sensitive technique that results in the enhancement of Raman scattering by molecules adsorbed on rough metal surfaces. The enhancement factor can be as much as 10"-10', which allows the technique to be sensitive enough to detect single molecules. [0066] "Raman scattering" or "Raman effect" is the inelastic scattering of a photon. When light is scattered from an atom or molecule, most photons are elastically scattered. The scattered photons have the same energy (frequency) and wavelength as the incident photons. However, a small fraction of the scattered light is scattered by an excitation, with 12 WO 2009/088408 PCT/US2008/012369 the scattered photons having a frequency different from, and usually lower than, the frequency of the incident photons. DESCRIPTION OF THE PREFERRED EMBODIMENTS [0067] Certain embodiments of the invention include microchips with microfluidic sample flow channels, active nanostructured surfaces, optical windows, and attached molecular probe arrays for multiplexed optical detection. Advantages include ultra small sample volume, high detection speed, throughput, sensitivity, reliability and completeness over the conventional molecular diagnostic method and devices, as well as two to three orders of magnitude lower cost. This may be applied to the molecular-level disease diagnosis in laboratory and clinical environments with unprecedented sensitivity, accuracy and affordability. [00681 Methods and devices are provided for a device for surface enhanced Raman scattering (SERS) detection from microchannels in silicon or plastic substrates. The silicon device can be formed by separately etching and machining different microstructures with appropriate masking and different protective coatings and layers, which may be individually removed prior to final etching to provide deep microstructures, and by chemical and physical surface roughening to generate unique nanostructures as SERS substrate. The device can accommodate parallel fluid streams, and allow focused laser light to illuminate the SERS substrate surface. For molding with polymeric materials, the silicon device may be replicated twice and used with polymers to obtain a desired result. [0069] The present invention demonstrates an integrated microscale fluidic chamber with sub-micro liter volume and a nanostructured surface for SERS spectroscopy. The microscale optical chamber has one transparent surface which allows for light to be transmitted in the chamber and illuminated onto a SERS substrate surface. This also allows Raman scattering light to be transmitted out of the chamber and collected. Compared to the conventional optical chamber or cuvette used for Raman measurements, the volume of this Raman detection fluidic chamber may be smaller than I pL. The shorter or shallower microchannel can allow for further miniaturization of the detection module in the chip. The SERS signal can be detected by a spectrometer camera but the required volume can be more than 1000 times smaller than that used in conventional Raman spectroscopy. The microscale dimensions of the optical chamber can enable integration of multiple individual optical chambers in one chip, such that multiplexed SERS spectroscopy 13 WO 2009/088408 PCT/US2008/012369 of 2, 3, 8, 16, 32, 48, 96, 192, 384, 768, and even 1536 samples can be accomplished using a single device which holds all the samples at once. [0070] Accordingly, certain embodiments present high sensitivity biomolecule detection on a chip with simultaneous detection of SERS spectra. The fluidic sample flow and reaction temperature in the microscale chamber may be controlled by external electronics, and/or mechanical micro-pumps. Due to the relatively small volume of the microchip and the fluidic sample, the flow rate and heating/cooling rate can be orders of magnitude higher than bulk scale counterparts, which enable many special applications, such as on-chip PCR and fast fluidic exchange. [0071] Particular embodiments include a monolithically fabricated nanostructured SERS substrate, also enclosed in a microfluidic chamber such that SERS spectral detection of a biological/chemical sample can be implemented in the microfluidic environment. The unique microfabrication, nanofabrication and packaging as described herein allows for the detection of SERS spectra in a simulated aqueous biological environment. 10072] Multiple biological or enzymatic substrate extensions, such as small peptides and nucleotides may be attached on the SERS substrate in the microfluidics chamber, and may also be specific to multiple kinds of biomarkers, such as enzymes, for example, which are related to cancer, cardiovascular disease, diabetes and neurological diseases. Human and animal fluidic samples can be introduced into the microfluidic chamber and reacted with the attached probes. The chemical change of the probes can be detected by SERS spectral detection. [00731 Conventionally, a chemical or biological sample is dropped on the SERS substrate and dried for Raman spectroscopic analysis. However, real time biological events may only occur in aqueous solutions. Particular embodiments of the present invention allow for the detection of biomolecule Raman signals in a simulated biofluidic environment for both static and dynamic biochemical reactions. [00741 Nanostructures may be on the surface of the microfluidics channel to provide enhancement of optical signals or to anchor enzymatic substrate extensions to capture target molecules or particulates for detection. Substrate extensions, such as antibodies, aptamers, DNA or RNA oligonucleotides and longer extensions, including peptides, polysaccharides, polymers, small molecules, etc., can be chemically linked to the surfaces of the microfluidic chamber in the chip. Enzymatic substrate extensions may also be 14 WO 2009/088408 PCT/US2008/012369 tethered to physically fabricated nanostructures to create nanobio-hybrid probes in the microfluidic chamber. [00751 Particular embodiments as described herein have applications in, inter alia, diagnostic tests and molecular diagnostics. For example, molecular diagnostics, and in particular molecular diagnostics that detect biomarkers related to cancer, measure biomarkers including small molecule metabolites or metabolic intermediates, nucleic acids, carbohydrates, proteins, protein fragments, protein complexes and/or derivatives or combinations thereof. Chemical assays such as analytical methods that employ spectroscopic detection systems may be used in the detection and quantification of such biomarkers, and may provide information about the interaction of biomarkers with test molecules such as small molecules, enzymes, carbohydrates, nucleic acid probes, nucleic acid or protein aptamers, peptide nucleic acids, peptides, or polyclonal or monoclonal antibodies. Such assay methods may be employed initially during the identification, characterization, and development of molecular diagnostics, and may also be employed as molecular diagnostic tests used to assay biological samples and thus measure the health status of patients or to provide information that may support medical decisions. 100761 Particular embodiments also have applications in, inter alia, molecular therapeutics. For example, identification and characterization of drug targets may involve detection and quantification of such drug targets in biological samples. Chemical assays and analytical methods that employ spectroscopic detection systems may be used to detect and quantify potential drug targets including proteins such as cell surface proteins, extracellular proteins, peptide hormones, transmembrane proteins, receptor proteins, signaling proteins, cytosolic proteins or enzymes, nuclear proteins, DNA-binding proteins, RNA molecules including messenger RNA or micro-RNAs, and/or DNA. Such assays and methods may also provide information about the interaction of drug targets with drugs such as small molecules, polyclonal or monoclonal antibodies, therapeutic proteins or therapeutic enzymes, antisense nucleic acids, small-interfering RNAs, nucleic acid or protein aptamers, peptide nucleic acids, or other drugs and potential drugs. Such assay methods may be employed initially during the identification, characterization, and development of molecular therapeutics, and may also be employed in tests to identify individual patients' responsiveness to treatment with drugs or potential drugs, and thus provide valuable information that may support medical decisions. 15 WO 2009/088408 PCT/US2008/012369 [0077] Silicon wafers are preferable to conventional antibody affinity binding assay substrates that can only detect concentration. Other semiconductor wafers (e.g., GaAs, InP, GaP, GaSb, InSb, InAs, CaF 2 , LaAI2O3, LiGaO2, MgO, SrTiOq, YSZ and ZnO) can also be used in certain embodiments. Suitable semiconductor materials for the wafer include, but are not limited to, elements of Groups II-VI (ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, HgS, HgSe, HgTe, MgS, MgSe, MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS, BaSe, BaTe, etc.) and III-V (GaN, GaP, GaAs, GaSb, InN, InP, InAs, InSb, etc.) and IV (Ge, Si, etc.) groups on the periodic table, and alloys or mixtures thereof. Suitable metals and metal oxides for the surface coating include, but are not limited to, Au, Ag, Co, Ni, Fe 2

