WO2017123781A1

WO2017123781A1 - Proteome-wide drug screening using mass spectrometric imaging (msi) of bead-arrays

Info

Publication number: WO2017123781A1
Application number: PCT/US2017/013218
Authority: WO
Inventors: Ying Zhou; Kenneth J. Rothschild; Mark J. Lim
Original assignee: Ambergen, Inc.
Priority date: 2016-01-12
Filing date: 2017-01-12
Publication date: 2017-07-20

Abstract

This invention relates to biology, molecular biology, biochemistry, cell biology, biomedicine, biomarkers and clinical diagnostics; proteomics, reverse proteomics and mass spectrometry; bio-molecular arrays, microarrays, bead-arrays, and bead-displays; multiplexed assays and bio-assays; and label-free bio-molecular detection. More specifically, the invention relates to detecting or imaging molecules or compounds on individual beads or particles using mass spectrometry as applied to the aforementioned fields.

Description

PROTEOME-WIDE DRUG SCREENING USING MASS SPECTROMETRY IMAGING

(MSI) OF BEAD-ARRAYS

CROSS-REFERENCE TO RELATED APPLICATIONS

The present application claims the benefit of U.S. Provisional Patent Application No.

62/277,589, filed on January 12, 2016, which is incorporated herein by reference.

FIELD OF THE INVENTION

BACKGROUND OF THE INVENTION

A. Drug discovery and ligand-target binding

An initial step in the drug discovery process depends on identifying and optimizing lead compounds which interact with high affinity and regulate the activity of specific biological drug targets (e.g. a protein receptor involved in a disease pathway). However, a second equally important step is to verify that these leads do not interact and interfere with the functioning of any of the vast number of other (non-target) proteins which comprise the human proteome [Kim, Pinto, Getnet, Nirujogi, Manda et al. (2014) Nature 509: 575-81]. This second step is critical since there are over 100,000 deaths per year in the U.S. that are caused by serious adverse drug reactions (SADRs), such as through off-target effects [Giacomini, Krauss, Roden, Eichelbaum, Hayden and Nakamura (2007) Nature 446: 975-7; Yamanishi, Pauwels and Kotera (2012) J Chem Inf Model 52: 3284-92]. Furthermore, the selection of lead compounds and their subsequent clinical testing is a long (-10-15 year) and expensive (>$1B) process [Hughes, Rees, Kalindjian and Philpott (2011) Br J Pharmacol 162: 1239-49]. On average only 30% of drugs reach Phase I with only 8% of drugs receiving FDA approval [DiMasi, Hansen and Grabowski (2003) J Health Econ 22: 151-85]. Thus, the ability to perform proteome-wide screening of drug-protein interactions to assist in selecting drugs, candidate drugs or lead drug compounds that have minimized off-target effects could result in a much faster and cheaper drug discovery process, as well as safer drugs (complementing but not replacing animal and human studies). Such proteome-wide screening could also be used to repurpose existing drugs [Bakhtiar and Nelson (2001) Mol Pharmacol 60: 405-15; Adams, Keiser, Basuino, Chambers, Lee, Wiest and Babbitt (2009) PLoS Comput Biol 5: el 000474; Bantscheff, Scholten and Heck (2009) Drug Discov Today 14: 1021-9; Oprea and Mestres (2012) AAPS J 14: 759-63].