O

3 , TiO 2 , and the like. Suitable carbon nanoparticles for surface coating include, e.g., carbon nanospheres, carbon nano-onions, carbon nanotubes, and fullerene. [00781 In particular embodiments, enzymatic activity, in addition to protein concentration may be detected. In the context of prostate tumors, for example, whereas prostate-specific antigen (PSA) concentration can now be detected, such assays do not necessarily clarify whether the antigen is active or not, and may provide a misleading measurement. An aspect of certain embodiments of the invention includes generating information regarding not only concentration, but also activity. Further, particular embodiments also include a detection system in lieu of a chip scanner. [0079] A system for liquid sample microspectroscopy in certain embodiments may generally include a detection apparatus (e.g., instrumentation portion) coupled to a microfluidics optical device (e.g., a chip or integrated circuit (IC) portion). The detection apparatus can include a light source for sending light through a liquid sample to be characterized, and a spectrograph and/or analysis unit to analyze the light (e.g., fluorescence, absorbance, etc.), which is affected by the molecules of the sample. The microfluidic optical device can be fabricated using semiconductor processing techniques, and may be packaged to protect the semiconductor therein and to accommodate inlet/outlet ports for the liquid sample. 10080] Referring now to FIGS. IA-I F, shown is an example fabrication process for a silicon based surface enhanced Raman scattering (SERS) substrate device in accordance with embodiments of the present invention. [00811 FIG. 1A shows thermal deposition of relatively thin layers of polycrystalline silicon 104-0 and 104-1 on top and bottom surfaces of single crystal wafer 102. For example, 16 WO 2009/088408 PCT/US2008/012369 polycrystalline silicon layers 104-0 and 104-1 can be in a range of from about 100 nm to about 500 nm thick, such as from about 200 nm to about 400 nm, and more specifically about 300 nm. [0082] FIG. lB shows laser drilling or chemical etching of via-holes 116 through wafer 102 and polycrystalline silicon 104-0/104-1. The etchant may be hot potassium hydroxide and a 30W carbon dioxide laser may be employed. In one embodiment, via-holes 116 may have a diameter/width of about 100 pm. Of course, any suitable width for these via-holes (e.g., within ranges of from about 80 pm to about 120 pm, or from about 50 pm to about 150 pm) can be utilized in particular embodiments. For example, these via-hole widths may also be configured to form a filtering function, such as by disallowing larger molecules from flowing into the microfluidic optical chamber, as will be discussed in more detail below. [0083] FIG. IC shows photoresist 106 applied on portions of polycrystalline silicon 104-0 to allow for photolithography patterning of to-be-etched areas. [0084] FIG. ID shows plasma etching 108 of polycrystalline layer 104-0 to form silicon nanostructures 110. Plasma etching 108 can include multiple steps in order to form geometric shapes or other suitable "roughness" on a surface of silicon nanostructure 110. For example, a nanopyramid array can be formed by application of a plasma treatment that includes HBr + 02 for less than about 10 seconds. Plasma etching with HBr for from about 10 seconds to about 20 seconds can form nanopillar arrays. Oxide portions can then be removed from the pillars by plasma etching that includes, e.g., SF 6 . Next, the surface can be plasma etched for from about 1 minute to about 2 minutes with HBr plasma. Such an approach can produce nanopyramids having a height of from about 50 nm to about 200 nm, and more specifically about 100 nm. [0085] Any suitable type of nanostructures can be implemented in certain embodiments. Any shape that accommodates an enhancement of certain frequencies inherent or appearing after modification of the substrate, such as by enzymatic substrate accommodation discussed below in further detail, can be utilized. Other example nanostructure may include different geometries with enhancement properties, nano rings, nano squares, nano wires, parallel wires, nano grooves, etc., and these structures can be formed using e-beam, lithography, or any suitable processing method. [0086] FIG. IE shows metal deposition 112 of a thin film 114. For example, the deposited metal 114 can include gold, silver, platinum, palladium, or copper, etc., and the thickness 17 WO 2009/088408 PCT/US2008/012369 of the thin film 114 can be from about 10 nm to about 80 nm, such as from about 20 nm to about 60 nm, and more specifically about 40 nm. [0087] FIG. IF shows the removal of photoresist 106 and annealing of thin metal nanoparticles 114 to form a smoothed metallic coating surface of layer 114. Suitable annealing temperatures may be from about 200-300*C, and more preferably 250*C. [0088] A surface of layer 114 in particular embodiments may be relatively rough, or may contain other geometrical properties, e.g., of sharp edges/points to make enhanced electromagnetic fields around such edges. [00891 Referring now to FIGS. 2A-2F, shown are process diagrams of printing various molecular probes on a SERS chip in accordance with embodiments of the present invention. Different types of peptides or nucleotides may be dropped on a metallized nanostructure SERS substrate using microscale contact pins or injectors. Formed enzymatic substrate extensions can covalently bond to the SERS substrate surface. [0090] FIG. 2A shows polycrystalline silicon 104-0 and 104-1 on either surface of single crystal wafer 102, with metal nanoparticles 114, and via-holes 116. Probe 204 can be positioned to apply a drop 202-0 of peptides or nucleotides. FIG. 2B shows enzymatic substrate extension 206-0 that is formed from a covalent bond between metal nanoparticles 114 and drop 202-0 of peptides/nucleotides. [00911 FIG. 2C shows a repositioning of probe 204 with a different drop 202-1, and FIG. 2D shows a corresponding enzymatic substrate extension 206-1. Probe 204 can be repositioned a number of times to create a plurality of enzymatic substrate extensions bonded to metal nanoparticles 114. [0092] FIG. 2E shows enzymatic substrate extensions 206-0, 206-1, 206-2, and 206-3. Probe 204 can then be repositioned to release drop 202-4 as shown. FIG. 2F shows a completed group of enzymatic substrate extensions in SERS substrate chip 210, including extension 206-4 corresponding to drop 202-4. In addition, an electromagnetic field around each enzymatic substrate extension may be altered, and metal 114 may serve as an enhancer for electromagnetic or photonic excitation of certain frequencies. [00931 Referring now to FIGS. 3A and 3B, shown is an example assembly process with a completed assembly of an example microfluidic molecular diagnostic device in accordance with embodiments of the present invention. Generally, three separated units can be included in the assembly process. A top layer can be formed with polydimethylsiloxane (PDMS) portions 306-0 and liquid sample inlet 302 and outlet 304. Because the optical 18 WO 2009/088408 PCT/US2008/012369 apparatus or instrumentation portion may be placed on an opposite chip side (e.g., the bottom side) relative to inlet/outlet channels (e.g., the top side), there is substantial leeway as to placing the inlet and outlet channels without interfering with the optical analysis aspects. A middle unit can include SERS substrate chip 210 with enzymatic substrate extensions. A bottom layer can include PDMS portions 306-1 and transparent window 310 to accommodate microfluidic channels therein. [0094] In particular embodiments, transparent window 310 can generally be relatively thin such that optical loss due to absorption in the window can be minimized (e.g., to under about 10%). Typical window implementations can be in a range of about 1-3 mm thick, whereas particular embodiments can allow for such a window thickness of from about 200 pm to about 300 pm. Further, a transparent window in certain embodiments can be formed of any suitable material that is transparent to the spectrum of light (e.g., Si0 2 , PDMS, cyclic olefin copolymer (COC) polymer, or any ultraviolet (UV) transparent plastics, etc.). [00951 FIG. 3B shows an example assembled discovery tool device. Bonding the three separated units shown in FIG. 3A into the assembly of FIG. 3B can include using covalent bonding between silicon dioxide on silicon surface (e.g., polycrystalline silicon layers 104 0, 104-1) and active siloxane groups on PDMS surfaces (e.g., 306-0 and 306-1). The assembly can also include formation of microfluidic optical chamber 318 for analysis of a sample fluid received via inlet 302 and output via outlet 304. [00961 Generally, certain embodiments can include an instrumentation portion discussed in more detail below, as well as an integrated circuit (IC) portion 210. Transparent window 310 may serve to isolate IC portion 210 from the instrumentation portion. The IC portion can include semiconductor material 102, with via-holes 116 therein to accommodate inlet 302 and outlet 304 ports as shown. Semiconductor material 102 can include any suitable semiconductor material, such as silicon (Si), germanium, silicon dioxide, gallium arsenide (GaAs), etc. Suitable semiconductor materials for the wafer include, but are not limited to, elements of Groups II-VI (ZnS, ZnSe, ZnTe, CdS, CdSe, CdTe, HgS, HgSe, HgTe, MgS, MgSe, MgTe, CaS, CaSe, CaTe, SrS, SrSe, SrTe, BaS, BaSe, BaTe, etc.) and Ill-V (GaN, GaP, GaAs, GaSb, InN, InP, InAs, InSb, etc.) and IV (Ge, Si, etc.) groups on the periodic table, and alloys or mixtures thereof. [0097] In certain embodiments, mixing of a sample solution can be controlled for optical chamber 318 in order to observe real-time reactions of different chemicals and/or multiple components being pumped into the inlet at the same time. Further, inlet 302 and/or outlet 19 WO 2009/088408 PCT/US2008/012369 304 can be coupled to any suitable type of tubing (e.g., plastic tubing), and the diameter of the via-holes can range from about 100 pm to about 1 mm. Further, sizes of the inlet and outlet channels or ports can be varied, thus providing a filtering function by allowing for different sample volumes, molecule sizes, etc., depending upon the particular application. [0098] In one embodiment, through-holes can provide ducts for a liquid sample flowing through microfluidic optical chamber 318, such that that liquid handling units can be installed on a side of the silicon chip other than the side where the microscale optical chambers are positioned. Without having the liquid handling units (e.g., reservoirs, connectors, tubings, or pumps) obstructing the microscale optical chamber, optical systems can have substantial exposure to chamber 318. Also, chamber 318 in certain embodiments may extend in length in a range of from about 10 pm to about 10 cm long, such as from about 500 gm to about 2 cm, and more specifically about 1 cm, to accommodate a variety of enzymatic substrate extensions 206. A depth of chamber 318 can range from about 10 pm to about 200 gm for providing a piL or sub-jiL sample volume. For example, chamber 318 may hold a sample volume in a range of from about 0.10 pL to about 2 pL of fluid. [0099] Inlet 302 and/or outlet 304 may be coupled to multiple channels, where these pathways can be routed, and may be arranged in an array format to allow easy loading via robots (e.g., to accommodate standard distances for such loading). A polymer bonding layer may also be used in the assembly, and may include any suitable layer of soft or hard plastic (e.g., PDMS, epoxy, adhesive rubber, a metal, etc.). The surface of the silicon device may also be oxidized by plasma enhanced chemical vapor deposition (PECVD), or electron beam evaporation. In addition, a liquid handling package can surround left and right edges of the structure, as well as covering the top portion along with a sealing material (e.g., epoxy, PDMS, rubber, glass, quartz, etc.). [00100] Referring now to FIG. 4A, an example top view of microfabrication masks for making two-channel devices in accordance with embodiments of the present invention is shown. In this example, a silicon wafer 402 can be defined with device masking, inlet/outlet reservoir 404 masking, microfluidic optical chamber 406 masking, and via-hole masking layers. As shown in the example close-up top view of the mask structures in FIG. 4B, via-hole masking layer 408 can be aligned with an edge of microfluidic optical chamber 406, and within the inlet/outlet reservoir 404 masking layer. [00101] Referring now to FIGS. 5A and 5B, shown are principles of protease and nuclease biomarker detections in an exemplary microfluidic SERS chip in accordance with 20 WO 2009/088408 PCT/US2008/012369 embodiments of the present invention. Different line types on the SERS substrate surface 114 represent exemplary peptide/nucleotide enzymatic substrate extensions, such as 206-3 and 206-4. The triangle pairs (e.g., 502 and 504) represent exemplary protease and/or nuclease biomarkers in biofluidic samples. [00102] FIG. 5B shows decomposed procedures of biomarker enzymatic reactions, following a sequence of 510 (introduction of biomarker enzymes 502 and 504), 512 (specific binding of biomarker enzymes 502 and 504 with enzymatic substrate extensions 206-3 and 206-4), 514 (restrictive cleavage of enzymatic substrate extensions), and 516 (washing of reaction residues to leave modified enzymatic substrate extensions 206-3' and 206-4'). [00103] Referring now to FIGS. 6A and 6B, shown are principles of kinase biomarker detection in another exemplary microfluidic SERS chip in accordance with embodiments of the present invention. Different line types on the SERS substrate surface 114 represent exemplary enzymatic substrate extensions, such as 206-1 and 206-2. The triangle pairs (e.g., 602 and 604) represent kinase biomarkers in biofluidic samples. It is noted that the substrate extensions are not limited to enzymes, but may include various other molecules mentioned herein, such as, for example, antibodies, aptamers, DNA or RNA oligonucleotides and longer extensions, including non-enzymatic peptides, polysaccharides, polymers, small molecules, etc., all of which may be acted upon and/or modified by molecules in the incoming biofluidic sample. All such substrate extensions are capable of being chemically linked to the surfaces of the microfluidic chamber in the chip. Likewise, 602 and 604 do not necessarily represent enzymatic biomarkers in all embodiments of the invention. Rather, incoming biomarkers to be analyzed may include nucleic acids (DNA and RNA), other non-enzymatic proteins, peptides, sugars/carbohydrates, metabolites and small chemical compounds. [00104] FIG. 6B shows decomposed procedures of exemplary biomarker enzymatic reactions, following a sequence of 610 (introduction of biomarker enzymes 602 and 604), 612 (specific binding biomarker enzymes 602 and 604 with enzymatic substrate extensions 206-1 and 206-2), 614 (phosphorylation 606 of enzymatic substrate extensions), and 616 (washing of reaction residues). [00105] Referring now to FIG. 7A, an example top view of an integrated well plate and silicon microfluidic device structure in accordance with embodiments of the present invention is shown. FIG. 7B shows a cross-section view of the example structure of FIG. 21 WO 2009/088408 PCT/US2008/012369 7A. Silicon device 704 can be topped by microfluidic network layer (e.g., PDMS) 706, and well plate 702. Thus, such a multichannel version can have access holes through to the top of the structure for a microfluidic channel or routing layer. In this fashion, a microfluidics optical chip can be integrated with 96, 384, 1536, etc., micro well plates that may comply with standard micro well plate dimensions. The assembly of the microfluidics optical chip with the micro well plates may then be compatible with standard robotic liquid handling systems. [00106] Referring now to FIG. 8, shown is an example configuration of a fluorescence detection system for a microfluidic protease/nuclease biomarker diagnostic device in accordance with embodiments of the present invention. The fluorescence enzymatic substrate extensions at a free end of each peptide/nucleotide may be removed with the proteolytic/nucleolytic reactions, and serve as optical beacons for biomarker diagnosis. [00107] In this fashion, enzymatic substrate extensions can provide targets for enzymes in the sample solution, whereby proteases may attach in dynamic recognition followed by catalysis. Thus, in particular embodiments, a chemical reaction occurs on enzymatic substrate extensions (e.g., 206-3, 206-4, etc.). In contrast, conventional approaches typically include a DNA probe on the surface, which measures other DNA in the solution, but does not actually change the substrate, but instead provides a binding or recognition result. In certain embodiments, initial binding occurs, however, this may be followed by an observed catalysis. This is due to the fact that an enzyme in the solution for analysis effectively changes the substrate (e.g., by removing a phosphate group from the substrate, for example). [00108] In FIG. 8, light source 802 can provide light beams that are filtered using fluorescence excitation filter 814. Filtered light beams can then be reflected by dichroic mirror 822, and passed via objective lens 820 for focusing and input to microfluidic optical chamber 318 through optically transparent window 310. Light source 802 can provide an illumination/excitation light beam that may be any suitable form of light, such as white light, laser light (e.g., visible laser, ultraviolet (UV) laser, near infrared (IR) laser, etc.), light emitting diode (LED), super luminescent diode, polarized light, halogen lamp generated light, continuous or pulsed Xenon Lamp, Mercury light source, Argon light source, Deuterium light source, Tungsten light source and Deuterium-Tungsten-Halogen mixed light source, etc. Generally, microfluidic optical chamber 318 can be populated by 22 WO 2009/088408 PCT/US2008/012369 molecules of a liquid or sample to be characterized, where the liquid is received via inlet port 302, and can also be discharged via outlet port 304. [00109] Once the light is reflected in microfluidic optical chamber 318 off a selected enzymatic substrate extension, absorbance can occur via objective lens 820, pass off mirror 822, and be sent to fluorescence emission filter 824, for receipt in detector 830. Detector 830 may also include a charge coupled device (CCD) for analysis of the various wavelengths contained in the received light beam. In this fashion, one or more characteristics of the sample found in chamber 318 can be determined based on analysis of received fluorescence and/or absorbance light in detector 830. Further, and as will be discussed in more detail below, the microscale dimensions of the optical chamber presented herein can allow for integration of multiple individual optical chambers in one chip, such that the multiplexed optical spectroscopy of 2, 96, and even 384 samples, can be accomplished. [00110] Referring now to FIG. 9, shown is an example configuration of a Raman detection system for an exemplary microfluidic protease/nuclease and/or kinase/phosphorylase biomarker diagnostic device in accordance with embodiments of the present invention. The Raman enzymatic substrate extensions at a free end of each peptide/nucleotide can be removed as a result of proteolytic/nucleolytic reactions. They may also be modified by phosphorylation/dephosphorylation reactions. As such, they may serve as optical beacons for biomarker diagnosis. [001111 In this particular example, a point detection method allows for the detection of one enzymatic substrate extension at a time. Therefore, the microfluidic optical device and/or the associated instrumentation may be translated for detection of each enzymatic substrate extension. Further, other microfluidic optical devices (e.g., arranged as shown in FIG. 4A) can also be accessed by translating or stepping an instrumentation portion. Here, the instrumentation portion includes laser 902, which can provide a laser beam for reflection off mirror 906. Beam splitter 908 can receive reflected laser beam from mirror 906, and may provide a split beam via lens 904 for microfluidic optical chamber 318. Reflected light is returned via lens 904, passed via beam splitter 908, mirrors 912 and 910, and then provided for analysis to spectrometer 914. [001121 In this example, spectrometer 914 shows a spectrum or range of wavelengths that show no reaction, while a different spectrum may show that there was a reaction on a particular enzymatic substrate extension. Determining whether a reaction has taken place, 23 WO 2009/088408 PCT/US2008/012369 or determining another characteristic of the liquid sample, can include an appearance of a new peak, disappearance of an existing peak, shifting of an existing peak, merging of multiple peaks, splitting of peaks, or any alteration as can be measured by spectrometry. In this fashion, chemical alterations can be detected using optical and/or electromagnetic properties of enzymatic substrate extensions and surrounding regions. Thus, fluorescence labeling of the enzyme substrates may not be required in certain embodiments. In such embodiments, detection of chemical, electromagnetic, acoustic, or any suitable properties possessing complex information for observation is utilized. [001131 Observable changes may be relatively subtle such that a combination of suitable nanostructures (e.g., nanopyramids on a surface of layer 114) may be added to enhance localized electromagnetic fields near the enzymatic substrate extensions (e.g., 206-3, 206 4, etc.) and thereby increase detection. In addition, the analysis in particular embodiments, while not necessarily utilizing a labeling step, may be performed in real-time. This is because the substrate may not need purification, and because time may not be needed to allow for any florescent reaction to take place. [00114] In one example, a tumor may be metastasized in the blood, affecting kinase activity profiles as compared to normal cells. Measuring kinase activity can convey the particular group or stage of the cancer, so that it may be treated with appropriate chemo- and/or immunotherapy, for example. In cancer, certain proteases may be upregulated. They may also exhibit altered enzymatic profiles, which can be identified using particular embodiments of the claimed invention. A biopsy may be placed in solution, and mild detergents used to lyse the cells, providing pL-range volumes for analysis in a lysate. A lysate may contain numerous enzymes (e.g., proteases, nucleases, kinases, phosphatases, etc.). In order to observe different enzymes, correspondingly different enzymatic substrate extensions are placed on the microarray (see, e.g., arrangement of FIG. 4A). Distinct enzymatic substrate extensions may be situated on the microarray in order to measure multiple enzymatic reactions simultaneously. Further, particular embodiments of the claimed invention can also measure binding reactions in addition to enzymatic reactions. In such embodiments, protein:protein binding and/or interactions may be detected using surface plasmon resonance (SPR), for example. [00115] Particular embodiments of the invention may also utilize an antibody array such that different antibodies can have different spectral signatures (e.g., peaks for different events, such as cleaving, different chemical reactions, binding and/or recognition events). 24 WO 2009/088408 PCT/US2008/012369 Particular embodiments can analyze any plasma or fluid (e.g., saliva, urine, spinal fluid, etc.) that can be used without substantial processing or sample preparation. However, the measurement of processes in prepared samples may be improved relative to corresponding unprepared samples due to possibly interfering fluid constituents. Spectrometer 914 supports a relatively large range which allows for the isolation of measurable signals from disturbing background noise. [00116] Referring now to FIG. 10, shown is an example configuration of a high throughput Raman detection system for a microfluidic protease/nuclease biomarker diagnostic device in accordance with embodiments of the present invention. A fast scanning mirror 1006 may be used in an optical path to convert a point-like laser excitation into a line-like laser excitation, such that multiple enzymatic substrate extensions on the SERS substrate surface can be excited and detected simultaneously by using a two-dimensional spectrograph 1014 to record the SERS spectra of the substrate extensions at a time. [00117] As discussed above, particular embodiments may also include a scanning platform in order to scan different enzymatic substrate extensions one by one. A scanning mirror 1006, as well as a moving stage for one or more components of the instrumentation portion, are included; each of which may be motor-step driven for high precision. Further, certain embodiments can also include autofocusing and/or other pattern recognition for proper light beam positioning relative to enzymatic substrate extensions for analysis. [001181 In certain embodiments, a digital light processing ("DLP") device can be used for fine adjustments of the light incident angle with computerized feedback control. For example, such a DLP can replace scanning mirror 1006 in the example configuration shown in FIG. 10. [001191 In addition to SERS, other spectroscopy modules and/or types of scattering may be employed, such as, for example, mechanical, electromagnetic and/or optical, etc.). For example, vibration of a molecule may change with different chemical reactions, where different frequencies of electromagnetic and acoustic ways, and IR may be used to measure rotation or tumbling as to an internal frequency for a molecule to be measured (e.g., from very low to very high, such as microwave frequencies). [00120] Referring now to FIG. 11, shown is an example Raman signal enhancement of peptide probes in kinase biomarker detection, in accordance with embodiments of the present invention. Because the SERS substrate in certain embodiments includes polysilicon and metal, the substrate with schematic substrate extensions is electrically 25 WO 2009/088408 PCT/US2008/012369 conductive. For phosphorylation detection, a positive DC voltage may be applied on the SERS substrate (e.g., metal portion 114), and a DC negative voltage can be applied in an associated reaction buffer. In 1102, positively charged peptide extensions may be repelled and straightened, while the negatively charged kinase enzymes are brought closer to the peptides. In 1104, kinase enzymes can bind to the peptide due to their proximity. In 1106, after the phosphorylation reaction, the peptides carry a negatively charged phosphate group and can thus be attracted to the SERS substrate surface, while the kinase enzymes lose negative charges and may be repelled away. The relatively large conformational change of the peptide after the phosphorylation reaction will likely induce more dramatic changes in the SERS spectra for analysis. [001211 In the detection or instrumentation module, absorbance and/or fluorescence of the supplied light can be analyzed. Typically, the fluorescence light is at higher wavelengths than the excitation light. Particular embodiments can also support photonic or multi photonic excitation, where the excitation wavelengths are higher than the emission wavelengths, as well as epi-fluorescence applications that may utilize a separate filter. [001221 Certain embodiments can also accommodate measurement of scattering light (e.g., X-ray small angle scattering spectroscopy). Measurements may also be taken using polarized light in circular dichrotomomy (CD) applications, which involves measurement of the response degree of angle movement of sample molecules. The fluorescence lifetimes can also be measured for Fourier transformed infrared (FTIR) applications, as well as Raman scattering, and luminescence. [00123] SPR and nuclear magnetic resonance (NMR) spectroscopy can also be accommodated in particular embodiments. For such applications, the illumination window can receive optically pumped hyper-polarized light, and such optical pumping, as well as the optical realization, can generally occur in close proximity. NMR may typically utilize a homogeneous field for measurement because this approach usually makes use of a metal coil, where the magnetic field can be reversed, and the optical pumping can be through chamber 318, where the magnetic field is around chamber 318. In this fashion, the microfluidic optical chamber can be optically activated. [00124] Other electromagnetic sources can also be incorporated for manipulating the material sample in the microfluidic optical chamber. For example, particular embodiments can allow for manipulation of sample physical properties using thermal, electromagnetic, optical, dielectric, inhomogeneality, etc. 26 WO 2009/088408 PCT/US2008/012369 [00125] Another aspect of a particular embodiment of the invention involves the relatively strong thermal conducting nature of silicon material 102, thus allowing the temperature of chamber 318 to be controlled by coupling to a thermal device (heating and/or cooling). For example, a metal block or junction can be used to measure sample material not only at room temperature, but as low as from about 0*C up to about 300*C, or as otherwise determined by the limits of the sample material itself. Thus, if a protein is active and in order to prevent denaturing at higher temperature, a sample measurement can be performed at about 37'C. In another embodiment, thermostable enzymes (e.g., Taq polymerase, and other thermal stable enzymes isolated or engineered from thermophilic microbes) can allow higher temperature (e.g., up to about 99'C) measurements. This type of measurement may not be possible with standard cuvettes without relatively bulky heating/cooling elements being coupled thereto. [00126] In particular embodiments, such temperature control and an associated sensing unit can be integrated with the microfluidics optical device. For example, such an integrated temperature control and sensing unit can be a Peltier junction heater or metal line resistance heater. This approach can allow for thermocycling analysis of samples at varying temperatures, such as relatively low temperatures to prevent heat-denaturation of proteins, and higher temperatures for real-time genetic amplification using polymerase chain reactions (PCR). [00127] In this fashion, measurement of chemical, biological, and/or physical reactions to temperature can be accommodated in chamber 318. Any temperature dependent characteristic can be isolated, such as measurement of the melting point of chemicals for assessing chemical purity. Further, some applications may also include a camera. PCR can include a cycling temperature (e.g., between about 55'C and about 95C), with observance of fluorescence in the reaction (e.g., about 10 ms per frame to about one second per frame) in order to observe a real-time PCR signal. In addition, the concentration and activities of any number of different enzymes such as, but not limited to, nucleases, proteases, kinases, polymerases, glycosylases, topoisomerases, ligases, and phosphatasess can be measured using the microfluidic optical chambers of particular embodiments of the invention. [00128] Referring now to FIG. 12, shown is a flow diagram of an example method of fabricating a structure for a microfluidic optical device in accordance with embodiments of the present invention. The flow begins (1202), and polycrystalline silicon layers may be 27 WO 2009/088408 PCT/US2008/012369 deposited on each side of a single crystal silicon wafer (1204). Via-holes can then be formed, such as by chemical etching or laser drilling (1206). Areas for subsequent etching on the front side of the wafer can then be pattern using photolithography (1208). Silicon nanostructures can then be etched (e.g., using plasma) in the patterned areas (1210). For example, such nanostructures can provide a surface roughness of any suitable shape, such as nanopyramidal arrays. Metal (e.g., gold, silver, etc.) can then be deposited on the etched areas (1212). Remaining photoresist can be removed, and the thin metal nanoparticles can be annealed (1214), completing the flow (1216). [00129] Referring now to FIG. 13, shown is a flow diagram of an example method of making a device for discovery of characteristics of a fluid sample in accordance with embodiments of the present invention. The flow begins (1302), and at least one enzymatic substrate extension may be placed on a metallized nanostructure surface (1304). A structure including the enzymatic substrate extensions can be inverted such that the extensions can reside in a microfluidic optical chamber (1306). A top layer having inlet and outlet ports can then be bonded to the structure (1308). A bottom layer having a transparent window to the structure to form a discovery device with an optical chamber for microfluidic analysis can then be bonded thereto (13 10), completing the flow (1312). [00130] Referring now to FIG. 14, shown is a flow diagram of an example method of using a discovery device for fluid sample analysis in accordance with embodiments of the present invention. The flow begins (1402), and a fluid sample can be received in a microfluidic optical chamber for analysis (1404). Excitation light (e.g., from a laser) can then be provided on an enzymatic substrate extension through a transparent window of the microfluidic optical chamber (1406). Return light from the enzymatic substrate extension can then be received (1408). For example, lenses, mirrors, and splitters can be employed to collect such return light. The return light can then be analyzed (e.g., using a spectrometer or spectrograph) to determine whether a reaction has occurred to modify the enzymatic substrate extension (1410), completing the flow (1412). [00131] Referring now to FIG. 15, shown is a flow diagram of an example method using a high speed system in accordance with embodiments of the invention. A motorized, rotating, glavo mirror (1506) allows for a quick scan of multiple coordinates on a SERS surface. Each coordinate may be bound by a different biomolecule (1518), which may be targeted by an enzyme or other molecule of interest, for example. Excitation light, e.g., from a laser (1502) contacts a mirror (1504) and is redirected to a rotating, glavo mirror 28 WO 2009/088408 PCT/US2008/012369 (1506). Light passes from here to a dichroic mirror (1508) and through to an objective lens (1510). A Raman filter (long pass) (1512) precedes a spectrograph (1514). Each biomolecule (1518) is tethered to a chip surface (1516). [00132] As depicted in FIG. 15, particular embodiments involve biomolecules that are tethered to the surface. For example, such biomolecules can include nucleic acids (DNA and RNA), proteins, peptides, sugar/carbohydrates, metabolites and small chemical compounds. Further, the surface-tethered biomolecules and chemical molecules may be patterned to form a microscale array of a biochemical assay. Various biochemical libraries may also be deposited on the surface of the microfluidics optical chamber for combinatorial detection. Functional groups can include reactive groups. Functional groups can also include bifunctional crosslinkers having two reactive groups capable of forming a bond with two or more different functional targets (e.g., peptides, proteins, macromolecules, surface coating/surface, etc.). In some embodiments, the bifunctional crosslinkers are heterobifunctional crosslinkers with two different reactive groups. To allow covalent conjugation of biomolecule to the surface, suitable reactive groups include, e.g., thiol (-SH), carboxylate (COOH), carboxyl (-COOH), carbonyl, amine (NH 2 ), hydroxyl (-OH), aldehyde (-CHO), alcohol (ROH), ketone (R 2 CO), active hydrogen, ester, sulfhydryl (SH), phosphate (-P0 3 ), or photoreactive moieties. Amine reactive groups can include, e.g., isothiocyanates, isocyanates, acyl azides, NHS esters, sulfonyl chlorides, aldehydes and glyoxals, epoxides and oxiranes, carbonates, arylating agents, imidoesters, carbodiimides, and anhydrides. Thiol-reactive groups include, e.g., haloacetyl and alkyl halide derivates, maleimides, aziridines, acryloyl derivatives, arylating agents, and thiol disulfides exchange reagents. Carboxylate reactive groups include, e.g., diazoalkanes and diazoacetyl compounds, such as carbonyldiimidazoles and carbodiimides. Hydroxyl reactive groups include, e.g., epoxides and oxiranes, carbonyldiimidazole, oxidation with periodate, N,N'-disuccinim idyl carbonate or N-hydroxylsuccimidyl chloroformate, enzymatic oxidation, alkyl halogens, and isocyanates. Aldehyde and ketone reactive groups include, e.g., hydrazine derivatives for schiff base formation or reduction amination. Active hydrogen reactive groups include, e.g., diazonium derivatives for mannich condensation and iodination reactions. Photoreactive groups include, e.g., aryl azides and halogenated aryl azides, benzophenones, diazo compounds, and diazirine derivatives. 29 WO 2009/088408 PCT/US2008/012369 [00133] In one embodiment, a heterobifunctional crosslinker includes two different reactive groups that form a heterocyclic ring that can interact with a substrate peptide. For example, a heterobifunctional crosslinker, such as cysteine, may include an amine reactive group and a thiol-reactive group that interacts with an aldehyde on a derivatized peptide. Additional combinations of reactive groups for heterobifunctional crosslinkers include, e.g., amine and sulfhydryl reactive groups, carbonyl and sulfhydryl reactive groups, amine and photoreactive groups, sulfhydryl and photoreactive groups, carbonyl and photoreactive groups, carboxylate and photoreactive groups, and arginine and photoreactive groups. [001341 Also in particular embodiments, the microfluidic optical chip can be automatically transported and aligned with an associated spectroscopic imaging system. For example, such transportation and/or alignment may be controlled by a computer using optimization algorithms. Also, special markers can be included on the microfluidic chips, and may be used in automated pattern recognition. [001351 Certain embodiments can also provide electrodes integrated into the channels such that a voltage potential can be applied across the microfluidics optical chamber to form a capillary electrophoresis system. For example, DNA and protein separation using electrophoresis and isoelectrical focusing can then be realized, and the optical spectra of the biomolecules can be monitored in real-time. [00136] Also in certain embodiments, the content within the microfluidic optical chamber can be gas phase material, rather than liquid. The optical properties of gas can be measured or monitored continuously in real-time. For example, concentration of particulates in the air can be monitored. [001371 In certain embodiments, antibodies are tethered to the chip surface. The presence and/or concentration of the corresponding antigen in a sample may be measured. Antibodies specific for a certain cancer biomarker are tethered to the surface in embodiments directed to cancer diagnosis. Among receptor tyrosine kinases, the EGF receptor gene family including EGFR and erb B2, which are most frequently implicated in human cancers. For example, amplification of EGFR and erb B2 genes for human gastric cancers has been determined at around 3-5% and 10-20% respectively (Albino et al., (1995) Eur. J. Surg. Oncol., 21:56-60; Sato et al., (1997) Pathol. Int., 47, 179-182; Hung and Lao, (1999) Semin. Oncol., 26:51-59). Coamplification of gastrin and erb B2 has been reported for intestinal-type gastric cancers (Vidgren et al., (1999) Genes Chromosomes Cancer, 24, 24-29). Thus, an increase in levels of EGFR and erb B2 proteins accompanied 30 WO 2009/088408 PCT/US2008/012369 by elevated levels of gastrin is indicative of intestinal cancer. The sensitivity of the instant invention facilitates detection of marginal increases in levels of these proteins. This improved sensitivity is significant as most gastric cancer is not diagnosed until the cancer has advanced to more serious stages. Moreover, measurement of the protein levels in the method of the invention requires minute sample volumes, making it suitable for testing biopsy samples. A multitude of antibodies suitable for use in the present invention are commercially available from vendors such as AbCam, BioMol, Sigma, etc. [00138] In particular embodiments, enzymatic activity and concentration may also be detected. The substrate for an enzyme is tethered to the nanostructure of the surface and a test sample comprising the enzyme passed over/incubated with the substrate in the conditions conducive to the occurrence of the catalytic reaction. The substrates can be those for proteases, kinases, phosphatases, nucleases, methyltransferases, acetyltransferases, acyltransferases, transaminases, glycosyltransferases, and the like. [00139] The substrates typically range in length from at least about four residues to up to about 10, 30, 50, 200 or 500 residues. Thus, the substrate for a protease is about four amino acids, and may be up to about 50, 200 or 500 amino acids. Such a substrate may have one or more recognition sequences recognized by the enzyme. Such a substrate may additionally be comprised of non-naturally occurring amino acid, nucleotide, and/or sugar residues. In addition, such a substrate may be modified by enzyme or chemical processes to add or remove functional groups. Detection of protease activity [00140] In particular embodiments, the present invention is used to detect protease activity. Proteases are required not only for maintenance of normal cellular functions but are often central to pathogenesis of a variety of human diseases. Parasitic, fungal, viral infections, cancer, inflammatory, respiratory, cardiovascular, and neurodegenerative diseases require proteolytic activity for progression. Detection of protease concentration and/or activity is valuable as a diagnostic /prognostic marker for the presence or likelihood of the disease. Further, detection of inhibition of protease activity is useful in screening for protease inhibitors for treatment of a number of pathologies. [001411 A "protease" that can be detected and/or quantitated according to the invention is an enzyme that typically hydrolyzes a peptide bond between a pair of amino acids in a protein/peptide, producing a shorter protein/peptide. This activity also referred to as 31 WO 2009/088408 PCT/US2008/012369 proteolysis. Proteolysis of the protein/peptide substrate is detectable by changes in spectrum obtained by SERS, electromagnetic resonance measurement or acoustic measurement. Proteases are typically defined by reference to the nucleophile in the catalytic center of the enzyme. The most common nucleophiles arise from the side chains of serine, aspartic acid and cysteine. Accordingly, proteases are classified into protease families such as serine proteases (Paetzel et al. (1997) Trends Biochem. Sci. 22:28-3 1), aspartyl proteases (Spinelli et al. (1991) Biochemie 73: 1391-1396), and cysteine proteases (Altschuh et al. (1994) Prot. Eng. 7:769-75, 1994). Metalloproteases usually contain a zinc catalytic metal ion at the catalytic center (Klimpel et al. (1994) Mol. Microbiol. 13: 1093 1100). A "protease recognition site" is a sequence of amino acids in a peptide or protein that contain a pair of amino acids that are hydrolyzed by a particular protease. The specific sequence of amino acids in the protease recognition site typically depends on the catalytic mechanism of the protease, which is defined by the nature of the functional group at the protease's active site. Thus, a protease such as trypsin hydrolyzes peptide bonds whose carbonyl function is donated by either a lysine or arginine residue, regardless of the length or amino acid sequence of the peptide/protein. Other proteases have a higher specificity, e.g., Factor Xa recognizes the sequence Ile-Glu-Gly-Arg and hydrolyses peptide bonds on the C-terminal side of the Arg. Various preferred protease recognition site include, but are not limited to protease recognition sites for proteases from the serine protease family, or from metallopproteases, or from cysteine proteases, and/or the aspartic acid protease family, and/or the glutamic acid protease family. Protease recognition sites are well known to those of skill in the art. Recognition sites have been identified for virtually all known proteases. Thus, for example, recognition sites (peptide substrates) for caspases are described by Earnshaw et al. (1999) Annu. Rev. Biochem. 68: 383-424, which is incorporated herein by reference. 100142] In certain embodiments, substrates for kinases or phosphatases are attached to the nanostructure surface of the device. The attachment is achieved via contact pins, injectors or covalent bonds. Different kinase or phosphatase substrates can be localized at specific locations on the surface, thereby providing an array for the detection of one or more kinases and/or phosphatases and/or the quantitation of the activity of one or more kinases and/or phosphatases. It will be recognized that while the apparatus, methods and 32 WO 2009/088408 PCT/US2008/012369 compositions are described with respect to detecting phosphorylation of a substrate, these apparatus, methods and compositions are also useful in detecting dephosphorylation of a substrate. Kinase/phosphatase activity detection [001431 Phosphorylation is a common posttranslational modification of proteins and plays a key role on protein structure and function and in all aspects of cell physiology. Protein kinases contain well conserved motifs and constitute the largest family of proteins in the human genome. Mutations of protein kinases are involved in carcinogenesis and several other pathological conditions. Phosphorylations of other biomolecules also play a critical role in the physiology and pathology of cells. Lipid kinases such as the phosphoinositide-3 kinase family members are key modulators of the cellular response to growth factors, hormones, and neurotransmitters and are involved in cancer. Nucleotide and nucleoside kinases regulate the intracellular levels of phosphate donors and nucleic acid precursors and are involved in the cellular response to injury and ischemia. Sugar kinases regulate the rates of sugar metabolism, energy generation, and transcription activation and are involved in the process of cellular transformation and apoptosis. Thus detecting and/or measuring kinase activity is useful in detecting changes in cell/tissue homeostasis, physiology, diagnosing disease conditions and the like. [00144] Any molecule that can be phosphorylated by a kinase and/or dephosphorylated by a phosphatase can be used as a kinase/phosphatase substrate in the apparatus, methods and compositions described herein. These molecules include proteins, peptides, sugars (e.g., hexose, glucose, fructose etc.), nucleic acids, acetate, butyrate, lipids, ceramide and the like. Table I provides an exemplary list of known kinases and their Enzyme Commission numbers (EC numbers), which can be detected by employing the methods of the invention. The name of the kinase usually identifies the substrate the enzyme acts upon. It is well known that most substrates that are modified by phosphorylation can be dephosphoryated by a phosphatase. Thus, a surface on which kinase substrates are attached can be used in a phosphatase assay by first modifying the substrates by phosphorylating them. [001451 Table 1. Illustrative kinases and corresponding Enzyme Commission (EC) Numbers 33 WO 2009/088408 PCT/US2008/012369 E.C. No. Kinase E.C.No. Kinase 2.7.1.32 Choline kinase 2.7.1.90 Diphosphate fructose-6 phosphate 1 phosphotransferase 2.7.1.37 Phosphorylase kinase 2.7.1.91 Sphinganine kinase 2.7.1.39 Homoserine kinase 2.7.1.107 Diacylglycerol kinase 2.7.1.67 -phosphatidylinositol 4- 2.7.1.138 Ceramide kinase kinase 2.7.1.72 Streptomycin 6-kinase 2.7.1.2 Glucokinase 2.7.1.82 Ethanolamine kinase 2.7.1.3 Ketohexokinase 2.7.1.87 Streptomycin 3 "-kinase 2.7.1.4 Fructokinase 2.7.1.95 Kanamycin kinase 2.7.1.11 6-phosphofructokinase 2.7.1.100 5-methylthioribose 2.7.1.15 Ribokinase kinase 2.7.1.103 Viomycin kinase 2.7.1.20 Adenosine kinase 2.7.1.109 [Hydroxymethylglutaryl- 2.7.1.35 Pyridoxal kinase CoA reductase (NADPH2)] kinase 2.7.1.112 Protein-tyrosine kinase 2.7.1.45 2-dehydfo-3 deoxygluconokinase 2.7.1.116 [Isocitrate 2.7.1.49 Hydroxymethylpyrimidine dehydrogenase kinase (NADP+)] kinase 2.7.1.117 [Myosin light-chain] 2.7.1.50 Hydroxyethylthiazole kinase kinase 2.7.1.119 Hygromycin-B kinase 2.7.1.56 1 -phosphofructokinase 2.7.1.123 Calcium/calmodulin 2.7.1.73 Inosine kinase dependent protein kinase 2.7.1.125 Rhodopsin kinase 2.7.1.92 5-dehydro-2 deoxygluconokinase 2.7.1.126 [Beta-ad renergic- 2.7.1.144 Tagatose-6-phosphate kinase receptor] kinase 2.7.1.129 [Myosin heavy-chain] 2.7.1.146 ADP-dependent kinase phosphofructokinase 2.7.1.135 [Tau protein] kinase 2.7.1.147 ADP-dependent glucokinase 2.7.1.136 Macrolide 2' -kinase 2.7.4.7 Phosphomethylpyrimidine kinase 2.7.1.137 1-phosphatidylinositol 3- 2.7.6.2 Thiamin pyrophosphokinase kinase 2.7.1.141 [RNA-polymerase] - 2.7.1.31 Glycerate kinase 34 WO 2009/088408 PCT/US2008/012369 subunit kinase 2.7.1.153 Phosphatidylinositol- 2.7.4.6 Nucleoside-diphosphate kinase 4,5-bisphosphate 3 kinase 2.7.1.154 Phosphatidylinositol-4- 2.7.6.3 2-amino-4-hydroxy-6 phosphate 3- kinase hydroxymethyldihydropteridine pyrophosphokinase 2.7.1.68 1 -phosphatidylinositol- 2.7.3.1 Guanidoacetate kinase 4-phosphate 5-kinase 2.7.1.127 ID-myo-inositol- 2.7.3.2 Creatine kinase trisphosphate 3-kinase 2.7.1.140 Inositol- 2.7.3.3 Arginine kinase tetrakisphosphate 5 kinase 2.7.1.149 1-phosphatidylinositol 5- 2.7.3.5 Lombricine kinase phosphate 4-kinase 2.7.1.150 1-phosphatidylinositol 3- 2.7.1.37 Protein kinase (Histidine phosphate 5-kinase kinase) 2.7.1.151 Inositol-polyphosphate 2.7.1.99 [Pyruvate multikinase dehydrogenase(Kpoamide)] kinase 2.7.4.21 Inositol- 2.7.1.115 [3-methyl-2-oxobutanoate hexakisphosphate kinase dehydrogenase (lipoamide)] kinase 2.7.1.134 Inositol- 2.7.1.1 Hexokinase tetrakisphosphate 1 kinase 2.7.9.1 Pyravate, phosphate 2.7.1.2 Glucokinase dikinase 2.7.9.2 Pyravate, water dikinase 2.7.1.4 Fructokinase 2.7.1.12 Gluconokinase 2.7.1.5 Rhamnulokinase 2.7.1.19 Phosphoribulokinase 2.7.1.7 Mannokinase 2.7.1.21 Thymidine kinase 2.7.1.12 Gluconokinase 2.7.1.22 Ribosylnicotinamide 2.7.1.16 L-ribulokinase kinase 2.7.1.24 Dephospho-CoA kinase 2.7.1.17 Xylulokinase 2.7.1.25 Adenylylsulfate kinase 2.7.1.27 Erythritol kinase 2.7.1.33 Pantothenate kinase 2.7.1.30 Glycerol kinase 2.7.1.37 Protein kinase (bacterial) 2.7. 1.33 Pantothenate kinase 2.7.1.48 Uridine kinase 2.7.1.47 D-ribulokinase 35 WO 2009/088408 PCT/US2008/012369 2.7.1.71 Shikimate kinase 2.7.1.51 L-fuculokinase 2.7.1.74 Deoxycytidine kinase 2.7.1.53 L-xylulokinase 2.7.1.76 Deoxyadenosine kinase 2.7.1.55 Allose kinase 2.7.1.78 Polynucleotide 5'- 2.7.1.58 2-dehydro-3 hydroxylkinase deoxygalactonokinase 2.7.1.105 6-phosphofructo-2- 2.7.1.59 N-acetylglucosamine kinase kinase 2.7.1.113 Deoxyguanosine kinase 2.7.1.130 Tetraacyldisaccharide 4'- 2.7. 1.60 N-acylmannosamine kinase kinase 2.7.1.145 Deoxynucleoside kinase 2.7.1.63 Polyphosphate-glucose ph 2.7.1.156 osphotransf erase Adenosylcobinamide kinase 2.7.4.1 Polyphosphate kinase 2.7.1.85 Beta-glucoside kinase 2.7.4.2 Phosphomevalonate kinase 2.7.4.3 Adenylate kinase 2.7.2.1 Acetate kinase 2.7.4.4 Nucleoside-phosphate 2.7.2.7 Butyrate kinase kinase 2.7.4.8 Guanylate kinase 2.7.2.14 Branched-ehain-fatty-acid kinase 2.7.4.9 Thymidylate kinase 2.7.2. Propionate kinase 2.7.4.10 Nucleoside-triphosphate 2.7.1.40 Pyravate kinase -adenylate kinase 2.7.4.13 (Deoxy)nucleoside- 2.7.1.36 Mevalonate kinase phosphate kinase 2.7.4.14 Cytidylate kinase 2.7.1.39 Homoserine kinase 2.7.4. Uridylate kinase 2.7.1.46 L-arabinokinase 2.7.1.37 Protein kinase (HPr 2.7.1.52 Fucokinase kinase/ phosphatase) 4.1.1.32 Phosphoenolpyruvate 2.7.1.71 Shikimate kinase carboxykinase (GTP) 4.1.1.49 Phosphoenolpyruvate 2.7.1.148 4-(cytidine 5'-diphospho)-2 carboxykinase (ATP) Cmethyl-D- erythritol kinase 2.7.2.3 Phosphoglycerate kinase 2.7.4.2 Phosphoraevalonate kinase 36 WO 2009/088408 PCT/US2008/012369 [001461 The substrate and/or the substrate consensus sequence for a majority of kinases and phosphatases are known. Short synthetic peptides based on consensus motifs are typically excellent substrates for kinases and phosphatases. Table 2 summarizes some of the known data about specific motifs for various well-studied protein kinases, along with examples of known phosphorylation sites in specific proteins, which can be detected by employing the methods of the invention. A more extensive list is present in Pearson and Kemp (1991) Meth. Enzymol., 200:68-82, which is incorporated herein by reference. [00147] Table 2. Recognition motifs and substrate sequences for some known kinases are listed. The amino acid phosphorylated by the corresponding kinase is underlined. Slash (/) indicates amino acids that can functionally substitute each other. Amino acids not contributing to the substrate recognition sequence are indicated by "X". Kinase Recognition Phosphorylation Sites Protein substrate Motifs) cAMP- R-X-S/T (SEQ ID Y 7 LRRASLAQLT (SEQ ID pyruvate kinase dependent NO:1) NO:3) Protein Kinase (PKA, cAPK) R-R/K-X-S/T FIRRLSIST (SEQ ID NO:4) phosphorylase kinase (SEQ ID NO:2) ax-chain

A

29 GARRKASGPP (SEQ ID histone HI, bovine NO:5) Casein Kinase I S(P)-X-X-S/T R 4 TLS(P)VSSLPGL (SEQ ID glycogen synthase, (CKI, CK-1) (SEQ ID NO:6) NO:7)

D

43 1GS(P)ES(P)TEDQ (SEQ ID rabbit muscle (asi NO:8) casein Casein Kinase n S/T-X-X-E (SEQ A 72 DSESEDEED (SEQ ID NO: PKA regulatory (CKII, CK-2) ID NO:9) 10) subunit, RI,

L

37 ESEEEGVPST (SEQ ID NO: p34 CZ, human 13) 37 WO 2009/088408 PCT/US2008/012369 acetyl-CoA carboxylase Glycogen S-X-X-X-S(P) S6 1 VPPSPSLS(P) (SEQ ID NO: glycogen synthase, Synthase Kinase (SEQIDNO:13) 14) human (site 3b) 3 (GSK-3)

S&

4 VPPS (P)PSLS(P) (SEQ ID NO: 15) glycogen synthase, human (site 3a) Cdc2 Protein SIT-P-X-R/K PI 3 AKUPVK (SEQ ID NO: 17) histone HI, calf thymus Kinase; CDK2- (SEQIDNO:16) cyclin A

H

1 22 STPPKKKRK (SEQ ED large T antigen NO: 18) Calmodulin- R-X-X-S/T R-X- N 2 YLRRRLSDSN synapsin (site 1) dependent X-S/T-V (SEQIDNO:19) Protein Kinase II (CaMKH) Klg 9 MARVFSVLR calcineurin (SEQIDNO:20) Mitogen- P-X-S-P (SEQ P 2 4 4LSP (SEQIDNO:23) c-Jun activated Protein ID NO:21) Kinase (Extracellular P92SSP (SEQ ED NO:24) cyclin B Signal-regulated X-X-S/T-P (SEQ Kinase) (MAPK, ID NO:2) Erk) V420LSP (SEQIDNO:25) Elk-] Abl Tyrosine I/V/L-Y-X-X-P/F Kinase (SEQ ED NO:26) [00148] Many kinase substrates are commercially available from various vendors such as Sigma, BioMol International, Bio-Rad, etc. Preferred kinase substrates include but are not limited to substrates for histidine, serine, threonine, and tyrosine kinases and/or the corresponding phosphatases. Multiple susbtrates for these kinases are well known in the art. In addition, methods are known for identification of substrates. For example, the program PREDIKIN is used to predict substrates for serine/threonine protein kinases based on the primary sequence of the kinase catalytic domain. Methods for using PREDIKIN to design substrates are described by Ross et al. (2003) PNAS, USA, 100 (1):74-79, which is 38 WO 2009/088408 PCT/US2008/012369 incorporated herein by reference. Other programs serving the same function are well known in the art. [001491 A number of substrates specific to a type of protein kinase are known. Table 3 lists well known tyrosine kinase substrates. [00150] Table 3. Partial list of known tyrosine kinase substrates and the position of the phosphorylated tyrosine residue is indicated. Shown are other post-translational protein modifications that can be detected by the methods of the invention. Substrate Phosphorylation Substrate Phosphorylation Site Site KDR Tyr996 PLCg Tyr771/775 STAT3 Tyr705 T-cell activation Tyr217 antigen cdc2 Tyr15 T-cell Receptor Tyrl52 Zeta chain JAKI Tyr1022/1023 ERK5 Tyr215/220 KDR Tyr1054/1059 GSK3 Tyr284 Paxillin TyrSI JNKI Tyri9O Pyk2 Tyr402 TrkC Tyr705 She Tyr317 Zinc Finger Tyr7O Protein 145 STATI Tyr701 TIF Tyr495 TrkA Tyr490 c-Kit (Y900) 64 TrkA Tyr785 PTPIB Tyr66 Tyk2 Tyr1054/1055 SHP-2 (Try542) 63 Zap70 Tyr493 P13K Tyr688 STAT6 Tyr641 Src Tyr416 HER2 Tyr1248 c-FGR Tyr412 39 WO 2009/088408 PCT/US2008/012369 STAT5 Tyr694 EGFR TyrlI73 CTD Tyr ERa Tyr537 FAK Tyr577 IRS] Tyr891 STAT4 Tyr693 ER.S2 Tyr766 PDGFR Tyr775 JAK2 TyrlOOS STAT2 Tyr690 PTEN Tyr315 JAKI Tyr023 c-Cbl Tyr700 Liver Glycogen Tyr637 Dynaminl/n Tyr231 Synthase NLK-1 TyrISI P62Dok Tyr398 PDGFR Tyr771 R-Ras Tyr66 Signal Tyrl60 PTEN Tyr336 Transduction Protein TLE2 Tyr226 VEGFRI Tyr 12 13 beta-adrenergic Tyr350 VEGFR2 TyrI212 receptor CSBP1 Tyr 182 Zap70 Tyr319 doublecortin Tyr345 c-CbI Tyr774 HER2 Tyr1248 Met Tyr 1349 Insulin Receptor Tyr992 Met Tyr1356 Precursor [00151] The foregoing kinase/phosphatase substrates are intended to be illustrative and not limiting. Using teachings provided herein and those well known in the art, other kinase substrates will be readily available to one of skill in the art for use in the apparatus, methods and compositions described herein. Attachment of kinase/phosphatase substrates to the SERS substrate device 40 WO 2009/088408 PCT/US2008/012369 [001521 The kinase and/or phosphatase substrates may be attached to nanoparticle(s) or to features present on a surface (e.g., a Raman active surface) by any of a number of methods well known to those of skill in the art. Such methods include but are not limited to using microscale contact pins or injectors or covalent bonds. [00153] For example, in certain embodiments that include a gold nanostructure, the kinase and/or phosphatase substrates are tethered onto a gold nanostructure by a covalent bond formed by a gold-thiol reaction between a cysteine group at the terminus of the substrate (e.g., peptide) and the gold surface. In various embodiments, the array surface and/or the kinase and/or phosphatase substrate can be derivatized with, for example, amine, carboxyl groups, alkyl groups, alkyene groups, hydroxyl groups, or other functional groups so that the peptide (or other substrate) can be linked directly to the surface or coupled through a linker. In other embodiments, the surface can be functionalized, e.g., with amine, carboxyl, or other functional groups for attachment to the kinase and/or phosphatase substrate(s). [00154] Suitable linkers include, but are not limited to hetero- or homo-bifunctional molecules that contain two or more reactive sites that may each form a covalent bond with the respective binding partner (kinase/phosphatase substrate, surface, or functional group thereon, etc.). Linkers suitable for joining such moieties are well known to those of skill in the art. For example, a protein molecule can readily be linked by any of a variety of linkers including, but not limited to a peptide linker, a straight or branched chain carbon chain linker, or by a heterocyclic carbon linker. Heterobifunctional cross-linking reagents such as active esters of N-ethylmaleimide have been widely used to link proteins to other moieties (see, e.g., Lerner et al. (1981) Proc. Nat Acad. Sci. (USA), 78: 3403-3407; Kitagawa et al. (1976) J. Biochem., 79: 233-236; Birch and Lennox (1995) Chapter 4 in Monoclonal Antibodies: Principles and Applications, Wiley-Liss, N.Y., and the like). [00155] In certain embodiment, the kinase and/or phosphatase substrate can be attached to the surface utilizing a biotin/avidin interaction. In certain embodiments, biotin or avidin, e.g., with a photolabile protecting group can be affixed to the surface and/or to the kinase/phosphatase substrate(s). Irradiation of the surface in the presence of the desired kinase and/or phosphatase substrate bearing the corresponding avidin or streptavidin, or biotin, results in coupling of the substrate to the surface. [00156] In various embodiments, multiple kinase and/or phosphatase substrates, usually at least about five, preferably at least ten, or at least 20, 50, 100, 500, 1000, 10,000 or 100, 1000 are attached to the surface. The kinase/phosphatase substrate can be a single substrate 41 WO 2009/088408 PCT/US2008/012369 attached in multiple copies on to the surface or attached in varying densities across the surface. Varying the density of the substrate will facilitate quantitation of the kinase/phosphatase activity. Thus, if a new peak appears upon the occurrence of a phopsphorylation reaction, the amplitude of the peak corresponding to different locations of the nanostrcuture surface will increase in accordance with the increase in density of the attached substrate. Alternatively, pluralities of substrates are attached at different locations on the surface. Thus, several positions are tethered with positive control substrates, at various densities and at other positions, negative control substrates, also at various densities. [00157] In certain embodiments, the surface provides a high density array of kinase and/or phosphatase substrates. In various embodiments, such an array can comprise at least 100 or at least 200 different substrates/cm 2 , preferably at least 300,400, 500, or 1000 different 22 substrates/cm2, and more preferably at least 1,500, 2,000,4,000,10,000, or 50,000, or 100,000 different substrates/cm 2 . [001581 Methods for patterning molecules on surfaces at high density are well known to those of skill in the art. Such methods include, for example, the use of high density microarray printers (See, e.g., Heller (2002) Ann. Rev. Biomed. Eng. 4: 129-153). Other microarray printers utilize "on-demand" piezoelectric droplet generators (e.g., inkjet printers) (see, e.g., U.S. Patent Nos. 6,395,562; 6,365,378; 6,228,659; and WO 95/251116 and WO/2003/028868) which are incorporated herein by reference. Other approaches involve de novo synthesis (see, e.g., Fodor et al. (1991) Science, 251:767-773 and U.S. Patent Nos. 6,269,846, 6,271,957 and 6,480,324 which are incorporated herein by reference). A number of printers are commercially available (see e.g., VERSA Mini Spot printing workstation from Aurora Biomed, BIOODYSSEY CALLIGRAPHER MiniArrayer from Bio-Rad, OmniGrid Accent from Genomic Solutions and the like). Substrate phosphorylation/dephosphorylation assay [00159] Where it is desirable to detect and/or measure the activity of a single type of kinase and/or phosphatase in a sample, a single type of substrate is tethered to the SERS surface of the microfluidic device. In embodiments pertaining to detection of a plurality of kinases and/or phosphatases in a sample, a plurality of substrates is tethered to the SERS surface of the microfluidic device. 42 WO 2009/088408 PCT/US2008/012369 [001601 The kinase and/or phosphatase activity detection/measurement described herein can be performed on any of a number of different samples. For example, in screening systems for the identification of kinase antagonists or agonists, cells/cell lines and/or lysates thereof, or appropriate buffer systems comprising the kinase(s) of interest can be contacted / administered as one or more test compounds. The samples derived therefrom can then be screened for kinase activity by identifying which test compounds show activty, e.g., as kinase inhibitors and/or phosphatase agonists, and which kinase/phosphatase enzymes they inhibit and/or agonize. [00161] In various diagnostic embodiments, the existence of the kinase and/or phosphatase enzyme(s), and/or concentration, and/or activity thereof, is determined in a biological sample. The biological sample can include essentially any biomaterial that is to be assayed. Such biomaterials include, but are not limited to biofluids such as blood or blood fractions, plasma, lymphatic fluid, tears, spinal and pulmonary fluid, cerebrospinal fluid, seminal fluid, urine, saliva and the like, tissue samples, cell samples, tissue or organ biopsies or aspirates, histological specimens, and the like. [00162] In certain embodiments the raw cell lysate can be directly introduced into the microfluidic device and the measurement can be done during the incubation. Samples are introduced into the reaction chamber through microfluidic channels. The total sample volume may be reduced to sub-microliter volume. [00163] Phosphorylation of a kinase substrate or dephosphorylation of a phosphatase substrate is detectable by changes in the spectrum obtained by SERS, electromagnetic resonance measurement, or acoustic measurement. Changes in the spectrum of the SERS surface compared to a control (no sample or control sample) may be indicative of kinase/phosphatase activity. The change in the spectrum could be appearance of a new peak accompanied by the disappearance of an existing peak, a shifting of peaks, as well as the merging and/or splitting of peaks. [001641 Such a surface provides an effective tool for real-time screening for the concentration and/or activity of one or a plurality of kinases and/or phosphatases and/or for quantification of the kinetics of one or more kinases and/or phosphatases. Such a surface can also be readily used to screen for kinase and/or phosphatase inhibitor activity of one or a plurality of test agents (e.g. a chemical library). [001651 In certain embodiments the kinase/phosphatase activity detection and/or measurements can be used in personalized molecular diagnostics for cancers by physicians 43 WO 2009/088408 PCT/US2008/012369 and hospital personnel. In one embodiment, the instant invention is used to detect the presence of molecular markers specific to a particular type of cancer. EXAMPLE 1 Detection of altered protease activity [001661 Real-time in situ detection of proteases is crucial for early-stage cancer screening as well as for assessing the efficacy of a treatment method. In one illustrative example, the instant invention is used to detect activity of a protease, prostate-specific antigen (PSA), in a biological sample. PSA levels are increased in prostate cancer. Thus, PSA serves as a biomarker for prostate cancer. Measurement of plasma PSA concentration does not differentiate prostate cancer patients from those with benign prostatic hyperplasia, leading to a high false-positive rate. Efforts to enhance the clinical value of PSA as an early detection marker for prostate cancer have included the characterization of various molecular isoforms of PSA. Among the various isoforms, the proteolytically active subpopulation of PSA is accepted as a more useful tumor marker and malignancy predictor than the serum PSA concentration (Wu et al. (2004) Prostate 58: 345-353; Wu et al. (2004) Clin. Chem., 50: 125-129). [00167] The peptide substrate used for detection of PSA protease activity incorporates the amino acid sequence of the active site of PSA-specific peptides with serine residues and flanking sequences that can be recognized by PSA. Thus, the peptide includes the sequence HSSKLQ-LAAAC which is known to have a very high specificity for proteolytically active PSA (Denmeade et al., (1997) Cancer Res 57:4924-4930). It has also been shown that HSSKLQ-L is cleaved by PSA but not by any other proteases in vivo in a mouse model (Denmeade et al., (2003) J. Natl. Cancer Inst. 95: 990-1000). Thus, a screen may be performed wherein multiple peptides are attached to the nanostructure of a SERS substrate surface, each having a random or known sequence portion, and the PSA specific sequence HSSKLQ-LAAAC or HSSKLQ-L. The PSA hydrolysis site is between Q and L. Proteolysis results in shortening of the peptide, which is detectable by changes in the spectrum associated with the peptides. This may then be observed in the resulting spectrograph. [001681 In this particular example, a SERS substrate surface has a gold nanostructure. The peptides are attached to the surface via a gold-thiol covalent bond formed between cysteine at the carboxyl terminus of the peptide and the gold nanostructure. The sample to be tested 44 WO 2009/088408 PCT/US2008/012369 is introduced into the microfluidic chamber where the temperature is maintained at 37*C. The sample is maintained in contact with the peptide substrates on the SERS surface in the device for about 2 hours. The spectrum obtained from the plasma sample from a patient with suspected prostate cancer is compared to that of an age matched non-afflicted person. Purified PSA is used as a positive control for the detection assay. [00169] Further, proteolysis dynamics may be monitored in real-time by time-resolved spectra acquisitions. Thus, the disappearance, appearance, shifting, merging, or splitting in peaks can be followed real-time. [001701 The use of a nanostructure facilitates the detection of changes in spectra associated with a particular molecule attached to the SERS surface. Thus, the fusion of an enzyme substrate to fluorescent or radioactive tags is not necessary. EXAMPLE 2 Detection of altered kinase activity [00171] Protein kinases represent approximately 1.7% of all human genes and not surprisingly are important cellular regulatory proteins (Manning et al. (2002) Science 298: 1912-1934). Most of the 30 known tumor suppressor genes and more than 100 dominant oncogenes are protein kinases (Futreal et al. (2001) Nature 409: 850-852). Tyrosine-kinase receptors are key molecules in signaling pathways leading to growth and differentiation of normal cells. Mutations leading to inactivation of certain tyrosine kinases and increased activity of others is a hallmark of tumor cells. The instant invention may be used to provide a tyrosine kinase activity profile associated with a certain tissue of interest. In this example, the tissue is a biopsy sample of the colon obtained from a person free of colon cancer (for obtaining a normal kinase activity profile) and from a patient afflicted with colon cancer (for obtaining a kinase activity profile from a positive control). Once the tyrosine kinase activity profile for normal tissue and control tissue is obtained, the same procedure is performed with a colon biopsy sample from a patient suspected of having colon cancer. A significant departure from the normal kinase activity profile spectrum and/or similarity to the positive control kinase activity profile spectrum is indicative of colon cancer. [001721 Biopsy samples are transferred to ceramic beads-containing special centritubes (Roche, Penzberg, Germany) with 0.1 mL of pre-chilled TLysis buffer. The tissue may be subjected to oscillation made by the MagNA Lyser machine at 6500 r/min for 120 45 WO 2009/088408 PCT/US2008/012369 seconds. The lysate is then centrifuged at 100,000 g for 1 h at 4"C, and the supernatant is saved and assayed for protein concentration (Lowry method). [00173] Tyrosine kinase substrates of Table 3 are tethered to the nanostructure surface of the instant invention. The tissue lysate may be introduced into the microfluidic chamber, which is maintained at 37"C. The lysate is incubated with tyrosine kinase substrates for 1 hour. The spectrum associated with the enzyme substrates attached to the nanostructure surface is measured before the introduction of the lysate, during the incubation and after washing away of the lysate. Thus, phosphorylation dynamics are monitored in real-time by time-resolved spectra acquisitions. This time-dependent tyrosine kinase activity profile increases the accuracy of data interpretation. EXAMPLE 3 Transcription factor activity profiling [00174] Gene expression profiling is increasingly used to characterize cell samples such as tumor biopsies. By measuring the levels of selected messenger RNAs in a sample, inferences may be drawn concerning the subtype or molecular profile of the sample, providing information that may support medical decisions, including treatment alternatives. A potentially more informative alternative to measuring RNA levels is to directly measure the activity of proteins in a tumor biopsy or other cell sample. DNA binding transcription factors are a class of proteins that are particularly informative for molecular profiling, providing information about the detailed transcriptional state of cells in a sample. [001751 In this example, the activity of DNA binding transcription factors in a cell sample are dynamically measured using a microfluidic SERS detection apparatus. The apparatus is prepared such that one or potentially many individually addressed oligonucleotide probes are attached to the nanostructure of the SERS substrate surface, with each oligonucleotide having a sequence comprising a binding site for a particular transcription factor of interest. For example, a 25-mer double stranded DNA oligonucleotide including the E-box hexamer sequence CACGTG may be used to interrogate the activity of a subclass of basic helix loop-helix transcription factors. Mismatch oligonucleotides may also be used as controls for nonspecific binding, and identical sequences may be redundantly arrayed to increase measurement accuracy. Evaluation of SERS spectra provides dynamic information about the binding of transcription factors to the oligonucleotide probes as well as the formation of 46 WO 2009/088408 PCT/US2008/012369 DNA-transcription factor super-complexes that may include additional transcription cofactors and TAF proteins. [001761 A needle biopsy containing Ix104 cells is taken and the nuclear extract isolated at 40 C using Sigma NXTRACT CELLYTIC NUCLEAR extraction kit. The nuclear extract is then resuspended in 19 tl cold 10mM Tris-HCL buffer containing 1mM DTT. I pl Sigma protease inhibitor cocktail P8340 is added, and the solution is transferred to the microfluidic SERS detection apparatus. At 250 C, the sample enters the microscale chamber and DNA binding events are measured in real-time using incident laser light and detection of transmitted SERS spectra. Transcription factor binding activity profiles are developed or calculated from one or more of the following measurements, for each oligonucelotide sequence: (1) the occupancy of bound oligonucleotides as a fraction of total available sites; (2) the average stability of DNA-protein complexes in seconds; and (3) the total number of binding events per unit time. Comparison of transcription factor binding activity profiles across tissue types and across diseased versus normal tissues characterize the molecular pathology of a tissue sample and are potentially diagnostic for treatment alternatives. [00177] Table 4. Additional proteases are presented, the concentration and activity of which may be detected and quantitated using embodiments of the methods of the invention. Protease Family Entrez Gene enzyme Map Location ID Descriptive Name (or default name) Gene Name ID ID (cytogenetic or I genetic location) PGA3 A01.001 643834 3.4.23.1 1 I1q2.2 pepsinogen 3, group I (pepsinogen A) PGA@ A01.001 5219 - 1q13 pepsinogen A gene cluster PGC AO 1.003 5225 3.4.23.3 6p21.3-p21.1 progastricsin (pepsinogen C) BACEl AOI.004 23621 - I1q23.2-q23.3 beta-site APP-cleaving enzyme I CYMP AO1.006 1542 - Ip13.

3 chymosin pseudogene 3.4.23.1 REN AO1.007 5972 5 1q32 renin cathepsin D (lysosomal aspartyl CTSD AO1.009 1509 3.4.23.5 11p15.5 protease) CTSE A01.010 1510 3.4.23.5 1q31 cathepsin E BACE2 AO1.041 25825 - 21q22.3 beta-site APP-cleaving enzyme 2 NAPSA AO1.046 9476 - 19q]3.33 napsin A aspartic peptidase PGA5 AO1.071 5222 3.4.23.1 l 1l3 pepsinogen 5, group I (pepsinogen A) napsin B aspartic peptidase NAPSB AO.POI 256236 - 19ql3.33 pseudogene SASP A02.059 151516 - 2p l3.3 hypothetical protein FLJ25084 47 WO 2009/088408 PCT/US2008/012369 Protease Family Entrez Gene enzyme Map Location ID Descriptive Name (or default name) Gene Name ID ID (cytogenetic or genetic location) DDII A02.xxx AK093336 - DDI2 A02.xxx BN000122 - NRIP2 A02.xxx 83714 - 12pl3.33 nuclear receptor interacting protein 2 NRIP3 A02.xxx 56675 - I lpl5.3 nuclear receptor interacting protein 3 PSENI A22.001 5663 - 14924.3 presenilin I (Alzheimer disease 3) PSEN2 A22.002 5664 - 1q31-q42 presenilin 2 (Alzheimer disease 4) HM13 A22.003 81502 - 20ql 1.21 histocompatibility (minor) 13 PSH4 A22.004 56928 - 19p13.3 signal peptide peptidase-like 2B PSH1 A22.005 121665 - 12q24.31 signal peptide peptidase 3 IMP5 A22.006 162540 - 17q21.31 intramembrane protease 5 putative intramembrane cleaving PSH5 A22.007 84888 - 15q21.2 protease PIP Axl.xxx 5304 - 7934 prolactin-induced protein CTSL2 CO 1.009 1515 - 9922.2 cathepsin L2 CTSZ C01.013 1522 - 20q]3 cathepsin Z CTSLL2 CO1.014 1517 - loq cathepsin L-like 2 CTSLL3 C01.015 1518 - 10q22.3-q23.1 cathepsin L-like 3 CTSF CO1.018 8722 - 11ql3 cathepsin F 3.4.22.1 CTSL C01.032 1514 5 9q21-922 cathepsin L 3.4.22.2 CTSS C01.034 1520 7 1q21 cathepsin S CTSO C01.035 1519 - 4q31-q32 cathepsin O CTSK C01.036 1513 - 1q21 cathepsin K (pycnodysostosis) CTSW C01.037 1521 - 11q13.1 cathepsin W (lymphopain) 3.4.22.1 CTSH C01.040 1512 6 15q24-q25 cathepsin H CTSB C01.060 1508 3.4.22.1 8p22 cathepsin B CTSC C01.070 1075 - 1Jq14.1-q4.3 cathepsin C BLMH C01.084 642 - 17q1 1.2 bleomycin hydrolase TINAG C01.973 27283 - 6p11.2-pl2 tubulointerstitial nephritis antigen LCN7 C01.975 64129 - 1p35.2 lipocalin 7 CTSLLI COI.P02 1516 - log cathepsin L-like 1 3.4.22.1 CAPNI C02.001 823 7 1lq13 calpain 1,(mu/1) large subunit 3.4.22.1 CAPN2 C02.002 824 7 1q41-q42 calpain 2, (m/Il) large subunit 3.4.22.1 CAPN3 C02.004 825 7 15q15.l-921.1 calpain 3, (p94) CAPN9 C02.006 10753 - Iq42.1 I-q42.3 calpain 9 CAPN8 C02.007 AA043093 CAPN7 C02.008 23473 - 3p24 calpain 7 SOLH C02.010 6650 - 16p13.3 small optic lobes homolog 48 WO 2009/088408 PCT/US2008/012369 Protease Family Entrez Gene enzyme Map Location ID Descriptive Name (or default name) Gene Name ID ID (cytogenetic or ______genetic location) (Drosophila) CAPN5 C02.011 726 - 11914 calpain 5 CAPNII C02.013 11131 - 6p12 calpain I _ CAPN12 C02.017 147968 - 19q13.2 calpain 12 CAPNIO C02.018 11132 - 2q37.3 calpain 10 CAPN13 C02.020 92291 - 2p22-p2l calpain 13 CAPN14 C02.021 440854 - 2 p 23 .1-p 2 1 calpain 14 CAPN6 C02.971 827 - xq23 calpain 6 chromosome 6 open reading frame C6orfl03 C02.972 79747 - 6q24.3 103 3.4.19.1 ubiquitin carboxyl-terminal esterase UCHLI C12.001 7345 2 4pl14 Li (ubiquitin thiolesterase) ubiquitin carboxyl-terminal esterase UCHL3 C12.003 7347 3.2.1.15 13q22.2 L3 (ubiquitin thiolesterase) BRCA1 associated protein-i (ubiquitin carboxy-terminal BAPI C12.004 8314 - 3p21.31-p2l.2 hydrolase) ubiquitin carboxyl-terminal hydrolase UCHL5 C12.005 51377 - lq32 L5 LGMN C13.004 5641 - 14q32.1 legumain PIGK C13.005 10026 - Ip31.1 phosphatidylinositol glycan, class K LGMN2P C13.PO1 122199 - 13q21.31 legumain 2 pseudogene caspase 1, apoptosis-related cysteine protease (interleukin 1, beta, CASPI C 14.001 834 - 11q23 convertase) caspase 3, apoptosis-related cysteine CASP3 C 14.003 836 - 4q34 protease caspase 7, apoptosis-related cysteine CASP7 C 14.004 840 - 10q25 protease caspase 6, apoptosis-related cysteine CASP6 C 14.005 839 - 4q25 protease caspase 2, apoptosis-related cysteine protease (neural precursor cell expressed, developmentally down CASP2 C14.006 835 - 7q34-q35 regulated 2) caspase 4, apoptosis-related cysteine CASP4 C14.007 837 - 11q22.2-q22.3 protease caspase 5, apoptosis-related cysteine CASP5 C 14.008 838 - 1 lq22.2-q22.3 protease caspase 8, apoptosis-related cysteine CASP8 C14.009 841 - 2q33-q34 protease caspase 9, apoptosis-related cysteine CASP9 C14.010 842 - Ip36.3-p36.1 protease caspase 10, apoptosis-related cysteine CASPIO C14.011 843 - 2q33-q34 protease caspase 14, apoptosis-related cysteine CASPI4 C14.018 23581 - l9p1 3 .1 protease mucosa associated lymphoid tissue MALT] C14.026 10892 - 18q21 lymphoma translocation gene 1 49 WO 2009/088408 PCT/US2008/012369 Protease Family Entrez Gene enzyme Map Location ID Descriptive Name (or default name) Gene Name ID ID (cytogenetic or genetic location) CASP8 and FADD-like apoptosis CFLAR C14.971 8837 - 2933-934 regulator CASP14L C14.975np 197350 - 16p13.3 hypothetical protein LOC197350 CASP12P1 Cl4.POI 120329 - l q22.3 caspase 12 pseudogene l PGPEPI C15.010 54858 3.4.19.3 19p13.ll pyroglutamyl-peptidase I PGPEP2 C15.011 145814 - 15q26.3 hypothetical protein LOC145814 ubiquitin specific protease 5 USP5 C19.001 8078 - 12p13 (isopeptidase T) ubiquitin specific protease 6 (Tre-2 USP6 C19.009 9098 - 17g11 oncogene) ubiquitin specific protease 4 (proto USP4 C19.010 7375 - 3p21.3 oncogene) USP8 C19.011 9101 - 15q21.2 ubiquitin specific protease 8 ubiquitin specific protease 13 USP13 C19.012 8975 - 3q26.2-q26.3 (isopeptidase T-3) USP2 C19.013 9099 - 1 1q23.3 ubiquitin specific protease 2 USP1I C19.014 8237 - xp l1.23 ubiguitin specific protease 11 ubiquitin specific protease 14 (tRNA USP14 C19.015 9097 - 18pl1.32 guanine transglycosylase) ubiquitin specific protease 7 (herpes USP7 C19.016 7874 - 16p13.3 virus-associated) ubiquitin specific protease 9, X-linked USP9X C19.017 8239 - xp 11.4 (fat facets-like, Drosophila) USPIO C19.018 9100 - 16q24.1 ubiquitin specific protease 10 USPI C 19.019 7398 - Ip32.1-p31.3 ubiquitin specific protease 1 ubiquitin specific protease 12 USP12 C19.020 9959 - 5q33-q34 pseudogene I USP16 C19.021 10600 - 21q22.11 ubiquitin specific protease 16 USP15 C19.022 9958 - 12ql4 ubiquitin specific protease 15 USP17 C19.023 391627 - 4p15 ubiquitin specific peptidase 17 USP19 C19.024 10869 - 3p2l.31 ubiquitin specific protease 19 USP20 C19.025 10868 - 9q34.11 ubiquitin specific protease 20 USP3 C 19.026 9960 - 15q22.3 ubiquitin specific protease 3 ubiquitin specific protease 9, Y-linked USP9Y C 19.028 8287 - yql 1.2 (fat facets-like, Drosophila) USP18 C19.030 11274 - 22q] 1.21 ubiquitin specific protease 18 USP21 C19.034 27005 - 1q22 ubiquitin specific protease 21 USP22 C19.035 23326 - 17p] 1.2 ubiquitin specific protease 22 USP33 C19.037 23032 - 1p3 1.1 ubiquitin specific protease 33 USP29 C 19.040 57663 - 19q13.43 ubiquitin specific protease 29 USP25 C19.041 29761 - 21ql 1.2 ubiquitin specific protease 25 USP36 C 19.042 57602 - 17q25.3 ubiquitin specific protease 36 USP32 C 19.044 84669 - 17q23.2 ubiguitin specific protease 32 USP26 C 19.046 83844 3.1.2.15 xq26.2 ubiquitin specific protease 26 50 WO 2009/088408 PCT/US2008/012369 Protease Family Entrez Gene enzyme Map Location ID Descriptive Name (or default name) Gene Name ID ID (cytogenetic or _____ genetic location) USP24 C 19.047 23358 - lp32.3 ubiquitin specific protease 24 USP42 C19.048 84132 - 7p22.1 ubiquitin specific protease 42 USP46 C19.052 64854 - 4q12 ubiquitin specific protease 46 USP37 C 19.053 57695 - 2q35 ubiquitin specific protease 37 USP28 C19.054 57646 - 11q23 ubiquitin specific protease 28 USP47 C19.055 55031 - ll p15.3 ubiquitin specific protease 47 USP38 C19.056 84640 - 493 1.1 ubiquitin specific protease 38 USP44 C19.057 84101 - 12q22 ubiquitin specific protease 44 USP50 C19.058 373509 - 15q21.1 ubiquitin specific protease 50 USP50 C19.058np A1990110 - - USP35 C19.059 57558 - 1I14.1 ubiquitin specific protease 35 USP30 C 19.060 84749 - 12q24.11 ubiquitin specific protease 30 USP45 C19.064 85015 - 6q16.3 ubiquitin specific protease 45 USPS1 C 19.065 158880 - xpl 1.22 ubiquitin specific protease 51 USP51 C19.065 BF741256 - - USP34 C 19.067 9736 - 2p15 ubiquitin specific protease 34 USP48 C19.068 84196 - lp36.12 ubiquitin specific protease 48 USP40 C19.069 55230 - 2q37.1 ubiquitin specific protease 40 USP41 C19.070 150200 - 22ql 1.21 ubiquitin specific peptidase 41 USP31 C19.071 57478 - l6pI2.1 ubiquitin specific protease 31 USP49 C19.073 25862 - 6p21 ubiquitin specific protease 49 ubiquitin specific protease 27, X USP27X C19.075 373504 - xpI1 linked USP27 C19.075 AW851065 - - USP54 C 19.080 159195 - 10922.2 ubiquitin specific protease 54 USP53 C19.081 54532 - 4q26 ubiquitin specific protease 53 USP39 C19.972 10713 - 2pl 1.2 ubiquitin specific protease 39 USP43 C19.976 124739 - 17p]3.1 ubiquitin specific protease 43 USP52 C19.978 9924 - 12ql3.2-ql3.3 ubiquitin specific protease 52 ubiquitin specific protease 8 USP8P C19.980 394216 - 6p2I pseudogene ubiquitin associated domain UBADCI C19.M01 10422 - 9q34.3 containing I NEK2P C19.POI 326302 - 14q11.2 NEK2 pseudogene USP17L C19.xxx BNO0016 gamma-glutamyl hydrolase (conjugase, folylpolygammaglutamyl GGH C26.001 8836 3.4.19.9 8q12.3 hydrolase) GMPS C26.950 8833 6.3.5.2 3q24 guanine monphosphate synthetase phosphoribosyl pyrophosphate PPAT C44.001 5471 2.4.2.14 4q12 amidotransferase GFPTI C44.970 2673 2.6.1.16 2p13 glutamine-fructose-6-phosphate 51 WO 2009/088408 PCT/US2008/012369 Protease Family Entrez Gene enzyme Map Location ID Descriptive Name (or default name) Gene Name ID ID (cytogenetic or genetic location) transaminase I glutamine-fructose-6-phosphate GFPT2 C44.972 9945 - 5q34-q35 transaminase 2 ASNS C44.974 440 6.3.5.4 7q21.3 asparagine synthetase SHH C46.002 6469 - 7q36 sonic hedgehog homolog (Drosophila) Indian hedgehog homolog IHH C46.003 3549 - 2q33-q35 (Drosophila) desert hedgehog homolog DHH C46.004 50846 - 12ql2-q13.1 (Drosophila) SENPI C48.002 29843 - 12q 13.1 SUMOI/sentrin specific protease I SUMO1/sentrin/SMT3 specific SENP3 C48.003 26168 - 17pI3 protease 3 SENP6 C48.004 26054 - 6ql3-ql4.3 SUMOI/sentrin specific protease 6 SUMO1/sentrin/SMT3 specific SENP2 C48.007 59343 - 3q27.2 protease 2 SENP5 C48.008 205564 - 3q29 SUMOl/sentrin specific protease 5 SENP7 C48.009 57337 - 3q12 SUMOl/sentrin specific protease 7 SUMO/sentrin specific protease SENP8 C48.011 123228 - 15q23 family member 8 3.4.22.4 extra spindle poles like I (S. ESPLI C50.001 9700 9 12q cerevisiae) APG4 autophagy 4 homolog A (S. ATG4A C54.002 115201 - xq22.1-q22.3 cerevisiae) APG4 autophagy 4 homolog B (S. ATG4B C54.003 23192 - 2q37.3 cerevisiae) APG4 autophagy 4 homolog C (S. ATG4C C54.004 84938 - 1p31.3 cerevisiae) APG4 autophagy 4 homolog D (S. ATG4D C54.005 84971 - 19p13.2 cerevisiae) Parkinson disease (autosomal PARK7 C56.002 11315 - lp36.33-p36.12 recessive, early onset) 7 phosphoribosylformylglycinamidine PFAS C56.972 5198 6.3.5.3 17pl3.1 synthase (FGAR amidotransferase) ZA20DI C64.001 56957 - Iq21.2 zinc finger, A20 domain containing 1 chromosome 15 open reading frame C15orfl6 C64.002 161725 - 15ql3.3 16 tumor necrosis factor, alpha-induced TNFAIP3 C64.003 7128 - 6q23 protein 3 zinc finger, RAN-binding domain ZRANBI C64.004 54764 - 10q26.13 containing I OTU domain, ubiquitin aldehyde OTUBI C65.001 55611 - 1 ql3.1 bindingI OTU domain, ubiquitin aldehyde OTUB2 C65.002 78990 - 14q32.13 binding 2 cylindromatosis (turban tumor CYLD C67.001 1540 - 16q]2.1 syndrome) SCRN I C69.003 9805 - 7pl4.3-pl4.1 secernin I SCRN2 C69.004 90507 - 17q21.32 secernin 2 SCRN3 C69.005 79634 - 2q3 1.1 secernin 3 52 WO 2009/088408 PCT/US2008/012369 Protease Family Entrez Gene enzyme Map Location ID Descriptive Name (or default name) Gene Name ID ID (cytogenetic or I __genetic location) OTUD4 Cxl.xxx 54726 - 4q31.21 HIV-1 induced protein HIN-1 HSHINIL Cxl.xxx BNOOO160 - CXorf45 Cxl.xxx 79868 - xq23 chromosome X open reading frame 45 HSHIN3 Cxl.xxx 23252 - 1p36.13 KIAA0459 protein OTUDI Cxl.xxx 220213 - 10p12.31 OTU domain containing I OTUD5 Cxl.xxx 55593 - xpl1.23 hypothetical protein DKFZp761AO52 OTUD6A Cx1.xxx 139562 - xq13.1 HIN-6 protease HSHIN7 Cxl.xxx B1829009 - OTUD6B Cxl.xxx 51633 - 8q2l.3 CGI-77 protein TTC28 Cx2.xxxnp 23331 - 22q12.1 KIAA1043 protein alanyl (membrane) aminopeptidase (aminopeptidase N, aminopeptidase M, microsomal aminopeptidase, ANPEP M01.001 290 3.4.11.2 15q25-q26 CD13, p150) glutamyl aminopeptidase ENPEP MO1.003 2028 3.4.11.7 4q25 (aminopeptidase A) LTA4H MO1.004 4048 3.3.2.6 12q22 leukotriene A4 hydrolase thyrotropin-releasing hormone TRHDE M01.008 29953 3.4.19.6 12ql5-q21 degrading ectoenzyme NPEPPS M01.010 9520 - 17q21 aminopeptidase puromycin sensitive LNPEP M01.011 4012 3.4.11.3 Sq15 leucyl/cystinyl aminopeptidase arginyl aminopeptidase RNPEP M01.014 6051 3.4.11.6 1q32 (aminopeptidase B) type 1 tumor necrosis factor receptor ERAPI M01.018 51752 - 5q15 shedding aminopeptidase regulator arginyl aminopeptidase RNPEPLI M01.022 57140 - 2q37.3 (aminopeptidase B)-like 1 leukocyte-derived arginine ERAP2 M01.023 64167 - 16 aminopeptidase AQPEP M01.027 BG623101 - C9orf3 MO1.028 84909 - 9q22.32 chromosome 9 open reading frame 3 TAF2 RNA polymerase 11, TATA box binding protein (TBP)-associated TAF2 M01.972 6873 - 8q24.12 factor, 150kDa angiotensin I converting enzyme ACE2 M02.006 59272 3.4.15.1 xp22 (peptidyl-dipeptidase A) 2 3.4.24.1 THOPI M03.001 7064 5 19ql3.3 thimet oligopeptidase 1 3.4.24.1 neurolysin (metallopeptidase M3 NLN M03.002 57486 6 5q12.3 family) 3.4.24.5 MIPEP M03.006 4285 9 13q12 mitochondrial intermediate peptidase 3.4.24.3 leishmanolysin-like (metallopeptidase LMLN M08.003 89782 6 3q29 M8 family) matrix metalloproteinase I (interstitial MMPI M10.001 4312 3.4.24.7 11q22.3 collagenase) 3.4.24.3 matrix metalloproteinase 8 (neutrophil MMP8 M10.002 4317 4 1 1q22.3 collagenase) 53 WO 2009/088408 PCT/US2008/012369 Protease Family Entrez Gene enzyme Map Location ID Descriptive Name (or default name) Gene Name ID ID (cytogenetic or _ ._genetic location) matrix metalloproteinase 2 (gelatinase 3.4.24.2 A, 72kDa gelatinase, 72kDa type IV MMP2 M10.003 4313 4 16ql3-q21 collagenase) matrix metalloproteinase 9 (gelatinase 3.4.24.3 B, 92kDa gelatinase, 92kDa type IV MMP9 M10.004 4318 5 20q11.2-q13.1 collagenase) 3.4.24.1 matrix metalloproteinase 3 MMP3 M10.005 4314 7 1 1q22.3 (stromelysin 1, progelatinase) 3.4.24.2 matrix metalloproteinase 10 MMP1O M10.006 4319 2 11q22.3 (stromelysin 2) matrix metalloproteinase I I MMP11 M10.007 4320 - 22q11.23 (stromelysin 3) 3.4.24.2 matrix metalloproteinase 7 MMP7 M10.008 4316 3 11q21-q22 (matrilysin, uterine) matrix metalloproteinase 12 MMP12 M10.009 4321 - I 1q22.3 (macrophage elastase) matrix metalloproteinase 13 MMP13 M10.013 4322 - 11q22.3 (collagenase 3) matrix metalloproteinase 14 MMP14 M10.014 4323 - 14qll-ql2 (membrane-inserted) matrix metalloproteinase 15 MMP15 M10.015 4324 - 16ql3-q21 (membrane-inserted) matrix metalloproteinase 16 MMP16 M10.016 4325 - 8q21 (membrane-inserted) matrix metalloproteinase 17 MMPI7 M10.017 4326 - 12q24.3 (membrane-inserted) matrix metalloproteinase 20 MMP20 M10.019 9313 - 11q22.3 (enamelysin) MMP19 M10.021 4327 - 12q14 matrix metalloproteinase 19 MMP23B M10.022 8510 - 1p36.3 matrix metalloproteinase 23B matrix metal loprotei nase 24 MMP24 M10.023 10893 - 20q 1l.2 (membrane-inserted) MMP25 M10.024 64386 - 16p]3.3 matrix metalloproteinase 25 MMP21 M10.026 118856 - 10q26.2 matrix metalloproteinase 21 MMP27 M10.027 64066 - 1 lq24 matrix metalloproteinase 27 MMP26 M10.029 56547 - 1lp15 matrix metalloproteinase 26 MMP28 M10.030 79148 - 17q1-q21.1 matrix metalloproteinase 28 MMP23A MIO.037 8511 - 1p36.3 matrix metalloproteinase 23A MMPLI M10.973 4328 - 16p13.3 matrix metalloproteinase-like 1 3.4.24.1 meprin A, alpha (PABA peptide MEPlA M12.002 4224 8 6pl2-pll hydrolase) 3.4.24.1 MEPIB M12.004 4225 8 18ql2.2-q12.3 meprin A, beta 3.4.24.1 BMPI M12.005 649 9 8p 2 l bone morphogenetic protein 1 TLLI M12.016 7092 - 4q32-q33 tolloid-like I TLL2 M12.018 7093 - 10q23-q24 tolloid-like2 a disintegrin-like and metalloprotease (reprolysin type) with ADAMTS9 M12.021 56999 - 3p14.3-p14.2 thrombospondin type I motif, 9 54 WO 2009/088408 PCT/US2008/012369 Protease Family Entrez Gene enzyme Map Location ID Descriptive Name (or default name) Gene Name ID ID (cytogenetic or genetic location) a disintegrin-like and metalloprotease ADAMTSI (reprolysin type) with 4 M12.024 140766 - 1092 thrombospondin type I motif, 14 a disintegrin-like and metalloprotease ADAMTSI (reprolysin type) with 5 M12.025 170689 - 11q25 thrombospondin type I motif, 15 a disintegrin-like and metalloprotease ADAMTSI (reprolysin type) with 6 M12.026 170690 - 5p15 thrombospondin type I motif, 16 a disintegrin-like and metalloprotease ADAMTSI (reprolysin type) with 7 M12.027 170691 - 15q24 thrombospondin type I motif, 17 a disintegrin-like and metalloprotease ADAMTSI (reprolysin type) with 8 M12.028 170692 - 16q23 thrombospondin type I motif, 18 a disintegrin-like and metalloprotease ADAMTSI (reprolysin type) with 9 M12.029 171019 - 5q31 thrombospondin type I motif, 19 a disintegrin and metalloproteinase ADAMI M12.201 8759 - 12q24.12-q24.13 domain I (fertilin alpha) pseudogene a disintegrin and metal loproteinase ADAM8 M12.208 101 - 10q26.3 domain 8 a disintegrin and metalloproteinase ADAM9 M12.209 8754 - 8pl 1.23 domain 9 (meltrin gamma) a disintegrin and metalloproteinase ADAMIO M12.210 102 - 15q22 domain 10 a disintegrin and metalloproteinase ADAM12 M 12.212 8038 - 10q26.3 domain 12 (meltrin alpha) a disintegrin and metalloproteinase ADAM19 M12.214 8728 - 5q32-q33 domain 19 (meltrin beta) a disintegrin and metalloproteinase ADAMI5 M12.215 8751 - 1q21.3 domain 15 (metargidin) a disintegrin and metalloproteinase domain 17 (tumor necrosis factor, ADAM17 M12.217 6868 - 2p25 alpha, converting enzyme) a disintegrin and metalloproteinase ADAM20 M12.218 8748 - 14q24.1 domain 20 ADAMDEC 1 M12.219 27299 - 8p21.2 ADAM-like, decysin 1 a disintegrin-like and metalloprotease (reprolysin type) with ADAMTS3 M12.220 9508 - 4q13.3 thrombospondin type I motif, 3 a disintegrin-like and metalloprotease (reprolysin type) with ADAMTS4 M12.221 9507 - 1q21-q23 thrombospondin type I motif, 4 a disintegrin-like and metalloprotease (reprolysin type) with ADAMTS1 M 12.222 9510 - 21q21.2 thrombospondin type I motif, I a disintegrin and metalloproteinase ADAM28 M12.224 10863 - 8p 2 1.