B. Approaches currently used for detection of small molecule-protein binding

Microarrays were first developed for genomics more than two decades ago [Fodor, Read, Pirrung, Stryer, Lu and Solas (1991) Science 251 : 767-73; Chee, Yang, Hubbell, Berno, Huang et al. (1996) Science 274: 610-4] and subsequently for proteomics 15 years ago [MacBeath and Schreiber (2000) Science 289: 1760-3; Zhu, Bilgin, Bangham, Hall, Casamayor et al. (2001) Science 293 : 2101-5]. Since microarrays facilitate massively parallel analysis of the interaction of prey molecules with thousands to millions of arrayed bait molecules, they are potentially well-suited for proteome-wide drug-protein screening. Protein microarrays can reach densities of >10,000 features on a single chip the size of a microscope slide [Harrison, Kumar, Lang, Snyder and Gerstein (2002) Nucleic Acids Res 30: 1083-90; (2004) Nature 431 : 931-45] and bead-arrays can reach millions to billions of features in the same footprint [Michael, Taylor, Schultz and Walt (1998) Anal Chem 70: 1242-8; Leamon, Lee, Tartaro, Lanza, Sarkis et al. (2003) Electrophoresis 24: 3769-77]. However, a common feature of these arrays is the use of fluorescence readout and hence the requirement for labeling the prey molecules. In the case where the prey is a drug compound, it is important to avoid labeling which can alter its binding specificity and activity. DNA microarrays [1] are now widely used and accepted by the scientific community, most commonly used for multiplexed, "genome-wide" analysis of the entire expressed mRNA complement of a cell, tissue or other biological sample. In this case, the microarray features are oligonucleotide bait molecules that bind complementary mRNA or cDNA from a complex biological sample. Other examples of DNA microarray applications include single nucleotide polymorphism (SNP) genotyping and mutation analysis [2], copy number variation (CNV) [3] and chromatin immunoprecipitation (ChIP) analyses (so called ChlP-on-Chip) [4].

Conversely, a variety of effective label-free methods exist which can measure drug-protein interactions. Two of the most widely used proximity based assays are fluorescence resonance energy transfer (FRET) (where the unlabeled drug competes for a labeled compound in the protein binding site) and surface plasmon resonance (SPR) [Mason, Spais, Husten, Prouty, Albom, Meyer, Ator and Angeles (2012) Assay Drug Dev Technol 10: 468-75]. However, these assays are not ideally suited for proteome-wide screening. FRET type assays generally require some a priori knowledge of binding or target activation mechanisms and are not easily generalized to the entire proteome (e.g. fluorescent tracers are used at the ATP binding site in time-resolved fluorescence resonance energy transfer [TR-FRET] based LanthaScreen® kinase assays [Mason, Spais et al. (2012) Assay Drug Dev Technol 10: 468-75]). Furthermore, such assays utilize microtiter plates, which although suitable for high throughput screening (HTS), remain impractical for routine proteome-wide screening. For example, although the proteome size (which is larger than human genome) has been difficult to estimate [Harrison, Kumar et al. (2002) Nucleic Acids Res 30: 1083-90; Kim, Pinto et al. (2014) Nature 509: 575-81], it would still require more than a dozen 1,536-well microtiter plates to perform a single screen even against the only 23,500 genes in the human genome [Marian (2014) Methodist Debakey Cardiovasc J 10: 3-6]. Likewise, existing commercial SPR systems offer limited multiplex capabilities, with Bio-Rad's ProteOn™ XPR36 instrument offering a 6 x 6 array (36 simultaneous measurements) and the BiacoreTM4000 system offering a 4 x 5 array (reporting throughput of 4,800 measurements but on only 16 targets and requiring 24 hours) [Helmerhorst, Chandler, Nussio and Mamotte (2012) Clin Biochem Rev 33 : 161-73]. Emerging methods of SPR imaging (SPRi) could increase capacity, with reports of arrays containing approximately 800-features [Lausted, Hu and Hood (2008) Mol Cell Proteomics 7: 2464-74]. However, even with expanded SPR multiplex capability, it is difficult for SPR to identify or characterize a binding drug when more than one compound is screened simultaneously.