2 domain 28 a disintegrin-like and metalloprotease (reprolysin type) with thrombospondin type I motif, 5 ADAMTS5 M12.225 11096 - 21q21.3 (aggrecanase-2) 55 WO 2009/088408 PCT/US2008/012369 Protease Family Entrez Gene enzyme Map Location ID Descriptive Name (or default name) Gene Name ID ID (cytogenetic or genetic location) a disintegrin-like and metalloprotease (reprolysin type) with ADAMTS8 M12.226 11095 - 1lq25 thrombospondin type I motif, 8 a disintegrin-like and metalloprotease (reprolysin type) with ADAMTS6 M 12.230 11174 - 5q]2 thrombospondin type I motif, 6 a disintegrin-like and metalloprotease (reprolysin type) with ADAMTS7 M12.231 11173 - 15924.2 thrombospondin type I motif, 7 a disintegrin and metalloproteinase ADAM30 M12.232 11085 - lp13-pl domain 30 a disintegrin and metalloproteinase ADAM21 M12.234 8747 - 14q24.1 domain 21 a disintegrin-like and metalloprotease ADAMTSI (reprolysin type) with 0 M12.235 81794 - l9p13.3-p13.2 thrombospondin type 1 motif, 10 a disintegrin-like and metalloprotease ADAMTSI (reprolysin type) with 2 M12.237 81792 - 5q35 thrombospondin type I motif, 12 a disintegrin-like and metalloprotease ADAMTS1 (reprolysin type) with 3 M12.241 11093 - 9q34 thrombospondin type I motif, 13 a disintegrin and metalloproteinase ADAM33 M12.244 80332 - 20p13 domain 33 3.4.24.2 astacin-like metalloendopeptidase ASTL M12.245 431705 1 2ql1.1 (M12 family) HAMET M12.245 AJ537600 - - a disintegrin-like and metalloprotease ADAMTS2 (reprolysin type) with 0 M12.246 80070 - 12ql2 thrombospondin type 1 motif, 20 a disintegrin-like and metalloprotease (reprolysin type) with ADAMTS2 M12.301 9509 - 5qter thrombospondin type 1 motif, 2 a disintegrin and metalloproteinase ADAM2 M12.950 2515 - 8p11.2 domain 2 (fertilin beta) a disintegrin and metalloproteinase ADAM7 M12.956 8756 - 8p2l.2 domain 7 a disintegrin and metalloproteinase ADAM18 M12.957 8749 - 8pl 1.22 domain 18 a disintegrin and metalloproteinase ADAM32 M12.960 203102 - 8p]l1.23 domain 32 a disintegrin and metalloproteinase ADAM3A M 12.974 1587 - 8p21-p12 domain 3a (cyritestin 1) a disintegrin and metalloproteinase ADAM3B M12.975 1596 - 16q12.1 domain 3b (cyritestin 2) a disintegrin and metalloproteinase ADAM I M12.976 4185 - 17q21.3 domain I I a disintegrin and metal loproteinase ADAM22 M12.978 53616 - 7q21 domain 22 a disintegrin and metalloproteinase ADAM23 M 12.979 8745 - 2q33 domain 23 a disintegrin and metalloproteinase ADAM29 M12.981 11086 - 4q34 domain 29 56 WO 2009/088408 PCT/US2008/012369 Protease Family Entrez Gene enzyme Map Location ID Descriptive Name (or default name) Gene Name ID ID (cytogenetic or _ _ _genetic location) membrane metallo-endopeptidase 3.4.24.1 (neutral endopeptidase, MME M13.001 4311 1 3q25.1-q25.2 enkephalinase, CALLA, CDIO) ECEl M13.002 1889 - lp36.1 endothelin converting enzyme I ECE2 M13.003 9718 - 3q28-q29 endothelin converting enzyme 2 ECELI M13.007 9427 - 2q36-q37 endothelin converting enzyme-like 1 MELLI M13.008 79258 - 1p36 mel transforming oncogene-like 1 KEL M13.090 3792 - 7q33 Kell blood group phosphate regulating endopeptidase homolog, X-linked (hypophosphatemia, vitamin D PHEX M13.091 5251 - xp22.2-p22.1 resistant rickets) CPAI M14.001 1357 3.4.17.1 7q32 carboxypeptidase A l (pancreatic) 3.4.17.1 CPA2 M14.002 1358 5 7932 carboxypeptidase A2 (pancreatic) CPBI M14.003 1360 3.4.17.2 3q24 carboxypeptidase BI (tissue) carboxypeptidase N, polypeptide 1, CPNI M14.004 1369 - 10q24.2 50kD 3.4.17.1 CPE M14.005 1363 0 4q32.3 carboxypeptidase E 3.4.17.1 CPM M14.006 1368 2 12q 14.3 carboxypeptidase M carboxypeptidase B2 (plasma, CPB2 M 14.009 1361 - 13q]4.11 carboxypeptidase U) CPA3 M14.010 1359 3.4.2.1 3q21-q25 carboxypeptidase A3 (mast cell) CPZ M14.012 8532 - 4p16.1 carboxypeptidase Z CPA4 M14.017 51200 - 7q32 carboxypeptidase A4 CPA6 M14.018 57094 - 8q13.2 carboxypeptidase A6 CPA5 M14.020 93979 - 7q32 carboxypeptidase AS CPO M14.021 130749 - 2q33.3 carboxypeptidase 0 AGBL3 M14.026 340351 - 7q33 hypothetical protein LOC340351 AGBL4 M14.027 84871 - lp3 3 hypothetical protein FLJ14442 AGTPBPl M14.028 23287 - 9q21.33 ATP/GTP binding protein 1 AGBL2 M14.029 79841 - 1 IpI 1.2 hypothetical protein FLJ23598 AEBPI M14.951 165 - 7p13 AE binding protein 1 CPXM M14.952 56265 - 20p13-p12.3 carboxypeptidase X (M14 family) carboxypeptidase X (M14 family), CPXM2 M14.954 119587 - 10q26.13 member 2 IDE M16.002 3416 - 10q23-q25 insulin-degrading enzyme peptidase (mitochondrial processing) PMPCB M16.003 9512 - 7q22-q32 beta nardilysin (N-arginine dibasic NRDI M16.005 4898 - lp32.2-p32.1 convertase) PITRM I M16.009 10531 - 10p15.2 pitrilysin metalloproteinase I peptidase (mitochondrial processing) PMPCA M16.971 23203 - 9934.3 alpha 57 WO 2009/088408 PCT/US2008/012369 Protease Family Entrez Gene enzyme Map Location ID Descriptive Name (or default name) Gene Name ID ID (cytogenetic or _I__ genetic location) ubiquinol-cytochrome c reductase UQCRCI M16.973 7384 1.10.2.2 3p21.3 core protein I ubiquinol-cytochrome c reductase UQCRC2 M16.974 7385 1.10.2.2 16p12 core protein II AMPP M16.976np 133083 - 4q22.2-q22.3 similar to PMPCA protein LAP3 M17.001 51056 3.4.11.1 4p15.32 leucine aminopeptidase 3 NPEPL1 M17.006 79716 - 20q13.32 aminopeptidase-like 1 DNPEP M18.002 23549 - 2q35 aspartyl aminopeptidase 3.4.13.1 DPEP1 M19.001 1800 1 16q24.3 dipeptidase I (renal) DPEP2 M19.002 64174 - 16q22.1 dipeptidase 2 DPEP3 M19.004 64180 - 16q22.1 dipeptidase 3 3.4.13.1 CNDP dipeptidase 2 CNDP2 M20.005 55748 8 18q22.3 (metallopeptidase M20 family) carnosine dipeptidase I CNDPI M20.006 84735 - 18q22.3 (metallopeptidase M20 family) ACY1L2 M20.971 135293 - 6q15 aminoacylase 1-like 2 ACY] M20.973 95 3.5.1.14 3p21.1 aminoacylase 1 3.4.24.5 OSGEP M22.003 55644 7 14q 11.2 0-sialoglycoprotein endopeptidase 0-sialoglycoprotein endopeptidase OSGEPLI M22.004 64172 - 2q32.2 like I METAPI M24.001 23173 - 4q23 methionyl aminopeptidase 1 METAP2 M24.002 10988 - 12q22 methionyl aminopeptidase 2 X-prolyl aminopeptidase (aminopeptidase P) 2, membrane XPNPEP2 M24.005 7512 3.4.11.9 xq25 bound PEPD M24.007 5184 3.4.13.9 19q12-g13.2 peptidase D X-prolyl aminopeptidase XPNPEPI M24.009 7511 3.4.11.9 10q25.3 (aminopeptidase P) 1, soluble XPNPEP3 M24.026 63929 - 22q13.31-q13.33 hypothetical protein LOC63929 MAPID M24.028 254042 - 2q31.1 methionine aminopeptidase ID PA2G4 M24.973 5036 - 12qI3 proliferation-associated 2G4, 38kDa suppressor of Ty 16 homolog (S. SUPT16H M24.974 11198 - 14q11.2 cerevisiae) folate hydrolase (prostate-specific FOLHI M28.010 2346 - I1pl 1.2 membrane antigen) 1 NAALADL N-acetylated alpha-linked acidic I M28.011 10004 - 1lql2 dipeptidase-like 1 N-acetylated alpha-linked acidic NAALAD2 M28.012 10003 - I 1q14.3-q21 dipeptidase 2 PGCP M28.014 10404 - 8q22.2 plasma glutamate carboxypeptidase glutaminyl-peptide cyclotransferase QPCTL M28.016 54814 - 19ql3.32 like KIAA1815 M28.018 79956 - 9p24 KIAA1815 TFRC M28.972 7037 - 3q29 transferrin receptor (p90, CD71) TFR2 M28.973 7036 - 7q22 transferrin receptor 2 58 WO 2009/088408 PCT/US2008/012369 Protease Family Entrez Gene enzyme Map Location ID Descriptive Name (or default name) Gene Name ID ID (cytogenetic or genetic location) glutaminyl-peptide cyclotransferase QPCT M28.974 25797 2.3.2.5 2p22.2 (glutaminyl cyclase) NAALADL N-acetylated alpha-linked acidic 2 M28.975 254827 - 3926.31 dipeptidase 2 NCLN M28.978 56926 - 19p 13.3 nicalin homolog (zebrafish) carbamoyl-phosphate synthetase 2, 2.1.3.2, aspartate transcarbamylase, and CAD M38.972 790 3.5.2.- 2p22-p21 dihydroorotase DPYS M38.973 1807 - 8q22 dihydropyrimidinase CRMP1 M38.974 1400 - 4p16.l-p15 collapsin response mediator protein 1 DPYSL2 M38.975 1808 - 8p 22 -p 2 l dihydropyrimidinase-like 2 DPYSL3 M38.976 1809 - 5q32 dihydropyrimidinase-like 3 DPYSL4 M38.977 10570 - 10926 dihydropyrimidinase-like 4 DPYSL5 M38.978 56896 - 2p23.3 dihydropyrimidinase-like 5 GDA M38.981 9615 - 9q21.11-21.33 guanine deaminase YME1LI M41.004 10730 - lOp14 YMEI-like I (S. cerevisiae) spastic paraplegia 7, paraplegin (pure SPG7 M41.006 6687 - 16q24.3 and complicated autosomal recessive) AFG3 ATPase family gene 3-like 2 AFG3L2 M41.007 10939 - 18p]l (yeast) AFG3 ATPase family gene 3-like 1 AFG3LI M41.010 172 - 16q24.3 (yeast) pregnancy-associated plasma protein PAPPA M43.004 5069 - 9933.2 A, pappalysin 1 PAPPA2 M43.005 60676 - Iq23-q25 pappalysin 2 procollagen (type III) N CHMP1A M47.001 5119 - 16q24.3 endopeptidase zinc metallopeptidase (STE24 ZMPSTE24 M48.003 10269 - Ip34 homolog, yeast) OMAI homolog, zinc OMA1 M48.017 115209 - 1p32.2-p32.1 metallopeptidase (S. cerevisiae) DPP3 M49.001 10072 3.4.14.4 11q l2-q13.1 dipeptidylpeptidase 3 membrane-bound transcription factor MBTPS2 M50.001 51360 - xp22.1-p22.2 protease, site 2 proteasome (prosome, macropain) PSMD14 M67.001 10213 - 2q24.2 26S subunit, non-ATPase, 14 COP9 constitutive photomorphogenic COPS5 M67.002 10987 - 8q13.2 homolog subunit 5 (Arabidopsis) associated molecule with the SH3 domain of STAM (AMSH) like STAMBPLI M67.003 57559 - 10q23.31 protein CXorf53 M67.004 79184 - xq28 chromosome X open reading frame 53 MYSMI M67.005 114803 - lp3 2 .1 KIAA1915 protein STAMBP M67.006 10617 - 2p13.1 STAM binding protein eukaryotic translation initiation factor EIF3S3 M67.971 8667 - 8q24.1 1 3, subunit 3 gamma, 40kDa COP9 constitutive photomorphogenic COPS6 M67.972 10980 - 7q22.1 homolog subunit 6 (Arabidopsis) 59 WO 2009/088408 PCT/US2008/012369 Protease Family Entrez Gene enzyme Map Location ID Descriptive Name (or default name) Gene Name ID ID (cytogenetic or .. __._genetic location) proteasome (prosome, macropain) 26S subunit, non-ATPase, 7 (Mov34 PSMD7 M67.973 5713 - 16q23-q24 homolog) eukaryotic translation initiation factor EIF3F M67.974 8665 - I Ip15.4 3, subunit 5 epsilon, 47kDa EIF3FP M67.975 83880 - 13p13 1FP38 MPND M67.xxx 84954 - 19p13.3 hypothetical protein FLJ14981 PRP8 pre-mRNA processing factor 8 PRPF8 M67.xxxnp 10594 - 17p]3.3 homolog (yeast) aspartoacylase (aminoacylase 2, ASPA Mx2.xxxnp 443 3.5.1.15 l7pter-p13 Canavan disease) ACY3 Mx2.xxxnp 91703 - Ilql3.2 aspartoacylase (aminocyclase) 3 angiotensin I converting enzyme ACE XM02-001 1636 3.4.15.1 17q23 (peptidyl-dipeptidase A) I CPD XM14-001 1362 - 17pl1.1-ql1.2 carboxypeptidase D granzyme B (granzyme 2, cytotoxic T-lymphocyte-associated serine GZMB SO1.010 3002 - 14q11.2 esterase 1) PRSS21 S01.011 10942 - 16p]3.3 protease, serine, 21 (testisin) TPSAB1 SO1.015 7177 - 16p13.3 tryptase alpha/beta I TPSB2 SO1.015 64499 - 16p13.3 tryptase beta 2 KLK5 S01.017 25818 - 19ql3.3-ql3.4 kallikrein 5 CORIN SO1.019 10699 - 4p13-p12 corn, serine protease KLK12 S01.020 43849 - 19q13.3-q13.4 kallikrein 12 TMPRSS11 E S01.021 28983 - 4q13.2 DESCI protein TPSG1 SO1.028 25823 - 16p13.3 tryptase gamma I KLK14 SO1.029 43847 - 19ql3.3-q13.4 kallikrein 14 HABP2 SO1.033 3026 - 10q25.3 hyaluronan binding protein 2 TMPRSS4 SO1.034 56649 - 11 q23.3 transmembrane protease, serine 4 TMPRSSI 1 D S01.047 9407 - 4q13.2 airway trypsin-like protease TPSD1 S1.054 23430 - 16p13.3 tryptase delta 1 TMPRSS7 S01.072 344805 - 3ql3.2 transmembrane serine protease 7 PRSS27 SO1.074 83886 - 16p13.3 pancreasin PRSS33 SO1.075 260429 - 16p1 3

.

3 protease, serine, 33 TESSP1 S01.076 BN000124 - - TMPRSS3 S01.079 64699 - 21q22.3 transmembrane protease, serine 3 KLK15 S01.081 55554 - 19q 13.41 kallikrein 15 TMPRSS13 S01.087 84000 - I 1q23 mosaic serine protease PRSSI S01.127 5644 3.4.21.4 7q34 protease, serine, I (trypsin 1) 3.4.21.3 ELA2 SO1.131 1991 7 19p13.3 elastase 2, neutrophil mannan-binding lectin serine protease MASPI SO1.132 5648 - 3q27-q28 1 (C4/C2 activating component of Ra 60 WO 2009/088408 PCT/US2008/012369 Protease Family Entrez Gene enzyme Map Location ID Descriptive Name (or default name) Gene Name ID ID (cytogenetic or ______genetic location) reactive factor) CTSG S01.133 1511 - 14q11.2 cathepsin G proteinase 3 (serine proteinase, neutrophil, Wegener granulomatosis PRTN3 S01.134 5657 - 19pI3.3 autoantigen) granzyme A (granzyme 1, cytotoxic T-lymphocyte-associated serine GZMA S01.135 3001 - 5q11-912 esterase 3) GZMM S01.139 3004 - 19 p]3.3 granzyme M (lymphocyte met-ase 1) 3.4.21.3 CMAI SO1.140 1215 9 14q11.2 chymase 1, mast cell granzyme K (serine protease, GZMK S01.146 3003 - 5ql1-q12 granzyme 3; tryptase II) granzyme H (cathepsin G-like 2, GZMH S01.147 2999 - 14q 11.2 protein h-CCPX) CTRB1 SO1.152 1504 3.4.21.1 16q23-q24.1 chymotrypsinogen B1 3.4.21.3 ELAI SO1.153 1990 6 12ql3 elastase 1, pancreatic ELA3A S01.154 10136 - lp36.12 elastase 3A, pancreatic (protease E) ELA2A SO1.155 63036 - Ip36.21 elastase 2A PRSS7 SO1.156 5651 - 21q21.1 protease, serine, 7 (enterokinase) CTRC S01.157 11330 - Ip36.21 chymotrypsin C (caldecrin) PRSS8 SO1.159 5652 - 16p 11.2 protease, serine, 8 (prostasin) 3.4.21.3 KLKI SO1.160 3816 5 19ql3.3 kallikrein 1, renal/pancreas/salivary 3.4.21.3 KLK2 S01.161 3817 5 19q13.41 kallikrein 2, prostatic KLK3 S01.162 354 - 19q13.41 kallikrein 3, (prostate specific antigen) PRSS3 SO1.174 5646 3.4.21.4 9p11.2 protease, serine, 3 (mesotrypsin) complement component 1, r CIRL SO1.189 51279 - 12p13.31 subcomponent-like D component of complement DF SO1.191 1675 - 19p13.3 (adipsin) 3.4.21.4 complement component 1, r CIR SO1.192 715 1 12p13 subcomponent 3.4.21.4 complement component 1, s CIS S01.193 716 2 12pl3 subcomponent C2 SO1.194 717 - 6p21.3 complement component 2 3.4.21.4 BF S01.196 629 7 6p21.3 B-factor, properdin 3.4.21.4 IF S01.199 3426 5 4q25 I factor (complement) ELA3B SO1.205 23436 - Ip36.1 2 elastase 3B, pancreatic ELA2B SO1.206 51032 - 1p36.

2 1 elastase 2B 3.4.21.3 coagulation factor X11 (Hageman F12 SO1.211 2161 8 5q33-qter factor) kallikrein B, plasma (Fletcher factor) KLKBI SO1.212 3818 - 4q34-q35 1 61 WO 2009/088408 PCT/US2008/012369 Protease Family Entrez Gene enzyme Map Location ID Descriptive Name (or default name) Gene Name ID ID (cytogenetic or I ,genetic location) 3.4.21.2 coagulation factor Xl (plasma Fl1 S01.213 2160 7 4q35 thromboplastin antecedent) coagulation factor IX (plasma 3.4.21.2 thromboplastic component, Christmas F9 S01.214 2158 2 xq27.1-q27.2 disease, hemophilia B) coagulation factor VII (serum F7 S01.215 2155 - 13q34 prothrombin conversion accelerator) F1O S01.216 2159 3.4.21.6 13q34 coagulation factor X F2 S01.217 2147 3.4.21.5 lp1 I -q 12 coagulation factor II (thrombin) protein C (inactivator of coagulation PROC S01.218 5624 - 2ql3-q4 factors Va and VIla) 3.4.21.1 ACR S01.223 49 0 22ql3.33 acrosin hepsin (transmembrane protease, HPN S01.224 3249 - 19q11-q13.2 serine 1) HGFAC S01.228 3083 3.4.21.- 4pl6 HGF activator mannan-binding lectin serine protease MASP2 S01.229 10747 - lp36.3-p36.