Mass spectrometry (MS), a central tool in proteomics [Nesvizhskii (2007) Methods Mol Biol 367: 87-119], has also been used extensively as a label -free technology in the drug discovery process [Bakhtiar and Nelson (2001) Mol Pharmacol 60: 405-15]. For example, methods using size exclusion or affinity chromatography separation followed by mass spectrometry, including affinity selection mass spectrometry [Muckenschnabel, Falchetto, Mayr and Filipuzzi (2004) Anal Biochem 324: 241-9; Ng, Yang, Kameyama, Palcic, Hindsgaul and Schriemer (2005) Anal Chem 77: 6125-33; Annis, Nickbarg, Yang, Ziebell and Whitehurst (2007) Curr Opin Chem Biol 11 : 518-26; Jonker, Kool, Irth and Niessen (2011) Anal Bioanal Chem 399: 2669-81] and frontal affinity chromatography mass spectrometry [Ng, Yang et al. (2005) Anal Chem 77: 6125-33], enable screening of multiple compounds against single protein targets. Parallel analysis of drug binding to individual proteins each located in a 96 or 384 mini -column format has been reported with these approaches [Ng, Yang et al. (2005) Anal Chem 77: 6125-33]. However, proteome-wide drug screening would require tens of thousands of such parallel columns. Alternatively, a mass spectrometry based chemical proteomics approach uses a competitive binding assay between the free drug of interest and kinases immobilized by broad-selectivity inhibitors [Bantscheff, Eberhard, Abraham, Bastuck, Boesche et al. (2007) Nat Biotechnol 25: 1035-44]. This versatile tool is valuable in drug discovery and as an "in-depth" approach to reveal the mechanisms of action of the inhibitors. However, it involves many experimental steps including protein digestion, identification and quantification. Furthermore, the method is specific to certain protein classes such as kinases since broad-specificity inhibitors are needed to immobilize the targets, and again, cannot be readily generalized to proteome-wide screening.

SUMMARY OF THE INVENTION

This invention is described in preferred embodiments in the following description with reference to the Figures, in which like numbers represent the same or similar elements. Reference throughout this specification to "one embodiment," "an embodiment," or similar language means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, appearances of the phrases "in one embodiment," "in an embodiment," and similar language throughout this specification may, but do not necessarily, all refer to the same embodiment.

In one embodiment, the invention relates to a label-free method to measure the binding of one or more prey molecules to a bead library consisting of bait molecules immobilized on beads comprising: a) Making a mass-tag encoded bait-bead library whereby each bead carries a unique bait molecule and a corresponding mass-tag; b) Incubating said prey molecules with the bead library; c) Making an array with said bead library; d) Measuring the mass spectrum from at least one bead from each member of the bait-bead library carrying a unique bait molecule in said array; and e) Determining from the mass spectral measurements in step (d) the relative binding of each prey molecule with each member of the bait-bead library. In one embodiment, said bait molecules are selected from the group consisting of proteins, polypeptides, peptides, glycoproteins, proteoglycans, carbohydrates, lipids, nucleic acids, DNA, RNA and macromolecular structures comprised of a combination of biomolecules such as ribosomes which contain proteins and nucleic acid. In one embodiment, said prey molecules are drug compounds, candidate drug compounds or lead drug compounds which are selected from the group consisting of small molecule compounds, organic chemical compounds, inorganic chemical compounds, proteins, glycoproteins, proteoglycans, antibodies, polypeptides, peptides, nucleic acids, lipids, carbohydrates, linear polymers, and branched polymers as well as biomimetics such as peptoids. In one embodiment, said array is formed in micro-well plates, each well sized to hold only a single bead. In one embodiment, said micro-well plates used to form the array comprise an indium tin oxide surface coating.

In one embodiment, the invention relates to a label-free method to screen drug compounds for on and off-target interactions comprising: a) Making a mass-tag encoded bait-bead library whereby each bead carries a unique target molecule and a corresponding mass-tag; b) Incubating said compound molecules with the bead library; c) Making an array with said bead library; d) Measuring the mass spectrum from at least one bead from each member of the target-bead library carrying a unique target molecule in said array; and e) Determining from the mass spectral measurements in step (d) the relative binding of each compound molecule with each member of the target -bead library. In one embodiment, said target molecules are selected from the group consisting of proteins, polypeptides, peptides, glycoproteins, proteoglycans, carbohydrates, lipids, nucleic acids, DNA, RNA and macromolecular structures comprised of a combination of biomolecules such as ribosomes which contain proteins and nucleic acid. In one embodiment, said compound molecules are drug compounds, candidate drug compounds or lead drug compounds which are selected from the group consisting of small molecule compounds, organic chemical compounds, inorganic chemical compounds, proteins, glycoproteins, proteoglycans, antibodies, polypeptides, peptides, nucleic acids, lipids, carbohydrates, linear polymers, and branched polymers as well as biomimetics such as peptoids. In one embodiment, said array is formed in micro-well plates, each well sized to hold only a single bead. In one embodiment, said micro-well plates used to form the array comprise an indium tin oxide surface coating.

In one embodiment, the invention relates to a label-free method to measure the binding of one or more prey molecules to a bead library consisting of bait molecules immobilized on beads comprising: a) Making a mass-tag encoded bait-bead library whereby each bead carries a unique bait molecule and a corresponding mass-tag; b) Making an array with said bead library; c) Incubating said prey molecules with the bead library; d) Measuring the mass spectrum from at least one bead from each member of the bait-bead library carrying a unique bait molecule in said array; and e) Determining from the mass spectral measurements in step (d) the relative binding of each prey molecule with each member of the bait-bead library. In one embodiment, said bait molecules are selected from the group consisting of proteins, polypeptides, peptides, glycoproteins, proteoglycans, carbohydrates, lipids, nucleic acids, DNA, RNA and macromolecular structures comprised of a combination of biomolecules such as ribosomes which contain proteins and nucleic acid. In one embodiment, said prey molecules are drug compounds, candidate drug compounds or lead drug compounds which are selected from the group consisting of small molecule compounds, organic chemical compounds, inorganic chemical compounds, proteins, glycoproteins, proteoglycans, antibodies, polypeptides, peptides, nucleic acids, lipids, carbohydrates, linear polymers, and branched polymers as well as biomimetics such as peptoids. In one embodiment, said array is formed in micro-well plates, each well sized to hold only a single bead. In one embodiment, said micro-well plates used to form the array comprise an indium tin oxide surface coating.

In one embodiment, the invention relates to a label-free method to screen drug compounds for on and off-target interactions comprising: a) Making a mass-tag encoded bait-bead library whereby each bead carries a unique target molecule and a corresponding mass-tag; b) Making an array with said bead library; c) Incubating said compound molecules with the bead library; d) Measuring the mass spectrum from at least one bead from each member of the target-bead library carrying a unique target molecule in said array; and e) Determining from the mass spectral measurements in step (d) the relative binding of each compound molecule with each member of the target -bead library. In one embodiment, said target molecules are selected from the group consisting of proteins, polypeptides, peptides, glycoproteins, proteoglycans, carbohydrates, lipids, nucleic acids, DNA, RNA and macromolecular structures comprised of a combination of biomolecules such as ribosomes which contain proteins and nucleic acid. In one embodiment, said compound molecules are drug compounds, candidate drug compounds or lead drug compounds which are selected from the group consisting of small molecule compounds, organic chemical compounds, inorganic chemical compounds, proteins, glycoproteins, proteoglycans, antibodies, polypeptides, peptides, nucleic acids, lipids, carbohydrates, linear polymers, and branched polymers as well as biomimetics such as peptoids. In one embodiment, said array is formed in micro-well plates, each well sized to hold only a single bead. In one embodiment, said micro-well plates used to form the array comprise an indium tin oxide surface coating.

In one embodiment, the invention relates to a method of measuring the interaction of prey molecules with bait molecules on an array of beads, comprising: a) providing a mixture comprising first and second beads, said first beads comprising first bait molecules and said first beads having a first mass tag attached, said second beads comprising second bait molecules and said second beads having a second mass tag attached, wherein said first and second bait molecules and said first and second mass tags are different; b) contacting said first and second beads with a solution comprising a population of prey molecules; c) making an array with said beads; d) subjecting said array to mass spectrometry analysis under conditions wherein said first and second beads are identified by detection of their corresponding mass tags, wherein said mass spectrometry analysis comprises detecting prey molecules arising from individual beads within said array; and e) Determining from the mass spectral measurements in step (d) the relative binding of prey molecules with said first and second beads. In one embodiment, said bait molecules are selected from the group consisting of proteins, polypeptides, peptides, glycoproteins, proteoglycans, carbohydrates, lipids, nucleic acids, DNA, RNA and macromolecular structures comprised of a combination of biomolecules such as ribosomes which contain proteins and nucleic acid. In one embodiment, said bait molecules are drug targets or potential off-targets. In one embodiment, said prey molecules are drug compounds, candidate drug compounds or lead drug compounds which are selected from the group consisting of small molecule compounds, organic chemical compounds, inorganic chemical compounds, proteins, glycoproteins, proteoglycans, antibodies, polypeptides, peptides, nucleic acids, lipids, carbohydrates, linear polymers, and branched polymers as well as biomimetics such as peptoids. In one embodiment, said prey molecules are hormones, vitamins or small molecule metabolites. In one embodiment, said array is formed in micro-well plates, each well sized to hold only a single bead. In one embodiment, said micro-well plates used to form the array comprise an indium tin oxide surface coating.