2 2 3.4.21.3 PLAU SO1.231 5328 1 10q24 plasminogen activator, urokinase PLAT S01.232 5327 - 8 p]2 plasminogen activator, tissue PLG S01.233 5340 - 6q26 plasminogen KLK6 S01.236 5653 - 19q13.3 kallikrein 6 (neurosin, zyme) protease, serine, 12 (neurotrypsin, PRSS12 SO1.237 8492 - 4q28.1 motopsin) KLK8 S01.244 11202 - 19ql3.3-q13.4 kallikrein 8 (neuropsin/ovasin) KLK1O S01.246 5655 - 19ql3.3-q13.4 kallikrein 10 TMPRSS2 S01.247 7113 - 21q22.3 transmembrane protease, serine 2 kallikrein 4 (prostase, enamel matrix, KLK4 SO1.251 9622 - 19q13.41 prostate) PRSS22 S01.252 64063 - 16p13.3 protease, serine, 22 CTRL S01.256 1506 - 16q22.1 chymotrypsin-like KLKI1 S01.257 11012 - 19ql3.3-gl3.4 kallikrein I1 PRSS2 S01.258 5645 - 7q34 protease, serine, 2 (trypsin 2) PRSSI1 S01.277 5654 - 10q26.3 protease, serine, 11 (IGF binding) PRSS25 S01.278 27429 - 2p]2 protease, serine, 25 HTRA3 SO1.284 94031 - 4 p16.1 HtrA serine peptidase 3 HTRA4 SO1.285 203100 - 8p 11.23 HtrA serine peptidase 4 TYSNDI SO1.286 219743 - 10q22.1 trypsin domain containing 1 TMPRSS12 SO1.291 283471 - 12q13.12 hypothetical protein MGC57341 TMPRSSII epidermal type II transmembrane A SO1.292 339967 - 4q13.2 serine protease HATLJ SO1.292 BN000133 - - TMPRSS8 SO1.298 AJ488946 - - kallikrein 7 (chymotryptic, stratum KLK7 SO1.300 5650 - 19q13.41 corneum) 62 WO 2009/088408 PCT/US2008/012369 Protease Family Entrez Gene enzyme Map Location ID Descriptive Name (or default name) Gene Name ID ID (cytogenetic or .I_._genetic location) suppression of tumorigenicity 14 ST14 SO1.302 6768 - 1 1q24-q25 (colon carcinoma, matriptase, epithin) KLK13 SO1.306 26085 - 19ql3.3-q13.4 kallikrein 13 KLK9 SO1.307 23579 - TMPRSS6 SO1.308 164656 - 22ql2.3-q13.1 transmembrane protease, serine 6 PRSS23 SO1.309 11098 - 11914.1 protease, serine, 23 transmembrane protease, serine 5 TMPRSS5 SO1.313 80975 - 19 (spinesin) TESSP2 SO1.317 AJ544583 - MPN2 SO1.318 BNOO0131 - PRSSL1 SO1.319 400668 - 19p13.3 protease, serine-like 1 OVCH2 SO1.320 341277 - 11p15.4 oviductin protease OVTN SO1.320 BNO00130 - TMPRSS11 F SO1.321 389208 - 4q13.2 FLJ16046 protein OVCHI SO1.322 341350 - l2p11.22 ovochymase I OVCH SO1.322 BN000128 - TMPRSS9 SO1.357 360200 - 19pl3.3 transmembrane serine protease 9 TMPRSSII hypothetical protein B SO1.365 132724 - 4q13.2 DKFZp686L1818 PRSS36 SO1.414 146547 - 16p11.2 polyserase-2 KLKBL2 SO1.415 203074 - 8p23.1 tryptophan/serine protease TESSP5 SO1.968np BN000137 - azurocidin 1 (cationic antimicrobial AZUI SO1.971 566 - 19pl3.3 protein 37) HP SO1.972 3240 - 16q22.1 haptoglobin HPR SO1.974 3250 - 16922.1 haptoglobin-related protein macrophage stimulating I (hepatocyte MST1 SO1.975 4485 - 3p21 growth factor-like) hepatocyte growth factor (hepapoietin HGF SO1.976 3082 - 7q2 1.1 A; scatter factor) protein Z, vitamin K-dependent PROZ SO1.979 8858 - 13q34 plasma glycoprotein TRYX2 SO1.989np 136242 - 7q34 similar to RIKEN cDNA 1700016G05 KLKBL4 SO1.992np 221191 - 16q21 hypothetical protein FLJ25339 TSP50 SO1.993np 29122 - 3p1 4 -p1 2 testes-specific protease 50 PRSS35 SO1.994 167681 - 6ql4.2 protease, serine, 35 regeneration associated muscle PROCL SO1.998np 25891 - llpl3 protease LPA SO1.999 4018 - 6q26-q27 lipoprotein, Lp(a) KLKPI SO .P08 606293 - 19q13.41 kallikrein pseudogene I vitamin K epoxide reductase complex, VKORCI SO I.xxx 79001 - 16p11.2 subunit I ESSPL SOl.xxx BN000134 - - 63 WO 2009/088408 PCT/US2008/012369 Protease Family Entrez Gene enzyme Map Location ID Descriptive Name (or default name) Gene Name ID ID (cytogenetic or genetic location) PRSS7L SOI.xxx BQ638967 - TMPRSS7 SOI.xxx BN000125 - proprotein convertase subtilisin/kexin PCSK9 S08.039 255738 - 1p32.3 type 9 membrane-bound transcription factor MBTPSI S08.063 8720 - 16q24 protease, site 1 furin (paired basic amino acid FURIN S08.071 5045 - 15q26.1 cleaving enzyme) proprotein convertase subtilisin/kexin PCSK1 S08.072 5122 - 5q15-921 type 1 proprotein convertase subtilisin/kexin PCSK2 S08.073 5126 - 2 0p11.

2 type2 proprotein convertase subtilisin/kexin PCSK4 S08.074 54760 - 19p13.3 type 4 proprotein convertase subtilisin/kexin PCSK6 S08.075 5046 - 15q26 type 6 proprotein convertase subtilisin/kexin PCSK5 S08.076 5125 - 9q21.3 type 5 proprotein convertase subtilisin/kexin PCSK7 S08.077 9159 - I Iq23-q24 type 7 3.4.14.1 TPP2 S08.090 7174 0 13q32-q33 tripeptidyl peptidase II 3.4.21.2 PREP S09.001 5550 6 6q22 prolyl endopeptidase dipeptidylpeptidase 4 (CD26, adenosine deaminase complexing DPP4 S09.003 1803 3.4.14.5 2q24.3 protein 2) APEH S09.004 327 3.4.19.1 3p21.31 N-acylaminoacyl-peptide hydrolase FAP S09.007 2191 - 2q23 fibroblast activation protein, alpha PREPL S09.015 9581 - 2p22.1 putative prolyl oligopeptidase DPP8 S09.018 54878 - 15q22 dipeptidylpeptidase 8 DPP9 S09.019 91039 - 19p13.3 dipeptidylpeptidase 9 C13orf6 S09.051 84945 - 13q33.3 chromosome 13 open reading frame 6 chromosome 19 open reading frame C19orf27 S09.052 81926 - 19p13.3 27 hypothetical protein from FAM108CI S09.053 58489 - 15q25.1 EUROIMAGE 588495 chromosome 20 open reading frame C20orf22 S09.054 26090 - 20pll.21 22 C9orf77 S09.055 51104 - 9q21.13 chromosome 9 open reading frame 77 chromosome 14 open reading frame C14orf29 S09.061 145447 - 14q22.1 29 ABHDIO S09.062 55347 - 3q13.2 abhydrolase domain containing 10 BAT5 S09.065 7920 - 6p 2 l.3 HLA-B associated transcript 5 DPP6 S09.973 1804 - 7q36.2 dipeptidylpeptidase 6 DPPIO S09.974 57628 - 2q14.1 dipeptidylpeptidase 10 chromosome 20 open reading frame C20orfl35 S09.976 140701 - 20q13.33 135 AFMID S09.977 125061 3.5.1.9 17q25.3 aryiformamidase 64 WO 2009/088408 PCT/US2008/012369 Protease Family Entrez Gene enzyme Map Location ID Descriptive Name (or default name) Gene Name ID ID (cytogenetic or ; genetic location) __ TG S09.978 7038 - 8q24.2-q24.3 thyroglobulin ACHE S09.979 43 3.1.1.7 7q22 acetyicholinesterase (YT blood group) BCH E S09.980 590 3.1.1.8 3q26.1-q26.2 butyrylcholinesterase carboxylesterase 1 (monocyte/macrophage serine CESI S09.982 1066 3.1.1.1 16ql3-q22.1 esterase 1) CES3 S09.983 23491 - 16 carboxylesterase 3 (brain) CES2 S09.984 8824 - 16q22.1 carboxylesterase 2 (intestine, liver) 3.1.1.3, carboxyl ester lipase (bile salt CEL S09.985 1056 3.1.1.13 9q34.3 stimulated lipase) CES4 S09.986 51716 - 16q12.2 carboxylesterase 4-like NLGN3 S09.987 54413 - xql3.1 neuroligin 3 NLGN4X S09.988 57502 - xp22.32-p22.31 neuroligin 4, X-linked NLGN4Y S09.989 22829 - yql 1.221 neuroligin 4, Y-linked esterase D/formylglutathione ESD S09.990 2098 3.1.1.1 13q14.1-q14.2 hydrolase AADAC S09.991 13 - 3921.3-q25.2 arylacetamide deacetylase (esterase) AADACLI S09.992 57552 - 3q26.31 KIAA 1363 protein LIPE S09.993 3991 3.1.1.- 19q13.2 lipase, hormone-sensitive NLGN1 S09.994 22871 - 3q26.31 neuroligin I NLGN2 S09.995 57555 - 17p13.1 neuroligin 2 protective protein for beta PPGB S10.002 5476 - 20q13.1 galactosidase (galactosialidosis) CPVL S10.003 54504 - 7p15-p14 carboxypeptidase, vitellogenic-like SCPEP1 S10.013 59342 - 17q23.2 serine carboxypeptidase 1 LACTB S12.004 114294 - 15q22.1 lactamase, beta CIpP caseinolytic protease, ATP dependent, proteolytic subunit CLPP S14.003 8192 - 1 9 p1 3 .3 homolog (E. coli) PRSS15 S16.002 9361 - 19p13.2 protease, serine, 15 LONP2 S16.006 83752 - 16q12.1 peroxisomal Ion protease SECIILI S26.009 23478 - 15925.3 SECIl-like 1(S. cerevisiae) SECI 1L3 S26.010 90701 - 18q21.32 SEC 1]-like 3 (S. cerevisiae) IMP2 inner mitochondrial membrane IMMP2L S26.012 83943 - 7q31 protease-like (S. cerevisiae) IMMPIL S26.013. 196294 - IIpI3 hypothetical protein FLJ25059 FREMI S26.xxx 158326 - 9p22.3 FRASI related extracellular matrix I prolylcarboxypeptidase PRCP S28.001 5547 - 1I1q14 (angiotensinase C) DPP7 S28.002 29952 - 9q34.3 dipeptidylpeptidase 7 PRSS16 S28.003 10279 - 6p 2 1 protease, serine, 16 (thymus) ABHD8 S33.011 79575 - 19pl3.11 abhydrolase domain containing 8 SERHL S33.012 253190 - 22ql3 kraken-like 65 WO 2009/088408 PCT/US2008/012369 Protease Family Entrez Gene enzyme Map Location ID Descriptive Name (or default name) Gene Name ID ID (cytogenetic or _____genetic location) ABHD4 S33.013 63874 - 14q1.2 abhydrolase domain containing 4 epoxide hydrolase 1, microsomal EPHXI S33.971 2052 3.3.2.3 1942.1 (xenobiotic) mesoderm specific transcript homolog MEST S33.972 4232 - 7q32 (mouse) EPHX2 S33.973 2053 - 8p21-p12 epoxide hydrolase 2, cytoplasmic ABHD7 S33.974 253152 - I p22.1 abhydrolase domain containing 7 ABHD5 S33.975 51099 - 3p2l abhydrolase domain containing 5 ABHDI I S33.976 83451 - 7q 11.23 abhydrolase domain containing 11 ABHD6 S33.977 57406 - 3p14.3 abhydrolase domain containing 6 ABHD9 S33.978 79852 - l 9 p 1 3.

12 abhydrolase domain containing 9 MGLL S33.980 11343 - 3q21.3 monoglyceride lipase ABHDI4A S33.981 25864 - 3p2l.1 DKFZP5640243 protein biphenyl hydrolase-like (serine hydrolase; breast epithelial mucin BPHL S33.982 670 - 6p25 associated antigen) NDRG4 S33.986 65009 - 16q21-q22.1 NDRG family member 4 NDRG3 S33.987 57446 - 20q 1.21-g 1.23 NDRG family member 3 NDRG1 S33.988 10397 - 8q24.3 N-myc downstream regulated gene I RBP3 S41.950 5949 - 1Og 11.2 retinol binding protein 3, interstitial TPPI S53.003 1200 - ipi15 tripeptidyl peptidase I RHBDL2 S54.002 54933 - lp34.3 rhomboid, veinlet-like 2 (Drosophila) RHBDLI S54.005 9028 - 16p13.3 rhomboid, veinlet-like 1 (Drosophila) RHBDL4 S54.006 162494 - 17ql 1.2 rhomboid, veinlet-like 4 (Drosophila) PSARL S54.009 55486 - 3q27.1 presenilin associated, rhomboid-like RHBDFI S54.952 64285 - 16p13.3 rhomboid family 1 (Drosophila) RHBDL6 S54.953 79651 - 17925.1 rhomboid, veinlet-like 6 (Drosophila) RHBDD2 S54.955 57414 - 7q 11.23 rhomboid, veinlet-like 7 (Drosophila) RHBDDI S54.xxx 84236 - 2q36.3 hypothetical protein DKFZp547E052 RHBDL7 S54.xxxnp AC005067 - - NUP98 S59.001 4928 - lI p15.5 nucleoporin 98kDa LTF S60.001 4057 - 3921-q23 lactotransferrin TF S60.972 7018 - 3q22.1 transferrin antigen p97 (melanoma associated) identified by monoclonal antibodies MFI2 S60.973 4241 - 3q28-q29 133.2 and 96.5 egf-like module containing, mucin EMR2 S63.001 30817 - l9pl3.1 like, hormone receptor-like 2 CD97 S63.002 976 - l9pl 3 CD97 antigen egf-like module containing, mucin EMR3 S63.003 84658 - 19pl 3 .l like, hormone receptor-like 3 egf-like module containing, mucin EMRI S63.004 2015 - 19p1 3

.

3 like, hormone receptor-like I 66 WO 2009/088408 PCT/US2008/012369 Protease Family Entrez Gene enzyme Map Location ID Descriptive Name (or default name) Gene Name ID ID (cytogenetic or genetic location) egf-like module containing, mucin EMR4 S63.008 326342 - 19p13.3 like, hormone receptor-like 4 cadherin, EGF LAG seven-pass G type receptor 2 (flamingo homolog, CELSR2 S63.009 1952 - 1p21 Drosophila) RELN Sxl.xxx 5649 7922 reeling HSP90B1 Sx2.xxx 7184 - 12q24.2-q24.3 tumor rejection antigen (gp96) 1 HSP90AAI Sx2.xxxnp 3320 - l4q32.33 heat shock 90kDa protein 1, alpha HSP90ABI Sx2.xxxnp 3326 - 6p1 2 heat shock 90kDa protein 1, beta TRAPI Sx2.xxxnp 10131 - 16p 13.3 TNF receptor-associated protein I proteasome (prosome, macropain) PSMB6 TO1.010 5694 - 17p]3 subunit, beta type, 6 proteasome (prosome, macropain) PSMB7 TO1.01 1 5695 - 9q34.1 1-q34.12 subunit, beta type, 7 proteasome (prosome, macropain) PSMB5 TO1.012 5693 - 149 11.2 subunit, beta type, 5 proteasome (prosome, macropain) subunit, beta type, 9 (large PSMB9 TO1.013 5698 - 6p21.3 multifunctional protease 2) proteasome (prosome, macropain) PSMB10 TO1.014 5699 - 16q22.1 subunit, beta type, 10 proteasome (prosome, macropain) subunit, beta type, 8 (large PSMB8 TO1.015 5696 - 6p21.3 multifunctional protease 7) LMP7L TO1.016 122706 - 14q11.2 similar to RIKEN cDNA 5830406J20 proteasome (prosome, macropain) PSMA6 TO1.971 5687 - 14ql3 subunit, alpha type, 6 proteasome (prosome, macropain) PSMA2 TO1.972 5683 - 7p14.1 subunit, alpha type, 2 proteasome (prosome, macropain) PSMA4 TO1.973 5685 - 15q25.1 subunit, alpha type, 4 proteasome (prosome, macropain) PSMA7 TO1.974 5688 - 20q13.33 subunit, alpha type, 7 proteasome (prosome, macropain) PSMA5 TO1.975 5686 - I1pl3 subunit, alpha type, 5 proteasome (prosome, macropain) PSMA3 TO1.976 5682 - Ilpl5.1 subunit, alpha type, 3 proteasome (prosome, macropain) PSMA3 TO1.977 5684 - 14q23 subunit, alpha type, 3 proteasome (prosome, macropain) PSMA8 TO1.978 143471 - 18q1.2 subunit, alpha type, 8 proteasome (prosome, macropain) PSMB3 TO1.983 5691 - 17912 subunit, beta type, 3 proteasome (prosome, macropain) PSM1B2 TO1.984 5690 - 6p34.2 subunit, beta type, 2 proteasome (prosome, macropain) PSMBI TO 1.986 5689 - 6q27 subunit, beta type, I proteasome (prosome, macropain) PSMB34 TO1.987 5692 - 1 q21 subunit, beta type, 4 proteasome (prosome, macropain) PSMB3P TO1.PO2 121131 - 12q]3.2 subunit, beta type, 3 pseudogene 67 WO 2009/088408 PCT/US2008/012369 Protease Family Entrez Gene enzyme Map Location ID Descriptive Name (or default name) Gene Name ID ID (cytogenetic or genetic location) AGA T02.001 175 3.5.1.26 4932-q33 aspartylglucosaminidase _ASRGLI T02.002 80150 - 11 q2.3 asparaginase like I chromosome 20 open reading frame TASPI T02.004 55617 3.4.25.- 20pl2.1 13 gamma-glutamyltransferase-like GGTLAI T03.002 2687 - 2291 1.23 activity I GGT1 T03.006 2678 2.3.2.2 22ql 1.23 gamma-glutamyltransferase I GGT2 T03.015 2679 - 22ql 1.23 gamma-glutamyltransferase 2 GGTL4 T03.016 91227 - 22ql 1.22 gamma-glutamyltransferase-like 4 GGTL3 T03.017 2686 - 20g 1.22 gamma-glutamyltransferase-like 3 RCE1 homolog, prenyl protein RCEI U48.002 9986 - 11q13 protease (S. cerevisiae) BDNF Uxx.xxx 627 - 1pl3 brain-derived neurotrophic factor cystatin C (amyloid angiopathy and CST3 Uxx.xxx 1471 - 20pl 1.21 cerebral hemorrhage) KNGI Uxx.xxx 3827 - 3927 kininogen I neural precursor cell expressed, NEDD8 Uxx.xxx 4738 - 14ql 1.2 developmentally down-regulated 8 platelet-derived growth factor alpha PDGFA Uxx.xxx 5154 - 7p22 polypeptide serine (or cysteine) proteinase inhibitor, clade F (alpha-2 antiplasmin, pigment epithelium SERPINF2 Uxx.xxx 5345 - l7p13 derived factor), member 2 splicing factor, arginine/serine-rich 2, SFRS2IP Uxx.xxx 9169 - 12q13.11 interacting protein BIRC8 Uxx.xxx 112401 - 19ql3.3-q13.4 baculoviral IAP repeat-containing 8 [001781 Table 5. Additional kinases are presented, the concentration and activity of which may be detected and quantitated using embodiments of the methods of the invention. Map Location ID Kinase Gene Entrez (cytogenetic or Name Family Gene ID enzyme ID genetic location) Descriptive Name (or default name) AGC,AKT,SK018, v-akt murine thymoma viral AKT1 AKT1 207 2.7.1.37 14q32.32 oncogene homolog 1 AGC,AKT,SKO19, v-akt murine thymoma viral AKT2 AKT2 208 2.7.1.37 19q13.1-q13.2 oncogene homolog 2 v-akt murine thymoma viral AGC,AKT,SKO20, oncogene homolog 3 (protein AKT3 AKT3 10000 2.7.1.37 Iq43-q44 kinase B, gamma) AGC,DMPK,SK69 citron (rho-interacting, CRIK 5,CRIK 11113 - 12q24 serine/threonine kinase 21 AGC,DMPK,GEK dystrophia myotonica-protein DMPK I ,SKI I I,DMPKI 1760 - 19ql3.3 kinase AGC,DMPK,GEK CDC42 binding protein kinase MRCKa ,SK299,MRCKa 8476 - 1q42.1 1 alpha (DMPK-like) AGC,DMPK,GEK CDC42 binding protein kinase beta MRCKb ,SK241,MRCKb 9578 - 14q32.3 (DMPK-like) 68 WO 2009/088408 PCT/US2008/012369 Map Location ID Kinase Gene Entrez (cytogenetic or Name Family Gene ID enzyme ID genetic location) Descriptive Name (or default name) AGC,DMPK,GEK CDC42 binding protein kinase DMPK2 ,SK1I2,DMPK2 55561 - 11q13.1 gamma (DMPK-like) AGC,DMPK,ROC Rho-associated, coiled-coil ROCKI K,SK331,ROCKI 6093 2.7.1.37 18ql1.1 containing protein kinase 1 AGC,DMPK,ROC Rho-associated, coiled-coil ROCK2 K,SK263,ROCK2 9475 2.7.1.37 2p24 containing protein kinase 2 AGC,GRK,BARK, BARKI SK045,BARK 1 156 - I1lql3 adrenergic, beta, receptor kinase I AGC,GRK,BARK, BARK2 SK478,BARK2 157 - 22q 12.1 adrenergic, beta, receptor kinase 2 AGC,GRK,GRK,S G protein-coupled receptor kinase GPRK4 KI56,GPRK4 2868 - 4 p] 6

.

3 4 AGC,GRK,GRK,S G protein-coupled receptor kinase GPRK5 K157,GPRK5 2869 - 10q24-qter 5 AGC,GRK,GRK,S G protein-coupled receptor kinase GPRK6 K158,GPRK6 2870 - 5q35 6 AGC,GRK,GRK,S G protein-coupled receptor kinase RHOK K327,RHOK 6011 2.7.1.125 13q34 1 AGC,GRK,GRK,S G protein-coupled receptor kinase GPRK7 K578,GPRK7 131890 - 3q21-q23 7 AGC,MAST,SK34 microtubule associated MAST] 5,MASTI 22983 - 19p13.2 serine/threonine kinase 1 AGC,MAST,SK19 microtubule associated MAST3 6,MAST3 23031 - 19p13.11 serine/threonine kinase 3 AGC,MAST,SK21 microtubule associated MAST2 6,MAST2 23139 - lp34.1 serine/threonine kinase 2 similar to microtubule associated AGC,MAST,SK70 testis specific serine/threonine MAST4 1,MAST4 375449 - 5ql2.3 protein kinase AGC,MAST,SK45 microtubule associated MASTL 5,MASTL 84930 - 10p12.1 serine/threonine kinase-like AGC,NDR,SK441, LATS, large tumor suppressor, LATSI LATS1 9113 - 6q24-q25.1 homolog I (Drosophila) AGC,NDR,SK249, NDRI NDRI 11329 - 6p21 serine/threonine kinase 38 AGC,NDR,SK500, NDR2 NDR2 23012 - 12p1 1.23 serine/threonine kinase 38 like AGC,NDR,SK442, LATS, large tumor suppressor, LATS2 LATS2 26524 - 13ql l-q12 homolog 2 (Drosophila) AGC,PDK1,SK27 3-phosphoinositide dependent PDK1 6,PDKI 5170 - 16p13.3 protein kinase-l AGC,PKA,SK300, protein kinase, cAMP-dependent, PKACa PKACa 5566 2.7.1.37 19p13.1 catalytic, alpha AGC,PKA,SK301, protein kinase, cAMP-dependent, PKACb PKACb 5567 2.7.1.37 1p36.1 catalytic, beta AGC,PKA,SK302, protein kinase, cAMP-dependent, PKACg PKACg 5568 2.7.1.37 9q13 catalytic, gamma AGC,PKA,SK313, PRKX PRKX 5613 - xp22.3 protein kinase, X-linked AGC,PKA,SK320, PRKY PRKY 5616 - yp 11.2 protein kinase, Y-linked AGC,PKC,Alpha, PKCa SK303,PKCa 5578 2.7.1.37 1 7q22-q23.2 protein kinase C, alpha PKCb AGC,PKC,Alpha, 5579 2.7.1.37 16p 11.2 protein kinase C, beta 1 69 WO 2009/088408 PCT/US2008/012369 Map Location ID Kinase Gene Entrez (cytogenetic or Name Family Gene ID enzyme ID genetic location) Descriptive Name (or default name) SK304,PKCb AGC,PKC,Alpha, PKCg SK307,PKCg 5582 2.7.1.37 19q13.4 protein kinase C, gamma AGC,PKC,Delta,S PKCd K305,PKCd 5580 2.7.1.37 3p21.31 protein kinase C, delta AGC,PKC,Delta,S PKCt K310,PKCt 5588 2.7.1.37 lOp15 protein kinase C, theta AGC,PKC,Eta,SK PKCe 306,PKCe 5581 2.7.1.37 2p21 protein kinase C, epsilon AGC,PKC,Eta,SK PKCh 270,PKCh 5583 2.7.1.37 14q22-q23 protein kinase C, eta AGC,PKC,Iota,SK PKCi 308,PKCi 5584 2.7.11.13 3q26.3 protein kinase C, iota AGC,PKC,Iota,SK PKCz 311,PKCz 5590 2.7.1.37 lp36.33-p36.2 protein kinase C, zeta AGC,PKG,SK073, protein kinase, cGMP-dependent, PKG1 PKG1 5592 2.7.1.37 1091l.2 type I AGC,PKG,SK075, protein kinase, cGMP-dependent, PKG2 PKG2 5593 2.7.1.37 4q13.1-q21.1 type II AGC,PKN,SK317, PKN1 PKN1 5585 - 19pl3.1-p12 protein kinase NI AGC,PKN,SK318, PKN2 PKN2 5586 - Ip22.2 protein kinase N2 AGC,PKN,SK511, PKN3 PKN3 29941 - 9q34.11 protein kinase N3 AGC,RSK,MSK,S ribosomal protein S6 kinase, MSK2 K243,MSK2 8986 - I IqI 1-q13 90kDa, polypeptide 4 AGC,RSK,MSK,S ribosomal protein S6 kinase, MSK1 K242,MSK1 9252 - 14q31-q32.1 90kDa, polypeptide 5 AGC,RSK,p70,SK ribosomal protein S6 kinase, p70S6K 265,p7OS6K 6198 - 17q23.2 70kDa, polypeptide 1 AGC,RSK,p70,SK ribosomal protein S6 kinase, p70S6Kb 266,p7OS6Kb 6199 - 1 1913.2 70kDa, polypeptide 2 AGC,RSK,RSK,S ribosomal protein S6 kinase, RSK3 K338,RSK3 6195 - 1p 90kDa, polypeptide 1 AGC,RSK,RSK,S ribosomal protein S6 kinase, RSK1 K336,RSKI 6196 - 6q27 90kDa, polypeptide 2 AGC,RSK,RSK,S ribosomal protein S6 kinase, RSK2 K337,RSK2 6197 - xp22.2-p22.1 90kDa, polypeptide 3 AGC,RSK,RSK,S ribosomal protein S6 kinase, RSK4 K518,RSK4 27330 - xq21 90kDa, polypeptide 6 AGC,RSKL,SK51 ribosomal protein S6 kinase, RSKLI 7,RSKL1 26750 - 1q41 52kDa, polypeptide I AGC,RSKL,SK47 RSKL2 3,RSKL2 83694 - 14q24.3 ribosomal protein S6 kinase-like I AGC,RSKR,SK49 SgK494 1,SgK494 124923 - 17ql1.2 hypothetical protein FLJ25006 AGC,SGK,SK346, serum/glucocorticoid regulated SGKI SGK 6446 - 6q23 kinase AGC,SGK,SK523, serum/glucocorticoid regulated SGK2 SGK2 10110 - 20q13.2 kinase 2 AGCSGK,SK525, serum/glucocorticoid regulated SGK3 SGK3 23678 - 8q12.3-8913.1 kinase-like YANK2 AGC,YANK,SK48 55351 - 4p16.2 serine/threonine kinase 32B 70 WO 2009/088408 PCT/US2008/012369 Map Location ID Kinase Gene Entrez (cytogenetic or Name Family Gene ID enzyme ID genetic location) Descriptive Name (or default name) 1,YANK2 AGC,YANK,SK62 YANKI 4,YANKI 202374 - 5q32 serine/threonine kinase 32A AGC,YANK,SK46 YANK3 9,YANK3 282974 - 10q26.3 serine/threonine kinase 32C Atypical,ABC1,A BCl- chaperone, ABCI activity of bcl ADCK3 A,SK609,ADCK3 56997 - 1q42.13 complex like (S. pombe) Atypical,ABCI,A BCl ADCK4 A,SK013,ADCK4 79934 - 19q 13.2 aarF domain containing kinase 4 Atypical,ABCI,A BCl ADCKI B,SK401,ADCK1 57143 - 14q24.3 aarF domain containing kinase I Atypical,ABCI,A

BCI

ADCK5 B,SK780,ADCK5 203054 - 8q24.3 aarF domain containing kinase 5 Atypical,ABC1,A