In one embodiment, the invention relates to a method of measuring the interaction of prey molecules with bait molecules on an array of beads, comprising: a) providing a mixture comprising first and second beads, said first beads comprising first bait molecules and said first beads having a first mass tag attached, said second beads comprising second bait molecules and said second beads having a second mass tag attached, wherein said first and second bait molecules and said first and second mass tags are different; b) contacting said first and second beads with a solution comprising first and second prey molecules; c) making an array with said beads; d) subjecting said array to mass spectrometry analysis under conditions wherein said first and second beads are identified by detection of their corresponding mass tags, wherein said first beads had bound to said first prey molecules and said second beads and bound to said second prey molecules, and wherein said mass spectrometry analysis comprises detecting prey molecules arising from individual beads within said array; and e) Quantifying from the mass spectral measurements in step (d) the binding of said first and second prey molecules with said first and second beads. In one embodiment, said bait molecules are selected from the group consisting of proteins, polypeptides, peptides, glycoproteins, proteoglycans, carbohydrates, lipids, nucleic acids, DNA, RNA and macromolecular structures comprised of a combination of biomolecules such as ribosomes which contain proteins and nucleic acid. In one embodiment, said bait molecules are drug targets or potential off-targets. In one embodiment, said prey molecules are drug compounds, candidate drug compounds or lead drug compounds which are selected from the group consisting of small molecule compounds, organic chemical compounds, inorganic chemical compounds, proteins, glycoproteins, proteoglycans, antibodies, polypeptides, peptides, nucleic acids, lipids, carbohydrates, linear polymers, and branched polymers as well as biomimetics such as peptoids. In one embodiment, said prey molecules are hormones, vitamins or small molecule metabolites. In one embodiment, said array is formed in micro-well plates, each well sized to hold only a single bead. In one embodiment, said micro-well plates used to form the array comprise an indium tin oxide surface coating.

In one embodiment, the invention relates to a method of measuring the interaction of prey molecules with bait molecules on an array of beads, comprising: a) providing a mixture comprising first and second beads, said first beads comprising first bait molecules and said first beads having a first mass tag attached, said second beads comprising second bait molecules and said second beads having a second mass tag attached, wherein said first and second bait molecules and said first and second mass tags are different; b) making an array with said beads; c) contacting said first and second beads with a solution comprising a population of prey molecules; d) subjecting said array to mass spectrometry analysis under conditions wherein said first and second beads are identified by detection of their corresponding mass tags, wherein said mass spectrometry analysis comprises detecting prey molecules arising from individual beads within said array; and e) Determining from the mass spectral measurements in step (d) the relative binding of prey molecules with said first and second beads. In one embodiment, said bait molecules are selected from the group consisting of proteins, polypeptides, peptides, glycoproteins, proteoglycans, carbohydrates, lipids, nucleic acids, DNA, RNA and macromolecular structures comprised of a combination of biomolecules such as ribosomes which contain proteins and nucleic acid. In one embodiment, said bait molecules are drug targets or potential off-targets. In one embodiment, said prey molecules are drug compounds, candidate drug compounds or lead drug compounds which are selected from the group consisting of small molecule compounds, organic chemical compounds, inorganic chemical compounds, proteins, glycoproteins, proteoglycans, antibodies, polypeptides, peptides, nucleic acids, lipids, carbohydrates, linear polymers, and branched polymers as well as biomimetics such as peptoids. In one embodiment, said prey molecules are hormones, vitamins or small molecule metabolites. In one embodiment, said array is formed in micro-well plates, each well sized to hold only a single bead. In one embodiment, said micro-well plates used to form the array comprise an indium tin oxide surface coating.