BCI

ADCK2 C,SK712,ADCK2 90956 - 7q32-q34 aarF domain containing kinase 2 Atypical,Alpha,SK AlphaKI 765,AlphaK1 57538 - 15925.2 alpha-kinase 3 Atypical,Alpha,SK AlphaK3 755,AlphaK3 80216 - 4q25 alpha-kinase I Atypical,Alpha,SK AlphaK2 754,AlphaK2 115701 - 18q21.31 alpha-kinase 2 Atypical,Alpha,Ch transient receptor potential cation ChaKI aK,SK423,ChaKl 54822 - 15921 channel, subfamily M, member 7 Atypical,Alpha,Ch transient receptor potential cation ChaK2 aK,SK746,ChaK2 140803 - 9q21.13 channel, subfamily M, member 6 Atypical,Alpha,eE F2K,SK117,eEF2 eukaryotic elongation factor-2 eEF2K K 29904 - 16p12.1 kinase Atypical,BCR,SKO BCR 47,BCR 613 - 22q 1.23 breakpoint cluster region Atypical,BRD,SK7 BRDT 64,BRDT 676 - 1p22.1 bromodomain, testis-specific Atypical,BRDSK7 BRD2 61,BRD2 6046 - 6p21.3 bromodomain containing 2 Atypical,BRD,SK7 BRD3 62,BRD3 8019 - 9q34 bromodomain containing 3 Atypical,BRD,SK7 BRD4 63,BRD4 23476 - 19p13.1 bromodomain containing 4 Atypical,FAST,SK FASTK 139,FASTK 10922 - 7q35 FAST kinase Atypical,G1 1,SK7 Gil 56,G 11 8859 - 6p21.3 serine/threonine kinase 19 Atypical,H 11 ,SK7 H l1 82,H 11 26353 - 12924.23 heat shock 22kDa protein 8 Atypical,PDHK,S branched chain ketoacid BCKDK K046,BCKDK 10295 - 16p 11.2 dehydrogenase kinase Atypical,PDHK,S pyruvate dehydrogenase kinase, PDHKI K277,PDHKI 5163 - 2q31.1 isoenzyme I PDHK2 Atypical,PDHK,S 5164 - 17q21.33 pyruvate dehydrogenase kinase, 71 WO 2009/088408 PCT/US2008/012369 Map Location ID Kinase Gene Entrez (cytogenetic or Name Family Gene ID enzyme ID genetic location) Descriptive Name (or default name) K278,PDHK2 isoenzyme 2 Atypical,PDHK,S pyruvate dehydrogenase kinase, PDHK3 K279,PDHK3 5165 - xp22.1 I isoenzyme 3 Atypical,PDHK,S pyruvate dehydrogenase kinase, PDHK4 K280,PDHK4 5166 - 7q21.3-q22.1 isoenzyme 4 ataxia telangiectasia mutated Atypical,PIKK,AT (includes complementation groups ATM M,SK038,ATM 472 - 11 q22-q23 A, C and D) Atypical,PIKK,AT ataxia telangiectasia and Rad3 ATR R,SK039,ATR 545 - 3q22-q24 related Atypical,PIKK,DN APK,SK1 13,DNA protein kinase, DNA-activated, DNAPK PK 5591 - 8g11 catalytic polypeptide Atypical,PIKK,FR FK506 binding protein 12 FRAP AP,SK152,FRAP 2475 - lp36.2 rapamycin associated protein 1 Atypical,PIKK,SM SMGI GI,SK665,SMGI 23049 - 16p12.3 PI-3-kinase-related kinase SMG-1 Atypical,PIKK,TR RAP,SK380,TRR transformation/transcription TRRAP AP 8295 - 7q21.2-q22.1 domain-associated protein Atypical,RIO,RIO RIOKI 1,SK615,RIOK1 83732 - 6p 2 4 .3 RIO kinase 1 (yeast) Atypical,RIO,RIO RIOK2 2,SK753,RIOK2 55781 - 5q15 RIO kinase 2 (yeast) Atypical,RIO,RIO RIOK3 3,SK606,RIOK3 8780 - 18ql 1.2 RIO kinase 3 (yeast) TAFI RNA polymerase 11, TATA Atypical,TAF1,SK box binding protein (TBP) TAF 772,TAF1 6872 - xql3.1 associated factor, 250kDa TAF 1-like RNA polymerase II, Atypical,TAF I,SK TATA box binding protein (TBP) TAFIL 781,TAF1L 138474 - 9p21.1 associated factor, 21OkDa Atypical,TIF1,SK7 transcriptional intermediary factor TIFla 83,TIFIa 8805 - 7q32-q34 1 Atypical,TIF1,SK7 TIFIb 84,TIF I b 10155 - 19q 13.4 tripartite motif-containing 28 Atypical,TIFI,SK7 TIFIg 85,TIFlg 51592 - 1pl3.1 tripartite motif-containing 33 CAMK,CAMK1,S calcium/calmodulin-dependent CaMK4 K061,CaMK4 814 2.7.11.17 5q21.3 protein kinase IV CAMKCAMKI,S calcium/calmodulin-dependent CaMKla K056,CaMKla 8536 - 3p25.3 protein kinase I CAMK,CAMKI,S calcium/calmodulin-dependent CaMKld K572,CaMKId 57118 - lOp13 protein kinase ID CAMK,CAMKI,S calcium/calmodulin-dependent CaMKIg K021,CaMKlg 57172 - 1q32-q41 protein kinase IG CAMK,CAMKI,S pregnancy upregulated non CaMK~b K662,CaMKIb 139728 - xq28 ubiquitously expressed CaM kinase calcium/calmodulin-dependent CAMK,CAMK2,S protein kinase (CaM kinase) II CaMK2a K057,CaMK2a 815 2.7.11.17 5q32 alpha CAMK,CAMK2,S calcium/calmodulin-dependent CaMK2b K058,CaMK2b 816 - 7pl4.3-pl4.1 protein kinase (CaM kinase) II beta CaMK2d CAMK,CAMK2,S 817 - 4q26 calcium/calmodulin-dependent 72 WO 2009/088408 PCT/US2008/012369 Map Location ID Kinase Gene Entrez (cytogenetic or Name Family Gene ID enzyme ID genetic location) Descriptive Name (or default name) K703,CaMK2d protein kinase (CaM kinase) II delta calcium/calmodulin-dependent CAMK,CAMK2,S protein kinase (CaM kinase) II CaMK2g K060,CaMK2g 818 - 10q22 gamma CAMK,CAMKL, AMPK,SK032,AM protein kinase, AMP-activated, AMPKal PKal 5562 - 5p12 alpha I catalytic subunit CAMK,CAMKL, AMPK,SK033,AM protein kinase, AMP-activated, AMPKa2 PKa2 5563 - 1p3I alpha 2 catalytic subunit CAMK,CAMKL,B RSK,SKO15,BRS BRSK2 K2 9024 - I1p15.5 BR serine/threonine kinase 2 CAMK,CAMKL,B RSK,SK598,BRS BRSKI KI 84446 - 19q 13.4 BR serine/threonine kinase 1 CAMK,CAMKL,C HK1,SK078,CHK CHKI checkpoint homolog (S. CHK1 1 1111 - 11q24-q24 pombe) CAMK,CAMKL, HUNK,SK502,HU hormonally upregulated Neu HUNK NK 30811 - 21922.1 associated kinase CAMK,CAMKL,L serine/threonine kinase 11 (Peutz LKBI KB,SK208,LKB1 6794 - 19p13.3 Jeghers syndrome) CAMK,CAMKL, MARK,SK120,M MAP/microtubule affinity MARK2 ARK2 2011 - 1 lql2-q13 regulating kinase 2 CAMK,CAMKL, MARK,SK215,M MAP/microtubule affinity MARKI ARKI 4139 - 1q41 regulating kinase I CAMK,CAMKL, MARK,SK096,M MAP/microtubule affinity MARK3 ARK3 4140 - 14q32.3 regulation kinase 3 CAMK,CAMKL, MARK,SK5] 5,M MAP/microtubule affinity MARK4 ARK4 57787 - 199 13.3 regulating kinase 4 CAMK,CAMKL, MELK,SK298,ME maternal embryonic leucine zipper MELK LK 9833 - 9p13.2 kinase CAMK,CAMKL, NIM1,SK449,NIM NIMI 1 167359 - 5pI2 hypothetical protein MGC42105 CAMK,CAMKL, NuaK,SK195,Nua AMP-activated protein kinase NuaKI KI 9891 - 12q23.3 family member 5 CAMK,CAMKL, NuaK,SK472,Nua likely ortholog of rat SNF I/AMP NuaK2 K2 81788 - lq32.1 activated protein kinase CAMK,CAMKL,P ASK,SK499,PAS PAS domain containing PASK K 23178 - 2q37.3 serine/threonine kinase CAMK,CAMKL, QIK QIK,SK513,QIK 23235 - 11q23.1 SNFI-like kinase 2 73 WO 2009/088408 PCT/US2008/012369 Map Location ID Kinase Gene Entrez (cytogenetic or Name Family Gene ID enzyme ID genetic location) Descriptive Name (or default name) CAM K,CAMKL, QSK QIK,SK50,QSK 23387 - 1 1q23.3 KIAA0999 protein CAMK,CAMKL, SIK QIK,SK604,SIK 150094 - 21q22.3 SNFI-like kinase CAMK,CAMKL,S NRK,SK625,SNR SNRK K 54861 - 3p22.1 SNF-I related kinase

CAMK,CAMK

Unique,SK463,ST STK33 K33 65975 - 1lp15.3 serine/threonine kinase 33

CAMK,CAMK

Unique,SK062,VA VACAMKL CAMKL 79012 - 3p21.31 hypothetical protein MGC8407 calcium/calmodulin-dependent CAMK,CASK,SK serine protein kinase (MAGUK CASK 064,CASK 8573 - xp 11.4 family) CAMK,DAPK,SK DAPKI 103,DAPKI 1612 - 9q34.1 death-associated protein kinase 1 CAMK,DAPK,SK DAPK3 716,DAPK3 1613 - 19p13.3 death-associated protein kinase 3 CAMK,DAPK,SK DAPK2 104,DAPK2 23604 - 15q22.31 death-associated protein kinase 2 CAMK,DAPK,SK serine/threonine kinase 17b DRAK2 487,DRAK2 9262 - 2q32.3 (apoptosis-inducing) CAMK,DAPK,SK serine/threonine kinase 17a DRAKI 486,DRAK1 9263 - 7p12-pl4 (apoptosis-inducing) CAMK,DCAMKL ,SK063,DCAMKL doublecortin and CaM kinase-like DCLKI 1 9201 - 13ql3 1 CAMK,DCAMKL ,SK459,DCAMKL doublecortin and CaM kinase-like DCLK3 3 85443 - 3p22.3 3 CAMK,DCAMKL ,SK527,DCAMKL doublecortin and CaM kinase-like DCLK2 2 166614 - 4q31.23 2 CAMK,MAPKAP MAPKAPK K,MAPKAPK,SK mitogen-activated protein kinase 3 213,MAPKAPK3 7867 - 3p21.3 activated protein kinase 3 CAMK,MAPKAP MAPKAPK K,MAPKAPK,SK mitogen-activated protein kinase 5 214,MAPKAPK5 8550 - 12q24.12 activated protein kinase 5 CAMK,MAPKAP MAPKAPK K,MAPKAPK,SK mitogen-activated protein kinase 2 212,MAPKAPK2 9261 - lq32 activated protein kinase 2 CAMK,MAPKAP K,MNK,SK236,M MAP kinase interacting MNK2 NK2 2872 - 19p13.3 serine/threonine kinase 2 CAMK,MAPKAP K,MNK,SK235,M MAP kinase interacting MNKI NKI 8569 - 1p33 serine/threonine kinase I CAMK,MLCK,SK smMLCK 231,smMLCK 4638 2.7.11.18 3q21 m osin, light polypeptide kinase CAMK,MLCK,SK TTN 372,TTN 7273 - 2q31 titin 74 WO 2009/088408 PCT/US2008/012369 Map Location ID Kinase Gene Entrez (cytogenetic or Name Family Gene ID enzyme ID genetic location) Descriptive Name (or default name) CAMK,MLCK,SK myosin light chain kinase 2, skMLCK 675,skMLCK 85366 2.7.11.18 20q13.31 skeletal muscle CAMK,MLCK,SK caMLCK 536,caMLCK 91807 - 16ql 1.2 myosin light chain kinase (MLCK) CAMK,MLCK,SK SgK085 709,SgK085 340156 - 6 p 2 5.

2 hypothetical protein LOC340156 CAMK,PHK,SK28 phosphorylase kinase, gamma 1 PHKgI 3,PHKgl 5260 2.7.1.38 7pl2-q21 (muscle) CAMK,PHK,SK28 phosphorylase kinase, gamma 2 PHKg2 4,PHKg2 5261 - 16p12.1-pIl.2 (testis) CAMK,PIM,SK29 PIMI l,PIM1 5292 - 6p2l.2 pim-1 oncogene CAMK,PIM,SK29 PIM2 2,PIM2 11040 - xpll.23 pim-2oncogene CAMK,PIM,SK20 PIM3 0,PIM3 415116 - 22q]3 pim-3 oncogene CAMK,PKD,SK30 PRKDI 9,PKD1 5587 2.7.1.37 14ql1 protein kinase D1 CAMK,PKD,SK48 PKD3 9,PKD3 23683 - 2p21 protein kinase D3 CAMK,PKD,SK48 PRKD2 0,PKD2 25865 - 19q13.3 protein kinase D2 CAMK,PSK,SK32 PSKH 1 2,PSKH I 5681 - 16q22.1 protein serine kinase HI CAMK,PSK,SK60 PSKH2 2,PSKH2 85481 - 8q21.3 protein serine kinase H2 CAMK,RAD53,S CHK2 checkpoint homolog (S. CHK2 K079,CHK2 11200 - 22ql2.1 pombe)

CAMK,CAMK

Unique,SK492,Sg SgK495 K495 83931 - lp34.3 Ser/Thr-like kinase CAMK,Trbl,SKO1 Trbl 4,Trbl 10221 - 8q24.13 tribbles homolog 1 (Drosophila) CAMK,Trbl,SK16 Trb2 0,Trb2 28951 - 2p24.3 tribbles homolog 2 (Drosophila) CAMK,Trbl,SK69 Trb3 4,Trb3 57761 - 2 0p13-pl 2

.

2 tribbles homolog 3 (Drosophila) CAMK,Trio,SK60 obscurin, cytoskeletal calmodulin Obscn 1,Obscn 84033 - 1q42.13 and titin-interacting RhoGEF CAMK,Trio,SK53 SPEG 7,SPEG 729871 - 2q35 SPEG complex locus CAMK,Trio,SK37 triple functional domain (PTPRF Trio 6,Trio 7204 - 5p15.1-pl4 interacting) CAMK,Trio,SK53 huntingtin-associated protein Trad 3,Trad 8997 - 3q21.l-q21.2 interacting protein (duo) CAMK,TSSK,SK4 serine/threonine kinase 22B TSSK2 74,TSSK2 23617 - 22ql 1.21 (spermiogenesis associated) CAMK,TSSK,SK4 serine/threonine kinase 22C TSSK3 71,TSSK3 81629 - 1p35-p34 (spermiogenesis associated) CAMK,TSSK,SK7 serine/threonine kinase 22D TSSKI 05,TSSK1 83942 - 5q22.2 (spermiogenesis associated) CAMK,TSSK,SK5 serine/threonine protein kinase SSTK 24,SSTK 83983 - 19p13.1 1 SSTK TSSK4 CAMK,TSSK,SK5 283629 - 149 11.2 chromosome 14 open reading 75 WO 2009/088408 PCT/US2008/012369 Map Location ID Kinase Gene Entrez (cytogenetic or Name Family Gene ID enzyme ID genetic location) Descriptive Name (or default name) 34,TSSK4 frame 20 CKICKI,CK 1 CKIa A,SK082,CKla 1452 - 5932 casein kinase 1, alpha I

CKI,CKI,CKI

CKIa2 A,SK541,CKla2 122011 - 13q]3.3 casein kinase 1, alpha 1-like CKI,CK1,CKI CKId D,SK083,CKId 1453 - l7q25 casein kinase 1, delta CKI,CK1,CKI CKle E,SK084,CKle 1454 - 22q13.1 casein kinase 1, epsilon CK1,CKI,CKI CK g2 G,SK086,CKlg2 1455 - 19p13.3 casein kinase 1, gamma 2

CKI,CKI,CKI

CKlg3 G,SK087,CKlg3 1456 - 5q23 casein kinase 1, gamma 3

CKI.CKI,CKI

CKIgl G,SK647,CKlg1 53944 - 15922.1-922.31 casein kinase 1, gamma 1 CK1,TTBK,SK526 TTBKI ,TTBK 1 84630 - 6p2 1.1 tau tubulin kinase I CKI,TTBK,SK453 TTBK2 ,TTBK2 146057 - 15ql5.2 tau tubulin kinase 2 CK1,VRK,SK389, VRKI VRKI 7443 - 14q32 vaccinia related kinase 1 CKI,VRK,SK390, VRK2 VRK2 7444 - 2pl6-p15 vaccinia related kinase 2 CK1,VRK,SK535, VRK3 VRK3 51231 - 19qI3 vaccinia related kinase 3 CMGC,CDK,SK4 CCRK 83,CCRK 23552 - 9q22.1 cell cycle related kinase CMGC,CDK,CDC cell division cycle 2, GI to S and CDC2 2,SK065,CDC2 983 - 10q21.1 G2 to M CMGC,CDK,CDC CDK2 2,SK067,CDK2 1017 - 12q13 cyclin-dependent kinase 2 CMGC,CDK,CDC CDK3 2,SK068,CDK3 1018 - 17q22-qter cyclin-dependent kinase 3 CMGC,CDK,CDK cyclin-dependent kinase (CDC2 CDK1O 10,SK294,CDK1O 8558 - 16q24 like) 10 CMGC,CDK,CDK CDK4 4,SK069,CDK4 1019 - 12qI4 cyclin-dependent kinase 4 CMGC,CDK,CDK CDK6 4,SK071,CDK6 1021 - 7q21-q22 cyclin-dependent kinase 6 CMGC,CDK,CDK CDK5 5,SK070,CDK5 1020 - 7q36 cyclin-dependent kinase 5 cyclin-dependent kinase 7 (MO15 CMGC,CDK,CDK homolog, Xenopus laevis, cdk CDK7 7,SK055,CDK7 1022 - 5ql2.1 activating kinase) CMGC,CDK,CDK CDK8 8,SK072,CDK8 1024 - 13q12 cyclin-dependent kinase 8 CMGC,CDK,CDK cell division cycle 2-like 6 (CDK8 CDKI I 8,SK443,CDKI 1 23097 - 6921 like) CMGC,CDK,CDK cyclin-dependent kinase 9 (CDC2 CDK9 9,SK295,CDK9 1025 - 9q34.1 related kinase) cell division cycle 2-like 5 CMGC,CDK,CRK (cholinesterase-related cell division CHED 7,SK076,CHED 8621 - 7pl3 controller) CRK7 CMGC,CDK,CRK 51755 - 17912 CDC2-related protein kinase 7 76 WO 2009/088408 PCT/US2008/012369 Map Location ID Kinase Gene Entrez (cytogenetic or Name Family Gene ID enzyme ID genetic location) Descriptive Name (or default name) 7,SK485,CRK7 CMGC,CDK,TAI RE,SK271,PCTAI PCTAIREI REI 5127 - xpl1.3-pl1.23 PCTAIRE protein kinase I CMGC,CDK,TAI RE,SK272,PCTAI PCTAIRE2 RE2 5128 - 12q23.1 PCTAIRE protein kinase 2 CMGC,CDK,TAI RE,SK273,PCTAI PCTAIRE3 RE3 5129 - Iq3 I-q32 PCTAIRE protein kinase 3 CMGC,CDK,TAI RE,SK282,PFTAI PFTAIRE1 REI 5218 - 7q21-q22 PFTAIRE protein kinase 1 CMGC,CDK,TAI amyotrophic lateral sclerosis 2 RE,SK462,PFTAI (juvenile) chromosome region, PFTAIRE2 RE2 65061 - 2q33.2 candidate 7 CMGC,CDK,PITS LRE,SK297,PITS cell division cycle 2-like 2 PITSLRE LRE 985 - lp36.3 (PITSLRE proteins) CMGC,CDKL,SK CDKL5 361,CDKL5 6792 - xp22 cyclin-dependent kinase-like 5 CMGC,CDKL,SK cyclin-dependent kinase-like 1 CDKLI 203,CDKLI 8814 - 14q21.3 (CDC2-related kinase) CMGC,CDKL,SK cyclin-dependent kinase-like 2 CDKL2 202,CDKL2 8999 - 4q2 1.1 (CDC2-related kinase) CMGC,CDKL,SK CDKL3 509,CDKL3 51265 2.7.11.22 5q31 cyclin-dependent kinase-like 3 CMGC,CDKL,SK CDKL4 466,CDKL4 344387 - 2p22.1 cyclin-dependent kinase-like 4 Other,CK2,SK088, casein kinase 2, alpha I CK2al CK2al 1457 - 20pl3 polypeptide Other,CK2,SK089, casein kinase 2, alpha prime CK2a2 CK2a2 1459 - 16p]3.3-pl3.

2 polypeptide CMGC,CLK,SKO9 CLK1 0,CLKI 1195 - 2q33 CDC-like kinase I CMGC,CLK,SKO9 CLK2 1,CLK2 1196 - 1q21 CDC-like kinase 2 CMGC,CLK,SKO9 CLK3 2,CLK3 1198 - 15924 CDC-like kinase 3 CMGC,CLK,SK48 CLK4 4,CLK4 57396 - 5q35 CDC-like kinase 4 CMGC,DYRK,DY dual-specificity tyrosine-(Y) RKI ,SK234,DYR phosphorylation regulated kinase DYRKIA KIA 1859 - 21922.13 1A CMGC,DYRK,DY dual-specificity tyrosine-(Y) RK ],SKI 14,DYR phosphorylation regulated kinase DYRKIB KIB 9149 - 19q12-13.1 1B CMGC,DYRK,DY RK2,SK488,DYR dual-specificity tyrosine-(Y) DYRK3 K3 8444 - lq32.1 phosphorylation regulated kinase 3 CMGC,DYRK,DY RK2,SK1 15,DYR dual-specificity tyrosine-(Y) DYRK2 K2 8445 - 12915 phosphorylation regulated kinase 2 DYRK4 CMGC,DYRK,DY 8798 - 12p13.32 dual-specificity tyrosine-(Y) 77 WO 2009/088408 PCT/US2008/012369 Map Location ID Kinase Gene Entrez (cytogenetic or Name Family Gene ID enzyme ID genetic location) Descriptive Name (or default name) RK2,SK I 16,DYR phosphorylation regulated kinase 4 K4 CMGC,DYRK,Hi homeodomain interacting protein HIPK3 PK,SK314,HIPK3 10114 - I1p13 kinase3 CMGC,DYRK,H I homeodomain interacting protein HIPK2 PK,SK495,HIPK2 28996 - 7q32-q34 kinase 2 CMGC,DYRK,H I homeodomain interacting protein HIPK4 PK,SK582,HIPK4 147746 - 19q 13.2 kinase 4 CMGC,DYRK,HI homeodomain interacting protein HIPK1 PK,SK169,HIPKI 204851 - lpI 3

.

2 kinase I CMGC,DYRK,PR PRP4 pre-mRNA processing factor PRP4 P4,SK321,PRP4 8899 - 6p25.2 4 homolog B (yeast) CMGC,GSK,SK16 GSK3A 2,GSK3A 2931 - 19q 13.2 glycogen synthase kinase 3 alpha CMGC,GSK,SK16 GSK3B 3,GSK3B 2932 - 3q13.3 glycogen synthase kinase 3 beta CMGC,MAPK,ER Erk2 K,SK135,Erk2 5594 2.7.1.37 22g1 1.21 mitogen-activated protein kinase 1 CMGC,MAPK,ER ErkI K,SK134,Erkl 5595 2.7.1.37 16p12-pl 1.2 mitogen-activated protein kinase 3 CMGC,MAPK,ER Erk4 K,SK137,Erk4 5596 - 18912-q21 mitogen-activated protein kinase 4 CMGC,MAPK,ER Erk3 K,SK136,Erk3 5597 - 15921 mitogen-activated protein kinase 6 CMGC,MAPK,ER Erk5 K,SK408,Erk5 5598 - 17pl 1.2 mitogen-activated protein kinase 7 CMGC,MAPK,Erk extracellular signal-regulated Erk7 7,SK465,Erk7 225689 - 8924.3 kinase 8 CMGC,MAPK,JN MAPK8 K,SK188,JNKI 5599 2.7.1.37 1Og11.22 mitogen-activated protein kinase 8 CMGC,MAPK,JN MAPK9 K,SK189,JNK2 5601 2.7.1.37 5q35 mitogen-activated protein kinase 9 CMGC,MAPK,JN mitogen-activated protein kinase MAPK1O K,SK190,JNK3 5602 2.7.1.37 4q22.1-q23 10 CMGC,MAPK,nm NLK o,SK255,NLK 51701 - 17911.2 nemo like kinase CMGC,MAPK,p3 mitogen-activated protein kinase p38a 8,SK264,p38a 1432 - 6p21.3-p2l.2 14 CMGC,MAPK,p3 mitogen-activated protein kinase p38b 8,SK342,p38b 5600 2.7.1.37 22q]3.33 11 CMGC,MAPK,p3 mitogen-activated protein kinase p38d 8,SK344,p38d 5603 2.7.1.37 6p2l.31 13 CMGC,MAPK,p3 mitogen-activated protein kinase p38g 8,SK343,p38g 6300 2.7.1.37 22913.33 12 CMGC,RCK,SK21 MAK I,MAK 4117 - 6q22 male germ cell-associated kinase CMGC,RCK,SK50 MOK 5,MOK 5891 - 14q32 renal tumor antigen CMGC,RCK,SK49 ICK 7,ICK 22858 - 6p1 2

.

3 -pl 1.2 intestinal cell (MAK-like) kinase CMGC,SRPK,SK3 SRPKI 58,SRPKI 6732 - 6p21.3-p21.2 SFRS protein kinase I CMGC,SRPK,SK3 SRPK2 59,SRPK2 6733 - 7922-93 1.1 SFRS protein kinase 2 78 WO 2009/088408 PCT/US2008/012369 Map Location ID Kinase Gene Entrez (cytogenetic or Name Family Gene ID enzyme ID genetic location) Descriptive Name (or default name) CMGC,SRPK,SK5 MSSK1 07,MSSK1 26576 - xq28 serine/threonine kinase 23 Other,AUR,SK407 AurA ,AurA 6790 - 20q 13.2-9 13.3 serine/threonine kinase 6 Other,AUR,SK043 AurC ,AurC 6795 - 19q 13.43 aurora kinase C Other,AUR,SK406 AurB ,AurB 9212 - 17p13.1 aurora kinase B Other,BUB,SK409 BUBI budding uninhibited by BUBI ,BUBI 699 - 2q14 benzimidazoles I homolog (yeast) BUBI budding uninhibited by Other,BUB,SK053 benzimidazoles 1 homolog beta BUBRI ,BUBRI 701 - 15q15 (yeast) Other,Bud32,SK46 PRPK 4,PRPK 112858 - 20q13.2 TP53 regulating kinase Other,CAMKK,M eta,SK482,CaMK calcium/calmodulin-dependent CaMKK2 K2 10645 - 12q24.2 protein kinase kinase 2, beta Other,CAMKK,M eta,SK697,CaMK calcium/calmodulin-dependent CaMKKI K1 84254 - 17p13.2 protein kinase kinase 1, alpha Other,CDC7,SK06 CDC7 cell division cycle 7 (S. CDC7 6,CDC7 8317 - Ip22 cerevisiae) Other,Haspin,SK6 Haspin 92,Haspin 83903 - 17pI3 germ cell associated 2 (haspin) Other,IKK,SKI75, conserved helix-loop-helix IKKa IKKa 1147 - 10q24-q25 ubiquitous kinase inhibitor of kappa light polypeptide Other,IKK,SK176, gene enhancer in B-cells, kinase IKKb IKKb 3551 - 8pl1.

2 beta inhibitor of kappa light polypeptide Other,IKK,SK193, gene enhancer in B-cells, kinase IKKe IKKe 9641 - 1q32.1 epsilon Other,IKK,SK53 1, TBK1 TBKI 29110 - 12q 14.1 TANK-binding kinase 1 Other.IRE,SK182,1 endoplasmic reticulum to nucleus IREl REI 2081 - 17q24.2 signalling I Other,IRE,SK498,1 endoplasmic reticulum to nucleus IRE2 RE2 10595 - 16p 12.2 signalling 2 Other,Other Unique,SK661,KI U2AF homology motif (UHM) KIS S 127933 - lq23.3 kinase 1 Other,MOS,SK237 v-mos Moloney murine sarcoma MOS ,MOS 4342 - 8q1 viral oncogene homolog Other,NAK,SK422 AAKI ,AAKI 22848 - 2p24.3-pl4 AP2 associated kinase I Other,NAK,SK704 BIKE ,BIKE 55589 - 4q21.21 BMP2 inducible kinase Other,NAK,SK155 GAK ,GAK 2580 - 4p16 cyclin G associated kinase Other,NAK,SK506 MPSKI ,MPSKI 8576 - 2q34-q37 serine/threonine kinase 16 Other,NEK,SK250 NIMA (never in mitosis gene a) NEKI ,NEKI 4750 - 4q33 related kinase I 79 WO 2009/088408 PCT/US2008/012369 Map Location ID Kinase Gene Entrez (cytogenetic or Name Family Gene ID enzyme ID genetic location) Descriptive Name (or default name) Other,NEK,SK252 NIMA (never in mitosis gene a) NEK3 ,NEK3 4752 - 13q14.13 related kinase 3 similar to Serine/threonine-protein Other,NEK,SK558 kinase Neki (NimA-related protein NEK5 ,NEK5 341676 - 13914.3 kinase 1) Other,NEK,SK645 NEK1O ,NEKIO 152110 - 3p24.1 hypothetical protein FLJ32685 Other,NEK,SK574 NIMA (never in mitosis gene a) NEKI1 ,NEKI 1 79858 - 3q21.3 related kinase 1I Other,NEK,SK251 NIMA (never in mitosis gene a) NEK2 ,NEK2 4751 - 1q32.2-q41 related kinase 2 Other,NEK,SK256 NIMA (never in mitosis gene a) NEK4 ,NEK4 6787 2.7.11.1 3p21.1 related kinase 4 Other,NEK,SK420 NIMA (never in mitosis gene a) NEK6 ,NEK6 10783 - 9933.3-q34.11 related kinase 6 Other,NEK,SK421 NIMA (never in mitosis gene a) NEK7 ,NEK7 140609 - 1q31.3 related kinase 7 Other,NEK,SK470 NIMA (never in mitosis gene a) NEK9 ,NEK9 91754 - 14q24.3 related kinase 9 Other,NEK,SK476 NIMA (never in mitosis gene a) NEK8 ,NEK8 284086 - 17g 11.1 related kinase 8 Other,NKF I,SK65 SBK 0,SBK 388228 - 16pl1.2 SH3-binding domain kinase 1 Other,NKF1,SK58 SgK069 1,SgK069 646643 Other,NKF2,SK45 PINKI 6,PINK1 65018 - 1p36 PTEN induced putative kinase I Other,NKF3,SK64 SgK269 9,SgK269 79834 - 15q24.3 KIAA2002 protein Other,NKF3,SK64 hypothetical protein SgK223 3,SgK223 157285 - 8p23.1 DKFZp761PO423 Other,NKF4,SK49 CLIKI 3,CLIKI 140901 - 20p13 serine/threonine kinase 35 Other,NKF4,SK45 CLIKI L 2,CLIKIL 149420 - lp36.11 PDLIM1 interacting kinase I like Other,NKF5,SK69 SgK307 9,SgK307 56155 - 17q23.2 testis expressed sequence 14 Other,NRBP,SK47 NRBP1 9,NRBP1 29959 - 2 p23 nuclear receptor binding protein Other,NRBP,SK52 NRBP2 0,NRBP2 340371 - 8q24.3 nuclear receptor binding protein 2 Other,Other- ribonuclease L (2',5' Unique,SK729,RN oligoisoadenylate synthetase RNAseL AseL 6041 - lq25 dependent) Other,Other Unique,SK652,Sg SgK396 K396 56164 - 7p15.3 serine/threonine kinase 31 Other,Other Unique,SK628,Sg SgK196 K196 84197 - 8p 1.21 hypothetical protein FLJ23356 Other,PEK,GCN2, GCN2 SK490,GCN2 440275 - 15q 15.1 similar to GCN2 elF2alpha kinase Other,PEK,SK496, eukaryotic translation initiation HRI HRI 27102 - 7p22 factor 2-alpha kinase I 80 WO 2009/088408 PCT/US2008/012369 Map Location ID Kinase Gene Entrez (cytogenetic or Name Family Gene ID enzyme ID genetic location) Descriptive Name (or default name) Other,PEK,PEK,S eukaryotic translation initiation PEK K281,PEK 9451 - 2pl2 factor 2-alpha kinase 3 Other,PEK,SK 119, eukaryotic translation initiation PKR PKR 5610 - 2p22-p21 factor 2-alpha kinase 2 Other,PLK,SK315, PLK1 PLKI 5347 - 16p12.1 polo-like kinase 1 (Drosophila) Other,PLK,SK316, PLK3 PLK3 1263 - 1p34.1 polo-like kinase 3 (Drosophila) Other,PLK,SK353, PLK2 PLK2 10769 - 5ql2.1-ql3.2 polo-like kinase 2 (Drosophila) Other,PLK,SK341, PLK4 PLK4 10733 - 4q27-q28 polo-like kinase 4 (Drosophila) Other,SCYI,SK47 SCYL2 5,SCYL2 55681 - 12q23.1 SCY I -like 2 (S. cerevisiae) Other,SCYI,SK46 SCYL3 8,SCYL3 57147 - 1q24.2 ezrin-binding partner PACE-I Other,SCYI,SK45 SCYLI 4,SCYLI 57410 - 1 lql3 SCYI-like I (S. cerevisiae) Other,Other Unique,SK521,Sg chromosome 9 open reading frame SgK071 K071 169436 - 9q34.2 96 Other,Other Unique,SK460,Sg SgK493 K493 91461 - 2p21 hypothetical protein BC007901 Other,Other Unique,SK516,Sg receptor interacting protein kinase SgK496 K496 25778 - 1q32.1 5 Other,Slob,SK528, PX domain containing Slob Slob 54899 - 3p14.3 serine/threonine kinase Other,TBCK,SK66 TBCK 4,TBCK 93627 - 4q24 hypothetical protein MGC16169 Other,TLK,SK373, TLKI TLK1 9874 - 2q3 1.1 tousled-like kinase I Other,TLK,SK374, TLK2 TLK2 11011 - 17q23 tousled-like kinase 2 Other,TOPK,SK52 PBK 9,PBK 55872 - 8p21.2 PDZ binding kinase Other,TTK,SK383, TTK TTK 7272 - 6ql3-q21 TTK protein kinase Other,ULK,SK199 serine/threonine kinase 36 (fused Fused ,Fused 27148 - 2q35 homolog, Drosophila) Other,ULK,SK387 ULKI ,ULKI 8408 - 12q24.3 unc-51-like kinase I (C. elegans) Other,ULK,SK388 ULK2 ,ULK2 9706 - 17p 11.2 unc-5 1-like kinase 2 (C. elegans) Other,ULK,SK450 ULK3 ,ULK3 25989 - 15q24.1 unc-5 1-like kinase 3 (C. elegans) Other,ULK,SK457 ULK4 ,ULK4 54986 - 3p22.1 unc-5 1-like kinase 4 (C. elegans) Other,VPS15,SK2 phosphoinositide-3-kinase, PIK3R4 62,PIK3R4 30849 - 3q21.3 regulatory subunit 4, p150 Other,WEE,SK391 Weel ,Wee] 7465 - IIpI5.3-p15.1 WEEI homolog (S. pombe) PKMYTI Other,WEE,SK248 9088 - 16p3.3 protein kinase, membrane 81 WO 2009/088408 PCT/US2008/012369 Map Location ID Kinase Gene Entrez (cytogenetic or Name Family Gene ID enzyme ID genetic location) Descriptive Name (or default name) ,MYTI associated tyrosine/threonine 1 Other,WEE,SK723 WeelB ,WeelB 285962 - 7q34 hypothetical protein FLJ40852 Other,Wnk,SK508, WNK lysine deficient protein Wnkl Wnkl 65125 - 12pl3.3 kinase I Other,Wnk,SK588, WNK lysine deficient protein Wnk4 Wnk4 65266 - 17q21-q22 kinase 4 Other,Wnk,SK641, WNK lysine deficient protein Wnk3 Wnk3 65267 - xpl 1.23-pi 1.21 kinase 3 Other,Wnk,SKO16, WNK lysine deficient protein Wnk2 Wnk2 65268 - 9q22.3 kinase 2 RGC,RGC,SK171, guanylate cyclase 2C (heat stable HSER HSER 2984 - 12p]2 enterotoxin receptor) RGC,RGC,SK099, CYGF CYGF 2986 - xq22 guanylate cyclase 2F, retinal RGC,RGC,SK097, guanylate cyclase 2D, membrane CYGD CYGD 3000 - 17p]3.1 (retina-specific) natriuretic peptide receptor A/guanylate cyclase A RGC,RGC,SK034, (atrionatriuretic peptide receptor ANPa ANPa 4881 - Iq21-q22 A) natriuretic peptide receptor RGC,RGC,SK035, B/guanylate cyclase B ANPb ANPb 4882 - 9p21-pl2 (atrionatriuretic peptide receptor B) STE,STEI 1,SK22 mitogen-activated protein kinase MAP3K5 5,MAP3K5 4217 - 6q22.33 kinase kinase 5 STE,STEI 1,SK50 mitogen-activated protein kinase MAP3K6 3,MAP3K6 9064 - 1p36.1 I kinase kinase 6 STE,STE11,SK68 mitogen-activated protein kinase MAP3K7 1,MAP3K7 389840 - xp22.12 kinase kinase 15 STE,STE l1,SK22 mitogen-activated protein kinase MAP3KI 1,MAP3KI 4214 - 5q11.2 kinase kinase I STE,STEl 1,SK57 MAP3K8 3,MAP3K8 80122 - 2q21.3 hypothetical protein FLJ23074 STE,STEI 1,SK22 mitogen-activated protein kinase MAP3K3 3,MAP3K3 4215 - 17923.3 kinase kinase 3 STE,STEl I,SK22 mitogen-activated protein kinase MAP3K2 2,MAP3K2 10746 - 2ql4.3 kinase kinase 2 STE,STE 11 ,SK22 mitogen-activated protein kinase MAP3K4 4,MAP3K4 4216 - 6q26 kinase kinase 4 STE,STE20,FRAY OXSR1 ,SK428,OSR1 9943 - 3p22-p21.3 oxidative-stress responsive 1 STE,STE20,FRAY serine threonine kinase 39 STLK3 ,SK432,STLK3 27347 - 2q24.3 (STE20/SPSI homolog, yeast) STE,STE20,KHS, mitogen-activated protein kinase MA P4K2 SK048,GCK 5871 - 113 kinase kinase kinase 2 STE,STE20,KHS, mitogen-activated protein kinase KHS2 SK427,KHS2 8491 - 2 p 2 2.1 kinase kinase kinase 3 STE,STE20,KHS, mitogen-activated protein kinase KHSI SK 191,KHS1 11183 - 14q I 1.2-q21 kinase kinase kinase 5 STE,STE20,KHS, mitogen-activated protein kinase HPK1 SK170,HPKI 11184 - 19q13.1-q13.4 kinase kinase kinase 1 STE,STE20,MSN, mitogen-activated protein kinase HGK SK437,ZCI 9448 - 2qI 1.2-912 kinase kinase kinase 4 82 WO 2009/088408 PCT/US2008/012369 Map Location ID Kinase Gene Entrez (cytogenetic or Name Family Gene ID enzyme ID genetic location) Descriptive Name (or default name) STE,STE20,MSN, TRAF2 and NCK interacting TNIK SK438,ZC2 23043 - 3q26.2 kinase STE,STE20,MSN, NRK SK440,ZC4 203447 - xq22.3 Nik related kinase STE,STE20,MSN, MINK SK439,ZC3 50488 - 17p13.2 misshapen-like kinase 1 (zebrafish) STE,STE20,MST, serine/threonine kinase 3 (STE20 MST2 SK245,MST2 6788 - 8q22.2 homolog, yeast) STE,STE20,MST, MST1 SK244,MST1 6789 - 20ql 1.2-q13.2 serine/threonine kinase 4 STE,STE20,NinaC MYO3A ,SK636,MYO3A 53904 - lop] 1.1 myosin IlIlA STE,STE20,NinaC MYO3B ,SK583,MYO3B 140469 - 2q31.1-q31.2 myosin IIIB STE,STE20,PAKA p21 /Cdc42/Rac 1-activated kinase I PAKI ,SK267,PAKI 5058 - 1 lql3-g4 (STE20 homolog, yeast) STE,STE20,PAKA PAK2 ,SK268,PAK2 5062 2.7.11.1 3q29 p21 (CDKN I A)-activated kinase 2 STE,STE20,PAKA PAK3 ,SK269,PAK3 5063 - xq22.3-q23 p21 (CDKN1A)-activated kinase 3 STE,STE20,PAKB PAK4 ,SK430,PAK4 10298 - 19313.2 p21(CDKN1A)-activated kinase 4 STE,STE20,PAKB PAK6 ,SK429,PAK6 56924 - 15914 p21(CDKN1A)-activated kinase 6 STE,STE20,PAKB PAK5 ,SK510,PAK5 57144 - 20p12 p21(CDKN1A)-activated kinase 7 STE,STE20,SLK,S LOK K426,LOK 6793 - 5q35.1 serine/threonine kinase 10 STE,STE20,SLK,S SLK K348,SLK 9748 - 10q25.1 STE20-like kinase (yeast) amyotrophic lateral sclerosis 2 STE,STE20,STLK (juvenile) chromosome region, STLK6 ,SK434,STLK6 55437 - 2q33-q34 candidate 2 STE,STE20,STLK STLK5 ,SK433,STLK5 92335 - 17q23.3 protein kinase LYK5 STE,STE20,TAO, TAO2 SK362,TA02 9344 - 16p1l.

2 TAO kinase 2 STE,STE20,TAO, TAO3 SK435,TAO3 51347 - 12q TAO kinase 3 STE,STE20,TAO, TAOI SK436,TAOI 57551 - 17q11.2 TAO kinase I STE,STE20,YSK, serine/threonine kinase 24 (STE20 MST3 SK246,MST3 8428 - 13q31.2-q32.3 homolog, yeast) STE,STE20,YSK, serine/threonine kinase 25 (STE20 YSKI SK395,YSKI 10494 - 2q37.3 homolog, yeast) STE,STE20,YSK, MST4 SK431,MST4 51765 - xq26.2 Mst3 and SOKI-related kinase STE,STE7,SK217, mitogen-activated protein kinase MAP2KI MAP2KI 5604 2.7.12.2 15q22.l-q22.33 kinase 1 STE,STE7,SK218, mitogen-activated protein kinase MAP2K2 MAP2K2 5605 2.7.12.2 19p 13.3 kinase 2 STE,STE7,SK238, mitogen-activated protein kinase MAP2K3 MAP2K3 5606 - 17q 11.2 kinase 3 MAP2K6 STE,STE7,SK220, 5608 - 17q24.3 mitogen-activated protein kinase 83 WO 2009/088408 PCT/US2008/012369 Map Location ID Kinase Gene Entrez (cytogenetic or Name Family | Gene ID enzme ID genetic location) Descriptive Name (or default name) MAP2K6 kinase 6 STE,STE7,SK239, mitogen-activated protein kinase MAP2K4 MAP2K4 6416 2.7.12.2 17p l.2 kinase 4 STE,STE7,SK219, mitogen-activated protein kinase MAP2K5 MAP2K5 5607 - 15923 kinase 5 STE,STE7,SK230, mitogen-activated protein kinase MAP2K7 MAP2K7 5609 2.7.12.2 1 9 pl3.3-p13.2 kinase 7

STE,STE

Unique,SK093,CO mitogen-activated protein kinase COT T 1326 - 1Op 11.23 kinase kinase 8

STE,STE

Unique,SK253,NI mitogen-activated protein kinase NIK K 9020 - 17q21 kinase kinase 14 TK,Abl,SK006,AB v-abl Abelson murine leukemia ABLI L 25 - 9q34.1 viral oncogene homolog I v-abl Abelson murine leukemia TK,Abl,SK037,AR viral oncogene homolog 2 (arg, ABL2 G 27 - Iq24-q25 Abelson-related gene) TK,Ack,SK009,A ACK CK 10188 - 3q29 tyrosine kinase, non-receptor, 2 TK,Ack,SK375,T TNK1 NKI 8711 - 17p13.1 tyrosine kinase, non-receptor, I TK,Alk,SK024,AL ALK K 238 - 2p23 anaplastic lymphoma kinase (Ki-1) TK,Alk,SK209,LT LTK K 4058 2.7.1.112 15ql5.1-q21.1 leukocyte tyrosine kinase TK,Axl,SK044,A AXL XL 558 2.7.1.112 19q13.1 AXL receptor tyrosine kinase TK,Axl,SK386,TY TYRO3 R03 7301 2.7.1.112 I Sq15.1-q21.1 TYRO3 protein tyrosine kinase TK,Axl,SK226,M c-mer proto-oncogene tyrosine MER ER 10461 - 2ql4.1 kinase TK,CCK4,SK411, CCK4 CCK4 5754 2.7.1.112 6p21.1-p12.2 PTK7 protein tyrosine kinase 7 TK,Csk,SK095,CS CSK K 1445 2.7.10.1 15q23-q25 c-src tyrosine kinase TK,Csk,SK418,CT megakaryocyte-associated tyrosine CTK K 4145 - 19pl3.3 kinase TK,DDR,SK400,D discoidin domain receptor family, DDRI DRI 780 2.7.1.112 6p2I.3 member 1 TK,DDR,SK41 0,D discoidin domain receptor family, DDR2 DR2 4921 2.7.1.112 1q12-q23 member 2 epidermal growth factor receptor TK,EGFR,SK118, (erythroblastic leukemia viral (v EGFR EGFR 1956 - 7p12 erb-b) oncogene homolog, avian) v-erb-b2 erythroblastic leukemia viral oncogene homolog 2, TK,EGFR,SK166, neuro/glioblastoma derived ErbB2 HER2 2064 - 17q21.1 oncogene homolog (avian) TK,EGFR,SK167, v-erb-b2 erythroblastic leukemia ErbB3 HER3 2065 - 12q13 viral oncogene homolog 3 (avian) TK,EGFR,SK168, v-erb-a erythroblastic leukemia ErbB4 HER4 2066 - 2q33.3-q34 viral oncogene homolog 4 (avian) EphA2 TK,Eph,SK122,Ep 1969 2.7.1.112 1p36 EPH receptorA2 84 WO 2009/088408 PCT/US2008/012369 Map Location ID Kinase Gene Entrez (cytogenetic or Name Family Gene ID enzyme ID genetic location) Descriptive Name (or default name) hA2 TK,Eph,SK121,Ep EphA1 hAl 2041 2.7.1.112 7q34 EPH receptor Al TK,Eph,SK123,Ep EphA3 hA3 2042 2.7.1.112 3pl 1.2 EPH receptor A3 TK,Eph,SK124,Ep EphA4 hA4 2043 2.7.1.112 2q36.1 EPH receptor A4 TK,Eph,SK125,Ep EphA5 hA5 2044 - 4q13.1 EPH receptor A5 TK,Eph,SK416,Ep EphA7 hA7 2045 - 6q16.1 EPH receptor A7 TK,Eph,SK126,Ep EphA8 hA8 2046 2.7.1.112 lp36.12 EPH receptor A8 TK,Eph,SK127,Ep EphBl hB1 2047 - 3q21-q23 EPH receptor BI TK,Eph,SK128,Ep EphB2 hB2 2048 2.7.1.112 Ip36.1-p35 EPH receptor B2 TK,Eph,SK129,Ep EphB3 hB3 2049 - 3q21-qter EPH receptor B3 TK,Eph,SK130,Ep EphB4 hB4 2050 - 7q22 EPH receptor B4 TK,Eph,SK132,Ep EphB6 hB6 2051 - 7q33-q35 EPH receptor B6 TK,Eph,SK627,Ep EphA10 hAlO 284656 - Ip3 4 .3 EPH receptor A10 TK,Eph,SK646,Ep EphA6 hA6 285220 - 3q11.2 EPH receptor A6 TK,FAK,SK424,P PTK2B protein tyrosine kinase 2 PYK2 YK2 2185 - 8p21.1 beta TK,FAK,SK138,F FAK AK 5747 2.7.1.112 8q24-qter PTK2 protein tyrosine kinase 2 TK,Fer,SK140,FE fer (fps/fes related) tyrosine kinase FER R 2241 2.7.1.112 5q21 (phosphoprotein NCP94) TK,Fer,SK142,FE FES S 2242 - 15926.1 feline sarcoma oncogene fibroblast growth factor receptor 1 TK,FGFR,SK143, (fins-related tyrosine kinase 2, FGFR1 FGFR1 2260 2.7.1.112 8p 1.2-p11.1 Pfeiffer syndrome) fibroblast growth factor receptor 3 TK,FGFR,SK145, (achondroplasia, thanatophoric FGFR3 FGFR3 2261 - 4p16.3 dwarfism) fibroblast growth factor receptor 2 (bacteria-expressed kinase, keratinocyte growth factor receptor, craniofacial dysostosis 1, Crouzon syndrome, Pfeiffer TK,FGFR,SK144, syndrome, Jackson-Weiss FGFR2 FGFR2 2263 - 10926 syndrome) TKFGFR,SK147, FGFR4 FGFR4 2264 - 5935.1-qter fibroblast growth factor receptor 4 TK,lnsR,SK174,I insulin-like growth factor I IGFIR GFIR 3480 - 15926.3 receptor TK,InsR,SK178,1 INSR NSR 3643 - 19p13.3-p13.2 insulin receptor 85 WO 2009/088408 PCT/US2008/012369 Map Location ID Kinase Gene Entrez (cytogenetic or Name Family Gene ID enzyme ID genetic location) Descriptive Name (or default name) TK,InsR,SK183,IR IRR R 3645 - 1q21-q23 insulin receptor-related receptor TK,JakA,SK185,J Janus kinase I (a protein tyrosine JAKI AKI 3716 2.7.1.112 1p32.3-p31.3 kinase) TK,JakA,SK186,J Janus kinase 2 (a protein tyrosine JAK2 AK2 3717 2.7.1.112 9p24 kinase) TK,JakA,SKI 87,J Janus kinase 3 (a protein tyrosine JAK3 AK3 3718 - 19p13.1 kinase, leukocyte) TK,JakA,SK385,T TYK2 YK2 7297 2.7.1.112 1 9 pl 3

.

2 tyrosine kinase 2 TK,Lmr,SK4I3,L apoptosis-associated tyrosine LMRI MR1 9625 - 17q25.3 kinase TK,Lmr,SK414,L LMR2 MR2 22853 - 7q21.3 lemur tyrosine kinase 2 TK,Lmr,SK415,L LMR3 MR3 114783 - 19q13.32 lemur tyrosine kinase 3 TK,Met,SK227,M met proto-oncogene (hepatocyte MET ET 4233 - 7931 growth factor receptor) TK,Met,SK332,R macrophage stimulating I receptor RON ON 4486 - 3p21.3 (c-met-related tyrosine kinase) TK,Musk,SK247, muscle, skeletal, receptor tyrosine MUSK MUSK 4593 - 9q3I.3-q32 kinase colony stimulating factor I receptor, formerly McDonough TK,PDGFR,SKO9 feline sarcoma viral (v-flis) FMS 4,FMS 1436 - 5933-935 oncogene homolog TK,PDGFR,SK14 FLT3 9,FLT3 2322 2.7.1.112 13q12 fins-related tyrosine kinase 3 TK,PDGFR,SK20 v-kit Hardy-Zuckerman 4 feline KIT 1,KIT 3815 - 4q] 1-q12 sarcoma viral oncogene homolog TK,PDGFR,SK27 platelet-derived growth factor PDGFRa 4,PDGFRa 5156 - 4ql I-q13 receptor, alpha polypeptide TK,PDGFR,SK27 platelet-derived growth factor PDGFRb 5,PDGFRb 5159 - 5q3l-q32 receptor, beta polypeptide ret proto-oncogene (multiple endocrine neoplasia and medullary TK,Ret,SK326,RE thyroid carcinoma 1, Hirschsprung RET T 5979 - 10911.2 disease) TK,Ror,SK333,RO receptor tyrosine kinase-like RORI RI 4919 - 1p32-p31 orphan receptor I TK,Ror,SK334,RO receptor tyrosine kinase-like ROR2 R2 4920 - 9q22 orphan receptor 2 TK,Ryk,SK340,R RYK YK 6259 2.7.1.112 3q22 RYK receptor-like tyrosine kinase TK,Sev,SK335,RO v-ros UR2 sarcoma virus oncogene ROS S 6098 - 6q22 homolog I (avian) TK,Src,SK419,FR FRK K 2444 2.7.1.112 6q21-q22.3 fyn-related kinase TK,Src,SK148,FG Gardner-Rasheed feline sarcoma FGR R 2268 - 1 p36.2-p36.1 viral (v-fgr) oncogene homolog TK,Src,SK153,FY FYN oncogene related to SRC, FYN N 2534 - 6q21 FGR, YES TK,Src,SK357,SR v-src sarcoma (Schmidt-Ruppin A SRC C 6714 - 20q12-q13 2) viral oncogene homolog (avian) 86 WO 2009/088408 PCT/US2008/012369 Map Location ID Kinase Gene Entrez (cytogenetic or Name Family Gene ID enzyme ID genetic location) Descriptive Name (or default name) TK,Src,SK393,YE v-yes-I Yamaguchi sarcoma viral YES S 7525 - 18p]1.31-pil1.21 oncogene homolog _ TK,Src,SK049,BL BLK K 640 - 8p23-p22 B lymphoid tyrosine kinase TK,Src,SKI64,HC HCK K 3055 - 20q] l-q12 hemopoietic cell kinase TK,Src,SK206,LC lymphocyte-specific protein LCK K 3932 2.7.1.112 Ip3 4 .3 tyrosine kinase TK,Src,SK210,LY v-yes-I Yamaguchi sarcoma viral LYN N 4067 - 8q 13 related oncogene homolog TK,Src,SK051,BR BRK K 5753 2.7.1.112 20q13.3 PTK6 protein tyrosine kinase 6 src-related kinase lacking C TK,Src,SK425,SR terminal regulatory tyrosine and N SRM M 6725 - 20ql3.33 terminal myristylation sites TK,Syk,SK363,SY SYK K 6850 - 9q22 spleen tyrosine kinase TK,Syk,SK397,ZA zeta-chain (TCR) associated ZAP70 P70 7535 - 2q12 protein kinase 70kDa TK,Tec,SK417,B BMX MX 660 - xp22.2 BMX non-receptor tyrosine kinase TK,Tec,SK052,BT Bruton agammaglobulinemia BTK K 695 2.7.1.112 xq21.33-q22 tyrosine kinase TK,Tec,SK184,IT ITK K 3702 - 5q31-q32 IL2-inducible T-cell kinase TK,Tec,SK366,TE TEC C 7006 - 4p]2 tec protein tyrosine kinase TK,Tec,SK384,TX TXK K 7294 2.7.1.112 4p]2 TXK tyrosine kinase TEK tyrosine kinase, endothelial TK,Tie,SK367,TIE (venous malformations, multiple TIE2 2 7010 - 9p21 cutaneous and mucosal) tyrosine kinase with TK,Tie,SK370,TIE immunoglobulin-like and EGF-like TIE] 1 7075 2.7.1.112 1p34-p33 domains 1

TK,TK

Unique,SK530,Su SuRTK106 RTKI06 55359 - 12p13.2 serine/threonine/tyrosine kinase 1 TK,Trk,SK377,TR neurotrophic tyrosine kinase, TRKA KA 4914 2.7.1.112 Ig21-q22 receptor, type I TK,Trk,SK378,TR neurotrophic tyrosine kinase, TRKB KB 4915 2.7.1.112 9922.1 receptor, type 2 TK,Trk,SK379,TR neurotrophic tyrosine kinase, TRKC KC 4916 2.7.1.112 15q25 receptor, type 3 fins-related tyrosine kinase I (vascular endothelial growth TK,VEGFR,SK15 factor/vascular permeability factor FLTI 0,FLTI 2321 2.7.1.112 13q12 receptor) TK,VEGFR,SK15 FLT4 IFLT4 2324 2.7.1.112 5934-q35 fins-related tyrosine kinase 4 TK,VEGFR,SK40 kinase insert domain receptor (a KDR 2,KDR 3791 2.7.1.112 4q 1-ql2 type III receptor tyrosine kinase) TKL,IRAK,SK179 interleukin-I receptor-associated IRAKI ,IRAKI 3654 - xq28 kinase I 87 WO 2009/088408 PCT/US2008/012369 Map Location ID Kinase Gene Entrez (cytogenetic or Name Family Gene ID enzyme ID genetic location) Descriptive Name (or default name) TKL,IRAK,SK180 interleukin-1 receptor-associated IRAK2 ,IRAK2 3656 - 3p25.3 kinase 2 TKL,IRAK,SKI81 interleukin-I receptor-associated IRAK3 ,IRAK3 11213 - 12q14.3 kinase 3 TKL,IRAK,SK257 interleukin-I receptor-associated IRAK4 ,IRAK4 51135 - 12q12 kinase 4 TKL,LISK,LIMK, LIMKI SK412,LIMKI 3984 - 7q 11.23 LIM domain kinase I TKL,LISK,LIMK, LIMK2 SK207,LIMK2 3985 - 22q 12.2 LIM domain kinase 2 TKL,LISK,TESK, EC, TESKI SK368,TESKI 7016 2.7.12.1 9p13 testis-specific kinase I TKL,LISK,TESK, TESK2 SK532,TESK2 10420 - lp32 testis-specific kinase 2 TKL,LRRK,SK69 LRRK1 8,LRRKI 79705 - 15q26.3 leucine-rich repeat kinase I TKL,LRRK,SK69 LRRK2 0,LRRK2 120892 - 12q12 leucine-rich repeat kinase 2 TKL,MLK,HH498 HH498 ,SK494,HH498 51086 - Ip3 1.1 TNNI3 interacting kinase TKL,MLK,ILK,S ILK K177,ILK 3611 - 1 lpI5.5-p15.

4 integrin-linked kinase TKL,MLK,LZK,S mitogen-activated protein kinase DLK K110,DLK 7786 - 12q13 kinase kinase 12 TKL,MLK,LZK,S mitogen-activated protein kinase LZK K398,LZK 9175 - 3q27 kinase kinase 13 TKL,MLK,MLK,S mitogen-activated protein kinase MLKI K232,MLK1 4293 - 14q24.3-q31 kinase kinase 9 TKL,MLK,MLK,S mitogen-activated protein kinase MLK2 K233,MLK2 4294 - 19ql3.2 kinase kinase 10 TKL,MLK,MLK,S mitogen-activated protein kinase MLK3 K356,MLK3 4296 2.7.10.1 1 lql3.1-q13.3 kinase kinase 11 TKL,MLK,MLK,S MLK4 K691,MLK4 84451 - 1q42 mixed lineage kinase 4 TKL,MLK,TAK1, mitogen-activated protein kinase TAKI SK364,TAKI 6885 - 6q16.1-q16.3 kinase kinase 7 TKL,MLK,MLK,S sterile alpha motif and leucine ZAK K504,ZAK 51776 - 2q24.2 zipper containing kinase AZK TKL,RAF,SK205, KSR1 KSRI 8844 - 17q 11.2 kinase suppressor of ras TKL,RAF,SK605, KSR2 KSR2 283455 - 12q24.22-q24.23 kinase suppressor of ras 2 TKL,RAF,SK036, v-raf marine sarcoma 3611 viral ARAF ARAF 369 - xpl 1.4-p 11.2 oncogene homolog TKL,RAF,SK050, v-raf marine sarcoma viral BRAF BRAF 673 - 7q34 oncogene homolog BI TKL,RAF,SK324, v-raf-I marine leukemia viral RAF] RAFI 5894 - 3p25 oncogene homolog I TKL,RIPK,SK328, receptor (TNFRSF)-interacting RIPKI RIPKI 8737 - 6p 2 5.

2 serine-threonine kinase I TKL,RIPK,SK329, receptor-interacting serine RIPK2 RIPK2 8767 - 8q21 threonine kinase 2 TKL,RIPK,SK330, receptor-interacting seine RIPK3 RIPK3 11035 - 14q11.2 threonine kinase 3 88 WO 2009/088408 PCT/US2008/012369 Map Location ID Kinase Gene Entrez (cytogenetic or Name Family Gene ID enzyme ID genetic location) Descriptive Name (or default name) TKL,RIPK,SK546, receptor-interacting serine ANKRD3 ANKRD3 54101 - 21q22.3 threonine kinase 4 TKL,RIPK,SK658, ankyrin repeat and kinase domain SgK288 SgK288 255239 - 11 q23.2 containing I TKL,STKR,TypeI ALK2 ,SK026,ALK2 90 - 2q23-q24 activin A receptor, type I TKLSTKR,Type1 ALK4 ,SK028,ALK4 91 - 12q13 activin A receptor, type IB TKL,STKR,Typel ALKI ,SK025,ALK1 94 - 12q1 1-ql4 activin A receptor type II-like I TKL,STKR,Typel bone morphogenetic protein BMPR1A ,SK027,BMPRIA 657 - 10q22.3 receptor, type IA TKL,STKR,Typel bone morphogenetic protein BMPRIB ,SK030,BMPRI B 658 - 4q22-q24 receptor, type IB transforming growth factor, beta TKL,STKR,Typel receptor I (activin A receptor type TGFbR1 ,SK029,TGFbRI 7046 - 9922 Il-like kinase, 53kDa) TKL,STKR,Type1 ALK7 ,SK405,ALK7 130399 - 2q24.1 activin A receptor, type IC TKL,STKR,Type2 ACVR2A ,SKO1O,ACTR2 92 - 2q22.2-q23.3 activin A receptor, type II TKL,STKR,Type2 ACTR2B ,SKOI1,ACTR2B 93 - 3p22 activin A receptor, type IlB TKL,STKR,Type2 anti-Mullerian hormone receptor, MISR2 ,SK228,MISR2 269 - 12q13 type II bone morphogenetic protein TKL,STKR,Type2 receptor, type II (serine/threonine BMPR2 ,SK365,BMPR2 659 - 2q33-q34 kinase) TKL,STKR,Type2 transforming growth factor, beta TGFbR2 ,SK369,TGFbR2 7048 - 3p22 receptor II (70/8OkDa)