In one embodiment, the invention relates to a method of measuring the interaction of prey molecules with bait molecules on an array of beads, comprising: a) providing a mixture comprising first and second beads, said first beads comprising first bait molecules and said first beads having a first mass tag attached, said second beads comprising second bait molecules and said second beads having a second mass tag attached, wherein said first and second bait molecules and said first and second mass tags are different; b) making an array with said beads; c) contacting said first and second beads with a solution comprising first and second prey molecules; d) subjecting said array to mass spectrometry analysis under conditions wherein said first and second beads are identified by detection of their corresponding mass tags, wherein said first beads had bound to said first prey molecules and said second beads and bound to said second prey molecules, and wherein said mass spectrometry analysis comprises detecting prey molecules arising from individual beads within said array; and e) Quantifying from the mass spectral measurements in step (d) the binding of said first and second prey molecules with said first and second beads. In one embodiment, said bait molecules are selected from the group consisting of proteins, polypeptides, peptides, glycoproteins, proteoglycans, carbohydrates, lipids, nucleic acids, DNA, RNA and macromolecular structures comprised of a combination of biomolecules such as ribosomes which contain proteins and nucleic acid. In one embodiment, said bait molecules are drug targets or potential off-targets. In one embodiment, said prey molecules are drug compounds, candidate drug compounds or lead drug compounds which are selected from the group consisting of small molecule compounds, organic chemical compounds, inorganic chemical compounds, proteins, glycoproteins, proteoglycans, antibodies, polypeptides, peptides, nucleic acids, lipids, carbohydrates, linear polymers, and branched polymers as well as biomimetics such as peptoids. In one embodiment, said prey molecules are hormones, vitamins or small molecule metabolites. In one embodiment, said array is formed in micro-well plates, each well sized to hold only a single bead. In one embodiment, said micro-well plates used to form the array comprise an indium tin oxide surface coating.

A fundamental challenge in the drug discovery process is to develop compounds with high efficacy and minimal side-effects. We describe a new approach to proteome-wide drug screening for detection of on- and off-target binding which combines the advantages of mass spectrometry (MS) with microarray technology. The method involves matrix-assisted laser desorption/ionization mass spectrometric imaging (MALDI-MSI) of agarose micro-beads randomly arrayed at high-density in custom micro-well plates. Each bead carries a unique protein target and a corresponding photocleavable mass-tag for coding (PC-Mass-Tag). Compounds bound to specific protein beads and a photo-released coding PC-Mass-Tag are detected simultaneously using MALDI-MSI. As an example of this approach, two kinase-targeted drugs, Dasatinib and Brigatinib (AP26113), were simultaneously screened against a model 50-member kinase-bead library. A MALDI-MSI scan performed at the equivalent density of 495,000 beads in the footprint of a microscope slide yielded 100% sensitivity for detecting known strong interactions with no false positives (see Experimental Examples for detail). This invention reports a new, convenient and simple approach for proteome-wide drug screening termed Bead-GPS which combines the advantages of microarray (bead-array) technology with label-free mass spectrometry readout (Fig. 1). Previously, we demonstrated that photocleavable peptides can be utilized as PC-Mass-Tags to encode random bead-arrays. In combination with correlated MALDI-MSI (MALDI mass spectrometric imaging) and fluorescence readout, this provided a label-based method for detecting protein-protein molecular interaction such as between a protein library and fluorescently labeled antibodies or other proteins [Lim, Liu, Braunschweiger, Awad and Rothschild (2014) Rapid Communications in Mass Spectrometery 28: 49-62]. This invention demonstrates a label-free method for detection of drug-protein interactions that uses PC-Mass-Tags and proteins attached to beads but does not require fluorescence detection. This approach not only allows screening of a drug against a large number of protein targets in a high density microarray format, but has the additional advantage that, unlike SPR, can simultaneously screen multiple drugs against all protein targets owing to the ability to identify each binding drug by mass spectrometry.

Bead-GPS has the added advantage that multiple drugs may be simultaneously screened against the entire protein library, for full drug-by-library multiplexing. We estimate that for a micro-well plate the size of a microscope slide such as used here (see Detailed Description of Invention and Experimental Examples), roughly 10,000 unique proteins, or 20 sub-arrays each containing for example the entire kinome (estimated at about 500 kinases [Yan, King, Zhou, Warmuth and Xia (2006) Drug Discov Today Technol 3 : 269-76]), could be analyzed with a 50-fold bead redundancy. High capacity coding of the array can be achieved by combinatorial PC-Mass-Tag schemes [Lim, Liu et al. (2014) Rapid Communications in Mass Spectrometery 28: 49-62]. For example, for a library encoded with only two PC -Mass-Tags per bead species, only 54 unique PC-Mass-Tags are needed to provide 1,431 unique codes (see Fig. 2). This is more than sufficient for the entire human kinome. Similarly, a library encoded with three PC-Mass-Tags per bead species using only 54 unique PC-Mass Tags is sufficient to encode 24,804 different proteins. Alternatively, a library encoded with only two PC-Mass-Tags per bead using 150 unique PC-Mass-Tags species, is sufficient to encode 11,175 proteins.

It should be noted that, although many drug targets are proteins, this invention is not intended to be restricted to any particular type of drug target as the bait molecule on the beads, since targets, potential targets or potential off-targets of the drug compounds, candidate drug compounds or lead drug compounds can for example also be polypeptides, peptides, glycoproteins, proteoglycans, carbohydrates, lipids, nucleic acids, DNA, RNA or macromolecular structures comprised of a combination of biomolecules such as ribosomes which contain proteins and nucleic acid. For example, traditional cytotoxic drugs used in cancer therapy often target DNA or macromolecular structures such as microtubules [5, 6]. Antibiotic drugs have been reported for example that target ribosomes [7]. Glycans have also been targeted by therapeutic drugs, such as in anti -viral therapy [8].

Furthermore, drug compounds, candidate drug compounds or lead drug compounds can for example be small molecule compounds, organic chemical compounds, inorganic chemical compounds, proteins, glycoproteins, proteoglycans, antibodies, polypeptides, peptides, nucleic acids, lipids, carbohydrates, linear polymers, and branched polymers as well as biomimetics such as peptoids. Although many drugs are small molecule organic compounds, such the Dasatinib and Brigatinib kinase inhibitors described here (see Experimental Examples) [9, 10], other examples of drugs include proteins such as antibodies or cytokines [11, 12], lipid modified drugs such as liposome encapsulated doxorubicin [13], nucleic acid based drugs such as siRNA or DNA used in gene therapy [14, 15], and organometallic drug compounds such as those containing platinum commonly used in cancer therapy [6]. The described features, structures, or characteristics of the invention may be combined in any suitable manner in one or more embodiments. In the following description, numerous specific details are recited to provide a thorough understanding of embodiments of the invention. One skilled in the relevant art will recognize, however, that the invention may be practiced without one or more of the specific details, or with other methods, components, materials, and so forth. In other instances, well-known structures, materials, or operations are not shown or described in detail to avoid obscuring aspects of the invention.

Other objects, advantages, and novel features, and further scope of applicability of the present invention will be set forth in part in the detailed description to follow, taken in conjunction with the accompanying drawings, and in part will become apparent to those skilled in the art upon examination of the following, or may be learned by practice of the invention. The objects and advantages of the invention may be realized and attained by means of the instrumentalities and combinations particularly pointed out in the appended claims.

DESCRIPTION OF THE FIGURES

The accompanying figures, which are incorporated into and form a part of the specification, illustrate several embodiments of the present invention and, together with the description, serve to explain the principles of the invention. The figures are only for the purpose of illustrating a preferred embodiment of the invention and are not to be construed as limiting the invention.