TKL,TKL

Unique,SK458,ML MLKL KL 197259 - 16q22.3 mixed lineage kinase domain-like ATP-binding cassette, sub-family ABCB10 others 23456 - 1q42 B (MDRIlAP), member 10 ATP-binding cassette, sub-family ABCB8 others 11194 - 7q36 B (MDR/TAP), member 8 ATP-binding cassette, sub-family ABCG1 others 9619 - 21q22.3 G (WHITE), member I ARP2 actin-related protein 2 ACTR2 others 10097 - 2p14 homolog (yeast) ADCY3 others 109 4.6.1.1 2p24-p22 adenylate cyclase 3 ADCY6 others 112 4.6.1.1 12q12-q13 adenylate cyclase 6 ADCY7 others 113 4.6.1.1 16ql2-ql3 adenylate cyclase 7 ADCY8 others 114 4.6.1.1 8q24 adenylate cyclase 8 (brain) ADCY9 others 115 4.6.1.1 16p]3.3 adenylate cyclase 9 ADK others 132 2.7.1.20 10q22 adenosine kinase AK3LI others 205 2.7.4.10 1 p31.3 adenylate kinase 3 aldehyde dehydrogenase 18 family, ALDH18A1 others 5832 - 10q24.3 member Al amyotrophic lateral sclerosis 2 (juvenile) chromosome region, ALS2CRI I others 151254 - 2q33.1 candidate I1 ALS2CRI2 others 130540 - 2933.1 amyotrophic lateral sclerosis 2 89 WO 2009/088408 PCT/US2008/012369 Map Location ID Kinase Gene Entrez (cytogenetic or Name Family Gene ID enzyme ID genetic location) Descriptive Name (or default name) (juvenile) chromosome region, candidate 12 amyotrophic lateral sclerosis 2 (juvenile) chromosome region, ALS2CRI3 others 150864 - 2q33.2 candidate 13 amyotrophic lateral sclerosis 2 (juvenile) chromosome region, ICAIL others 130026 - 2q33.2 candidate 15 amyotrophic lateral sclerosis 2 (juvenile) chromosome region, PARD3B others 117583 - 2q33.3 candidate 19 amyotrophic lateral sclerosis 2 (juvenile) chromosome region, TRAK2 others 66008 - 2q33 candidate 3 amyotrophic lateral sclerosis 2 (juvenile) chromosome region, ALS2CR4 others 65062 - 2q33.2 candidate 4 amyotrophic lateral sclerosis 2 (juvenile) chromosome region, ALS2CR8 others 79800 - 2q33.2 candidate 8 DBF4 others 10926 - 7q21.3 activator of S phase kinase MAGIl others 9223 - 3p]4.1 BAII-associated protein 1 BUB3 budding uninhibited by BUB3 others 9184 - 10q26 benzimidazoles 3 homolog (yeast) chromosome 9 open reading frame IPPK others 64768 - 9q21.33-q22.31 12 caspase recruitment domain family, CARD 11 others 84433 - 7p22 member 11 caspase recruitment domain family, CARD14 others 79092 - 17q25 member 14 CARKL others 23729 2.7.1.14 l7p13 carbohydrate kinase-like CHKB others 1120 - 22q13.33 choline kinase beta cyclin-dependent kinase 2 CINP others 51550 - 14932.32 interacting protein CKB others 1152 2.7.3.2 14q32 creatine kinase, brain CKM others 1158 2.7.3.2 19q13.2-q13.3 creatine kinase, muscle CKMTIA others 548596 2.7.3.2 15q15 creatine kinase, mitochondrial IA creatine kinase, mitochondrial 1 CKMTIB others 1159 2.7.3.2 159]5 (ubiquitous) creatine kinase, mitochondrial 2 CKMT2 others 1160 2.7.3.2 5q13.3 (sarcomeric) CDC28 protein kinase regulatory CKSIB others 1163 - lq21.2 subunit lB CDC28 protein kinase regulatory CKS2 others 1164 - 9q22 subunit 2 CMPK others 51727 2.7.4.14 lp3 4 .1-p33 UMP-CMP kinase connector enhancer of kinase CNKSR2 others 22866 - xp22.12 suppressor of Ras 2 COASY others 80347 2.7.7.3 17q12-q21 Coenzyme A synthase collagen, type IV, alpha 3 (Goodpasture antigen) binding COL4A3BP others 10087 - 5q]3.3 protein coatomer protein complex, subunit COPB1 others 1315 - lIpl5.2 beta 90 WO 2009/088408 PCT/US2008/012369 Map Location ID Kinase Gene Entrez (cytogenetic or Name Family Gene ID e me genetic location) Descriptive Name (or default name) coatomer protein complex, subunit COPB2 others 9276 - 3q23 beta 2 (beta prime) DCK others 1633 2.7.1.74 4ql3.3-q21.1 deoxycytidine kinase DEAD (Asp-Glu-Ala-Asp) box DDX1 others 1653 - 2p24 polypeptide I DGKA others 1606 2.7.1.107 12q13.3 diacylglycerol kinase, alpha 80kDa DGKB others 1607 2.7.1.107 7p21.3 diacylglycerol kinase, beta 90kDa diacylglycerol kinase, delta DGKD others 8527 - 2q37.1 130kDa diacylglycerol kinase, epsilon DGKE others 8526 - 17q22 64kDa DGKH others 160851 - 13q14.11 diacylglycerol kinase, eta diacylglycerol kinase, gamma DGKG others 1608 2.7.1.107 3q27-q28 90kDa DGKI others 9162 - 7q32.3-q33 diacylglycerol kinase, iota diacylglycerol kinase, theta DGKQ others 1609 - 4p16.3 I 10kDa DGKZ others 8525 - 1 IpI 1.2 diacylglycerol kinase, zeta 104kDa DGUOK others 1716 2.7.1.113 2 p]3 deoxyguanosine kinase discs, large homolog 1 DLGI others 1739 - 3q29 (Drosophila) discs, large homolog 2, chapsyn DLG2 others 1740 - 11q21 110 (Drosophila) discs, large homolog 3 DLG3 others 1741 - xq13.1 (neuroendocrine-dig, Drosophila) discs, large homolog 4 DLG4 others 1742 - 17p13.1 (Drosophila) discs, large homolog 5 DLG5 others 9231 - 10q23 (Drosophila) deoxythymidylate kinase DTYMK others 1841 2.7.4.9 2q37.3 (thymidylate kinase) ETNK1 others 55500 - l2pl2.1 ethanolamine kinase I EVIl others 2122 - 3q24-q28 ecotropic viral integration site I ETNK2 others 55224 - lq32.1 ethanolamine kinase 2 OXSM others 54995 2.3.1.41 3p24.2 hypothetical protein FLJ20604 FN3K others 64122 - 17q25.3 fructosamine 3 kinase FXN others 2395 - 9ql3-q21.1 frataxin GALK2 others 2585 2.7.1.6 15q21.1 galactokinase 2 GK others 2710 2.7.1.30 xp21.3 glycerol kinase GK2 others 2712 - 4q13 glycerol kinase 2 glucosamine (UDP-N-acetyl)-2 epimerase/N-acetylmannosamine GNE others 10020 - 9p13.2 kinase guanylate cyclase 1, soluble, alpha GUCYIA2 others 2977 4.6.1.2 11 q21-q22 2 guanylate cyclase 1, soluble, alpha GUCYIA3 others 2982 4.6.1.2 4q31.1-q31.2 3 GUCY I B3 others 2983 4.6.1.2 4q31.3-q33 guanylate cyclase 1, soluble, beta 3 GUKI others 2987 2.7.4.8 1q32-q4l guanylate kinase I IHPK2 others 51447 - 3p21.31 inositol hexaphosphate kinase 2 inhibitor of kappa light polypeptide gene enhancer in B-cells, kinase IKBKAP others 8518 - 9q3I complex-associated protein CNKSRI others 10256 - 1p36.11 connector enhancer of kinase 91 WO 2009/088408 PCT/US2008/012369 Map Location ID Kinase Gene Entrez (cytogenetic or Name Family Gene ID enzyme ID genetic location) Descriptive Name (or default name) suppressor of Ras I MAP3K12 binding inhibitory MBIP others 51562 - 14913.3 protein I potassium voltage-gated channel, KCNEI others 3753 - 21q22.12 Isk-related family, member 1 membrane protein, palmitoylated MPPI others 4354 - xq28 1, 55kDa membrane protein, palmitoylated 2 (MAGUK p55 subfamily member MPP2 others 4355 - 17q12-q21 2) membrane protein, palmitoylated 3 (MAGUK p55 subfamily member MPP3 others 4356 - 17ql2-q21 3) membrane protein, palmitoylated 4 (MAGUK p55 subfamily member MPP4 others 58538 - 2q33.2 4) membrane protein, palmitoylated 5 (MAGUK p55 subfamily member MPP5 others 64398 - 14q23.3 5) membrane protein, palmitoylated 6 (MAGUK p55 subfamily member MPP6 others 51678 - 7pl5 6) membrane protein, palmitoylated 7 (MAGUK p55 subfamily member MPP7 others 143098 - lop 1 2

.

1 7) mevalonate kinase (mevalonic MVK others 4598 2.7.1.36 12q24 aciduria) NAGK others 55577 2.7.1.59 2pl3.3 N-acetylglucosamine kinase NADH dehydrogenase (ubiquinone) I alpha subcomplex, NDUFAIO others 4705 - 2q37.3 10, 42kDa non-metastatic cells 1, protein NMEI others 4830 - 17q21.3 (NM23A) expressed in non-metastatic cells 2, protein NME2 others 4831 - 17q21.3 (NM23B) expressed in non-metastatic cells 3, protein NME3 others 4832 - 16913 expressed in non-metastatic cells 4, protein NME4 others 4833 - 16pl3.3 expressed in non-metastatic cells 5, protein expressed in (nucleoside NME5 others 8382 - 5q31 diphosphate kinase) non-metastatic cells 6, protein expressed in (nucleoside NME6 others 10201 - 3p 2 1 diphosphate kinase) non-metastatic cells 7, protein expressed in (nucleoside NME7 others 29922 - 1q24 diphosphate kinase) natriuretic peptide receptor C/guanylate cyclase C NPR3 others 4883 - 5p14-p13 (atrionatriuretic peptide receptor C) NSF others 4905 - 17q21 N-ethylmaleimide-sensitive factor nucleotide binding protein I (MinD NUBPI others 4682 - 16p13.13 homolog, E. coli) 92 WO 2009/088408 PCT/US2008/012369 Map Location ID Kinase Gene Entrez (cytogenetic or Name Family Gene ID enzyme ID genetic location) Descriptive Name (or default name) nucleotide binding protein 2 (MinD NUBP2 others 10101 - l6p13.3 homolog, E. coli) protein kinase C and casein kinase PACSIN1 others 29993 - 6p21.3 substrate in neurons 1 PANKI others 53354 - 10q23.31 pantothenate kinase 1 pantothenate kinase 2 PANK2 others 80025 - 20p13 (Hallervorden-Spatz syndrome) PANK3 others 79646 - 5q34 pantothenate kinase 3 PANK4 others 55229 - lp36.3 2 pantothenate kinase 4 Y-phosphoadenosine 5' PAPSSI others 9061 2.7.7.4, 2.7 4q24 phosphosulfate synthase 1 2.7.7.4, 3'-phosphoadenosine 5' PAPSS2 others 9060 2.7.1.25 10q23-q24 phosphosulfate synthase 2 phosphoenolpyruvate PCKI others 5105 4.1.1.32 20q13.31 carboxykinase 1 (soluble) phosphoenolpyruvate PCK2 others 5106 4.1.1.32 14q 11.2 carboxykinase 2 (mitochondrial) pyridoxal (pyridoxine, vitamin B6) PDXK others 8566 2.7.1.35 21q22.3 kinase PFKL others 5211 2.7.1.11 21q22.3 phosphofructokinase, liver PFKM others 5213 2.7.1.11 12q13.3 phosphofructokinase, muscle PFKP others 5214 2.7.1.11 10p15.3-pl5.2 phosphofructokinase, platelet phosphatidylinositol 4-kinase type PI4K2B others 55300 - 4p15.2 II beta phosphatidylinositol 4-kinase type PI4K2A others 55361 - 10q24 II phosphoinositide-3-kinase, class 2, PIK3C2A others 5286 2.7.1.137 11 lpl5.5-pl4 alpha polypeptide phosphoinositide-3-kinase, class 2, PIK3C2B others 5287 2.7.1.137 1q32 beta polypeptide phosphoinositide-3-kinase, class 2, PIK3C2G . others 5288 2.7.1.137 12p12 gamma polypeptide PIK3C3 others 5289 - 18q12.3 phosphoinositide-3-kinase, class 3 phosphoinositide-3-kinase, PIK3CA others 5290 2.7.1.137 3q26.3 catalytic, alpha polypeptide phosphoinositide-3-kinase, PIK3CB others 5291 2.7.1.137 3q22.3 catalytic, beta polypeptide phosphoinositide-3-kinase, PIK3CD others 5293 - lp36.2 catalytic, delta polypeptide phosphoinositide-3-kinase, PIK3CG others 5294 2.7.1.137 7q22.3 catalytic, gamma polypeptide phosphoinositide-3-kinase, PIK3R2 others 5296 - 19q13.2-q13.4 regulatory subunit 2 (p 85 beta) phosphatidylinositol 4-kinase, PIK4CA others 5297 - 22q 11.21 catalytic, alpha polypeptide phosphatidylinositol 4-kinase, PIK4CB others 5298 - 1q21 catalytic, beta polypeptide phosphatidylinositol-4-phosphate PIP5KIA others 8394 - Iq22-q24 5-kinase, type I, alpha phosphatidylinositol-4-phosphate PIP5K I B others 8395 - 9q13 5-kinase, type 1, beta phosphatidyl inositol-4-phosphate PIP5K2A others 5305 - IlOp12.32 5-kinase, type 11, alpha PIP5K2B others 8396 2.7.1.149 1 7q 12 phosphatidylinositol-4-phosphate 93 WO 2009/088408 PCT/US2008/012369 Map Location ID Kinase Gene Entrez (cytogenetic or Name Family Gene ID enzyme ID genetic location) Descriptive Name (or default name) 5-kinase, type II, beta phosphatidylinositol-4-phosphate PIPSK2C others 79837 - 12q 13.3 5-kinase, type 11, gamma polycystic kidney disease 1 PKD1 others 5310 - 16p13.3 (autosomal dominant) polycystic kidney disease 2 PKD2 others 5311 - 4q21-q23 (autosomal dominant) EXOSC1O others 5394 - 1p36.22 exosome component 10 PMVK others 10654 2.7.4.2 1p13-q23 phosphomevalonate kinase protein kinase, AMP-activated, PRKAG3 others 53632 - 2q35 gamma 3 non-catalytic subunit PRP4 pre-mRNA processing factor PRPF4 others 9128 - 9q31-q33 4 homolog (yeast) phosphoribosyl pyrophosphate PRPSI others 5631 2.4.2.17 xq21-q27 synthetase I phosphoribosyl pyrophosphate PRPS2 others 5634 2.4.2.17 xp22.3-p22.2 synthetase 2 phosphoribosyl pyrophosphate PRPSAPI others 5635 - 17q24-925 synthetase-associated protein I phosphoribosyl pyrophosphate PRPSAP2 others 5636 - 17p l 1.2-p]2 synthetase-associated protein 2 LONPI others 9361 - 19p13.2 protease, serine, 15 TWFI others 5756 - 12q12 PTK9 protein tyrosine kinase 9 PTK9L protein tyrosine kinase 9 TWF2 others 11344 - 3p21.1 like (A6-related protein) protein tyrosine phosphatase, PTPRN others 5798 - 2q35-q36.1 receptor type, N protein tyrosine phosphatase, PTPRT others 11122 - 20ql2-q13 receptor type, T Rap guanine nucleotide exchange RAPGEF4 others 11069 - 2q3 1-q32 factor (GEF) 4 RBM19 others 9904 - 12q24.13-q24.21 RNA binding motif protein 19 RBKS others 64080 2.7.1.15 2p23.3 ribokinase RCE1 homolog, prenyl protein RCE1 others 9986 - I1q13 protease (S. cerevisiae) RECQL5 others 9400 - 17q25.2-q25.3 RecQ protein-like 5 RFK others 55312 - 9q21.13 riboflavin kinase solute carrier family 6 (amino acid SLC6AI4 others 11254 - xq23-q24 transporter), member 14 SPHKI others 8877 - 17q25.2 sphingosine kinase 1 SPHK2 others 56848 - 19qI3.2 sphingosine kinase 2 SEPHSI others 22929 - 10p14 selenophosphate synthetase 1 SEPHS2 others 22928 - 16p 11.2 selenophosphate synthetase 2 mitogen-activated protein kinase MAP3K7IP kinase kinase 7 interacting protein I others 10454 - 22q]3.l 1 mitogen-activated protein kinase MAP3K71P kinase kinase 7 interacting protein 2 others 23118 - 6q25.1-q25.3 2 TAS2R14 others 50840 - 1 2 p13 taste receptor, type 2, member 14 tight junction protein I (zona TJPI others 7082 - 15913 occludens 1) tight junction protein 2 (zona TJP2 others 9414 - 9ql3-q21 occludens 2) 94 WO 2009/088408 PCT/US2008/012369 Map Location ID Kinase Gene Entrez (cytogenetic or Name Family Gene ID enzyme ID genetic location) Descriptive Name (or default name) tight junction protein 3 (zona TJP3 others 27134 - 19p13.3 occludens 3) TKI others 7083 2.7.1.21 l7q23.2-q25.3 thymidine kinase 1, soluble TK2 others 7084 2.7.1.21 16q22-q23.1 thymidine kinase 2, mitochondrial TPKI others 27010 - 7q34-q35 thiamin pyrophosphokinase I thyroid hormone receptor TRIP13 others 9319 - Sp15.33 interactor 13 UCK2 others 7371 2.7.4.- 1 q23 uridine-cytidine kinase 2 UCKLI others 54963 - 20q 13.33 uridine-cytidine kinase 1-like 1 xylulokinase homolog (H. XYLB others 9942 - 3p22-p21.3 influenzae) MAG12 others 9863 - 7q21 atrophin-1 interacting protein 1 ADPGK others 83440 - 15q24.1 ADP-dependent glucokinase AGK others 55750 2.7.1.94 7q34 multiple substrate lipid kinase AKI others 203 2.7.4.3 9q34.1 adenylate kinase 1 AK2 others 204 2.7.4.3 1p34 adenylate kinase 2 AK3 others 50808 - 9p24.1-p24.3 adenylate kinase 3 like 1 AK5 others 26289 - 1p3l adenylate kinase 5 AK7 others 122481 - 14q32.2 adenylate kinase 7 calmodulin 2 (phosphorylase CALM2 others 805 - 2p21 kinase, delta) cyclin-dependent kinase 5, CDK5Rl others 8851 - 17911.2 regulatory subunit 1 (p35) cyclin-dependent kinase 5, CDK5R2 others 8941 - 2q35 regulatory subunit 2 (p39) cyclin-dependent kinase inhibitor 3 (CDK2-associated dual specificity CDKN3 others 1033 - 14q22 phosphatase) CERK others 64781 - 22q13.31 ceramide kinase CERKL others 375298 - 2q31.3 ceramide kinase-like CHKA others 1119 2.7.1.32 1 q13.2 choline kinase alpha DAK others 26007 - I Iq12.2 DKFZP586B1621 protein DCAKD others 79877 - 17q21.31 hypothetical protein FLJ22955 DGKK others 139189 - xp 11.22 similar to CI30007D14 protein DOLK others 22845 - 9q34.1 1 transmembrane protein 15 FASTKD1 others 79675 - 2q31 hypothetical protein FLJ21901 FASTKD2 others 22868 - 2q33.3 KIAA0971 FASTKD3 others 79072 - 5pl5.

3 -pl5.

2 hypothetical protein MGC5297 FASTKD5 others 60493 - 20p13 hypothetical protein FLJ13149 FUK others 197258 2.7.1.52 16q22.1 fucokinase glucokinase (hexokinase 4, 2.7.1.2, maturity onset diabetes of the GCK others 2645 2.7.1.1 7 pl5.3-p15.1 young 2) HKI others 3098 2.7.1.1 10q22 hexokinase I HK2 others 3099 2.7.1.1 2 pl3 hexokinase 2 HK3 others 3101 2.7.1.1 5q35.2 hexokinase 3 (white cell) HKDCI others 80201 - 10q22.1 hypothetical protein FLJ22761 IHPKI others 9807 - 3p 2 1.31 inositol hexaphosphate kinase I IHPK3 others 117283 - 6p 2 1.31 inositol hexaphosphate kinase 3 IPMK others 253430 - 10q21.1 inositol polyphosphate multikinase inositol 1,3,4-triphosphate 5/6 ITPKI others 3705 - 14q31 kinase ITPKA others 3706 2.7.1.- 15q14-q21 inositol 1,4,5-trisphosphate 3 95 WO 2009/088408 PCT/US2008/012369 Map Location ID Kinase Gene Entrez (cytogenetic or Name Family Gene ID enzyme ID genetic location) Descriptive Name (or default name) kinase A inositol 1,4,5-trisphosphate 3 ITPKB others 3707 2.7.1.- 1942.13 kinase B inositol 1,4,5-trisphosphate 3 ITPKC others 80271 - 199 13.1 kinase C NADK others 65220 - 1p36.33-p36.21 NAD kinase PHKB others 5257 2.7.1.38 16q12-q3 phosphorylase kinase, beta phosphatidylinositol-4-phosphate PIP5KIC others 23396 - 19p 13.3 5-kinase, type I, gamma phosphatidylinositol-4-phosphate PIP5KLI others 138429 - 9q34.1 1 5-kinase-like 1 PKLR others 5313 2.7.1.40 1q21 pyruvate kinase, liver and RBC PKM2 others 5315 2.7.1.40 15q22 pyruvate kinase, muscle PLAU others 5328 3.4.21.31 10q24 plasminogen activator, urokinase chromosome 10 open reading PSTK others 118672 - 10q26.13 frame 89 UCKI others 83549 2.7.1.48 9q34.13 uridine-cytidine kinase 1 calmodulin I (phosphorylase CALMI others 801 2.7.1.38 14q24-q31 kinase, delta) calmodulin 3 (phosphorylase CALM3 others 808 - 19q13.2-q 13.3 kinase, delta) CSNK2B others 1460 2.7.1.37 6p21.3 casein kinase 2, beta polypeptide GALKI others 2584 2.7.1.6 17q24 galactokinase I KHK others 3795 2.7.1.3 2p23.3-p23.2 ketohexokinase (fructokinase) membrane-associated guanylate MAGI3 others 260425 - lp12-pl1.2 kinase-related (MAGI-3) 2.7.1.105, 6-phosphofructo-2-kinase/fructose PFKFBI others 5207 3.1.-.- xpl 1.21 2,6-biphosphatase 1 2.7.1.105, 6-phosphofructo-2-kinase/fructose PFKFB2 others 5208 3.1.-.- 1q31 2,6-biphosphatase 2 6-phosphofructo-2-kinase/fructose PFKFB3 others 5209 - Op14-p15 2,6-biphosphatase 3 6-phosphofructo-2-kinase/fructose PFKFB4 others 5210 - 3p22-p2I 2,6-biphosphatase 4 PGKI others 5230 2.7.2.3 xgl3 phosphoglycerate kinase I PGK2 others 5232 - 6p12.3 phosphoglycerate kinase 2 phosphorylase kinase, alpha 1 PHKAl others 5255 2.7.1.38 xql2-q13 (muscle) phosphorylase kinase, alpha 2 PHKA2 others 5256 2.7.1.38 xp22.2-p22.1 (liver) protein kinase, AMP-activated, PRKAB1 others 5564 - 12924.1 beta I non-catalytic subunit protein kinase, AMP-activated, PRKA1B2 others 5565 - 1q21.1 beta 2 non-catalytic subunit protein kinase, AMP-activated, PRKAGI others 5571 - 12ql2-q 14 gamma I non-catalytic subunit protein kinase, AMP-activated, PRKAG2 others 51422 - 7q35-q36 gamma 2 non-catalytic subunit protein kinase, cAMP-dependent, regulatory, type I, alpha (tissue PRKARIA others 5573 2.7.1.37 17q23-q24 specific extinguisher 1) protein kinase, cAMP-dependent, PRKAR2A others 5576 2.7.1.37 3p21.3-p21.2 regulatory, type II, alpha PRKAR2B others 5577 2.7.1.37 7q22 protein kinase, cAMP-dependent, 96 WO 2009/088408 PCT/US2008/012369 Map Location ID Kinase Gene Entrez (cytogenetic or Name Family Gene ID enzyme ID genetic location) Descriptive Name (or default name) regulatory, type II, beta PRKRA others 11108 - 12923-924.1 PR domain containing 4 protein-kinase, interferon-inducible double stranded RNA dependent inhibitor, repressor of (P58 PRKRIR others 5612 - 11q13.5 repressor) cell division cycle 2-like 2 CDC2L2 others 728642 - lp36.33 (PITSLRE proteins) phosphatidylinositol-3 phosphate/phosphatidylinositol 5 PIP5K3 others 200576 - 2q33.3 kinase, type 111 similar to cAMP-dependent protein kinase type I-beta regulatory PRKARIB others 645590 - - subunit CDC28 protein kinase regulatory CKSIA others 137529 - 8q21.13 subunit IA Fc fragment of IgG, low affinity FCGR3A others 2214 - 1q23 Ila, receptor (CD16a) branched chain aminotransferase 2, BCAT2 others 587 2.6.1.26 19913 mitochondrial CCNA2 others 890 - 4q25-q3I cyclin A2 CCNE1 others 898 - 19q12 cyclin El glucokinase (hexokinase 4) GCKR others 2646 - 2p23 regulator CCND2 others 894 - 1 2 p13 cyclin D2 menage a trois 1 (CAK assembly MNAT . others 4331 - 14q23 factor) RAD17 others 5884 - 5ql3 RAD17 homolog (S. pombe) SHB (Src homology 2 domain SHB others 6461 - 9pl2-pl1 containing) adaptor protein B SHC (Src homology 2 domain SHCI others 6464 - lq21 containing) transforming protein I secretory leukocyte protease SLPI others 6590 - 20q]2 inhibitor (antileukoproteinase) carbamoyl-phosphate synthetase 2, 2.1.3.2, aspartate transcarbamylase, and CAD others 790 3.5.2.- 2p22-p2I dihydroorotase MYTI others 4661 - 20ql3.33 myelin transcription factor I v-crk sarcoma virus CT 10 CRK others 1398 - 17pl3.3 oncogene homolog (avian) general transcription factor IIH, GTH2HI others 2965 - I 1p15.1-p14 polypeptide 1, 62kDa ZRANB2 others 9406 - 1p31 zinc finger protein 265 BACE2 others 25825 - 21q22.3 beta-site APP-cleaving enzyme 2 CCNB1 others 891 - 5q]2 cyclin BI OSRI others 130497 - 2p24.1 odd-skipped related I (Drosophila) AAA743 MAPKNS others 01 - MAP kinase AAA365 AAA36585 others 85 - rac protein kinase-beta AAB050 AAB05036 others 36 - p38B MAP kinase AAC162 mitogen-activated protein kinase AAC16273 others 73 - - kinase 7b 97 WO 2009/088408 PCT/US2008/012369 Map Location ID Kinase Gene Entrez (cytogenetic or Name Family Gene ID enzyme ID genetic location) Descriptive Name (or default name) AAC247 AAC24716 others 16 - - p21 activated kinase IB AAC989 AAC98920 others 20 - - cell cycle related kinase AAH 130 AAH 13051 others 51 - - LIM domain kinase 2 AA0127 AA012758 others 58 - - casein kinase I gamma I isoform BAB629 BAB62909 others 09 - - testicular protein kinase 2 BAD186 BAD18671 others 71 - NME1 NME2 others 654364 - 17921.3 NMEI-NME2 protein tyrosine phosphatase, non receptor type 11 (Noonan PTPNI I others 5781 - 12924 syndrome 1) serine/threonine kinase 22A TSSKIA others 23752 - 22q 1.21 (spermiogenesis associated) [00179] The preceding merely illustrates the principles of the invention. It will be appreciated that those skilled in the art will be able to devise various arrangements which, although not explicitly described or shown herein, embody the principles of the invention and are included within its spirit and scope. Furthermore, all examples and conditional language recited herein are principally intended to aid the reader in understanding the principles of the invention and the concepts contributed by the inventors to furthering the art, and are to be construed as being without limitation to such specifically recited examples and conditions. Moreover, all statements herein reciting principles, aspects, and embodiments of the invention as well as specific examples thereof, are intended to encompass both structural and functional equivalents thereof. Additionally, it is intended that such equivalents include both currently known equivalents and equivalents developed in the future, i.e., any elements developed that perform the same function, regardless of structure. The scope of the present invention, therefore, is not intended to be limited to the exemplary embodiments shown and described herein. Rather, the scope and spirit of present invention is embodied by the appended claims. 98

Claims (29)

1. An apparatus configured for analysis of a sample, the apparatus comprising: a chamber configured to receive the sample via an inlet port, and to discharge the sample via an outlet port, wherein the inlet and outlet ports are positioned on a first side of the chamber; a plurality of enzymatic substrate extensions coupled to a surface on the first side of the chamber, the surface having a nanoparticle structure; an illuminator positioned on a second side of the chamber, the second side being opposite the first side, the illuminator being positioned to provide an excitation beam to a selected one of the plurality of enzymatic substrate extensions; and an analysis module configured to receive a reflected beam from the selected enzymatic substrate extension, and to determine therefrom whether a modification of the selected enzymatic substrate extension by the sample has occurred.

2. The apparatus of claim 1, further comprising a step control motor configured to position the illuminator and the analysis module relative to the selected enzymatic substrate extension.

3. The apparatus of claim 1, wherein the analysis module comprises a mirror and a spectrometer.

4. The apparatus of claim 3, wherein a waveform peak in the spectrometer indicates modification of the selected enzymatic substrate extension by the sample.

5. The apparatus of claim 1, wherein the nanoparticle structure comprises a metal deposited on a nanopyramid array.

6. The apparatus of claim 1, wherein the excitation beam comprises a laser. 99 WO 2009/088408 PCT/US2008/012369

7. The apparatus of claim 1, wherein the analysis module comprises a digital light processor (DLP).

8. The apparatus of claim 1, wherein at least one of the plurality of enzymatic substrate extensions comprises a polypeptide.

9. The apparatus of claim 1, wherein at least one of the plurality of enzymatic substrate extensions comprises a nucleic acid.

10. The apparatus of claim 1, wherein at least one of the plurality of enzymatic substrate extensions comprises a polysaccharide.

11. The apparatus of claim 1, wherein the modification comprises a phosphorylation event between the selected enzymatic substrate extension and the enzyme from the sample.

12. The apparatus of claim 1, wherein the modification comprises a dephsophorylation event between the selected enzymatic substrate extension and the enzyme from the sample.

13. The apparatus of claim 1, wherein the modification comprises a cleavage event between the selected enzymatic substrate extension and the enzyme from the sample.

14. A method of making a microfluidic optical device, comprising: depositing polycrystalline silicon layers on each side of a silicon wafer; forming via-holes through the silicon wafer; patterning a frontside of the silicon wafer; etching silicon nanostructures in areas formed by the patterning of the frontside; depositing metal in areas formed by the etched silicon nanostructures; removing remaining photoresist and annealing the deposited metal; and integrating a chip separated from the silicon wafer with handling units and a transparent window coupled to a chamber in the microfluidic optical device.

15. The method of claim 14, wherein the forming of the via-holes comprises using chemical etching. 100 WO 2009/088408 PCT/US2008/012369

16. The method of claim 14, wherein the forming of the via-holes comprises using laser drilling.

17. The method of claim 14, wherein the integrating of the chip comprises coupling inlet and outlet ports to the via-hole formation.

18. A method of characterizing a liquid sample, comprising: receiving the liquid sample via an inlet port, and discharging the sample via an outlet port, wherein the inlet and outlet ports are positioned on a first side of the chamber; providing an excitation beam to a selected one of a plurality of enzymatic substrate extensions, the enzymatic substrate extensions being coupled to a surface on the first side of the chamber, the surface having a nanoparticle structure; receiving a reflected beam from the selected enzymatic substrate extension in an analysis module; and determining from the received reflected beam whether a modification of the selected enzymatic substrate extension by the sample has occurred.

19. The method of claim 18, further comprising adjusting a voltage proximate to the selected enzymatic substrate extension.

20. The method of claim 18, further comprising positioning the analysis module relative to the selected enzymatic substrate extension.

21. A method for determining the activity of a target biomolecule using a surface enhanced Raman spectroscopy (SERS) system, comprising: introducing a fluid sample into a microfluidic optical chamber wherein said optical chamber comprises a Raman active surface with a plurality of substrates extending therefrom; allowing for specific interaction between a biomolecule in the fluid sample and a plurality of said substrates; 101 WO 2009/088408 PCT/US2008/012369 directing a laser at the fluid sample, wherein the interaction of the laser with the fluid sample produces a SERS signal that is specific for the interaction between the biomolecule and the substrate; and detecting the activity of the biomolecule by detecting a change in the Raman scattering spectrum of the biomolecule as compared to the Raman scattering spectrum of a control sample.

22. The method of claim 21 wherein the target biomolecule is a protein.

23. The method of claim 21 wherein the target biomolecule is an enzyme.

24. The method of claim 21 wherein the target biomolecule is a kinase.

25. The method of claim 21 wherein the target biomolecule is an antibody.

26. The method of claim 21 wherein the target biomolecule is a substrate for an enzymatic reaction.

27. The method of claim 21 wherein the target biomolecule is a DNA binding protein and the substrate is a nucleic acid.

28. The method of claim 21 wherein the interaction between the target biomolecule the plurality of substrates is a protein-ligand binding interaction.

29. The method of claim 21 wherein the interaction between the target biomolecule the plurality of substrates is a protein-protein binding interaction. 102