Figure 1 shows major steps in proteome-wide drug-protein screening using Bead-GPS. Unlike conventional HTS methods, Bead-GPS enables rapid, proteome-wide screening of entire compound libraries against large protein libraries enabling both on- and off-target drug interactions to be identified. In the example shown, the kinase-directed drug Dasatinib (structure shown in at top of figure) interacts with a particular protein residing on a bead in the array. MALDI-MSI scanning of the bead-array is used to both simultaneously decode the beads and detect the bound drugs. Blue curved line represents a Mass-Tag which is released by the MALDI-MS laser; Yellow oval represents a photocleavable linker which in this case was PC-Biotin.

Figure 2A&B show high capacity combinatorial PC-Mass-Tag barcoding of bead libraries. Figure 2A shows color-coded MALDI-MS image of a bead-array containing 85 unique bead species coded with 2 PC-Mass-Tags each (54 unique PC-Mass-Tags used in total). For simplicity, the image was created by assigning unique colors to only 8 of the 54 PC-Mass-Tags (the overlay of 2 colors per bead species also creates a unique color/shade), with the remainder of the PC-Mass-Tags all colored pink. However, all 85 unique bead species were detected in the array region shown. Figure 2B shows color-coded spectra are shown for 8 representative single beads (the beads noted with white circles and numbered in Panel Figure 2A. Masses of the 8 different tags are indicated at the top of the compiled spectra, rounded to the nearest integer for simplicity. The red asterisk indicates a control peptide spiked into the matrix solution which is sprayed over the entire bead-array. The numbering of each spectrum corresponds to the bead numbering in Figure 2A. As an example metric to demonstrate correct pairing of PC-Mass-Tags on the beads in the array, a pixel -by-pixel linear regression correlation between the grayscale MALDI-MS images of 2 PC-Mass-Tags expected to be found uniquely on only 1 bead species (i.e. no other bead species in the library contains either of these 2 mass-tags) yielded an R = 0.86. Conversely, regression analysis of 2 unique PC-Mass-Tags which were used for coding beads in the array, but were not paired on any of the 85 different bead species, returned an R = 0.02. Note that even at this coding level, >1,000 unique codes can be created, more than sufficient to code an entire kinome comprised of the approximate 500 kinases in the human proteome.

Figure 3A-C show the production of a 50-member kinase bead-library. Figure 3A shows the The 50-member kinase bead-library was probed with fluorescent antibodies against both the FLAG epitope (common to all kinases) and a specific kinase (LCK in this example; different fluorescent colors on each antibody). A 2-color fluorescence image overlay is shown of the bead-array where red is the FLAG detection (all kinases) and yellow the specific kinase detection (LCK). The array was also imaged by MALDI-MS where blue is the overlaid MALDI-MS image of the PC-Mass-Tag which codes the LCK beads (aligns with yellow fluorescence). Figure 3B shows colorized MALDI-MS images of the same array are shown for 9 additional kinase bead species, overlaid with the fluorescence image of the FLAG antibody detection (shown as white in this case). Figure 3C shows color-coded and overlaid MALDI spectra from representative single beads (the beads circled and labeled with kinase gene names in Figure 3 A and Figure 3B.

Figure 4A-C show a bead-GPS drug screening with a 50-member kinase bead-array. The 50-member kinase bead-library was treated simultaneously with two drugs, Dasatinib and Brigatinib. The beads were then arrayed and the array scanned by MALDI-MSI. Binding results are expressed as average monoisotopic peak area for the drug on all PC-Mass-Tag positive beads for each kinase. Figure 4A shows bead-GPS binding results for the drug Dasatinib for each kinase, where the Kd values are known. Figure 4B shows bead-GPS binding results for the drug Brigatinib for each kinase, where the IC50 values are known. Drug-kinase interactions with known Kd or IC50 values are categorized as follows: Weak >100 nM; Medium <100 nM but >10nM; Strong <10 nM. The black dotted line is the cutoff for scoring hits, set at 3 standard deviations above the mean of the known weak drug-kinase interactions. Figure 4C shows bead-GPS binding results for the drugs Dasatinib & Brigatinib for all kinases. Blue Bars are for Dasatinib; Orange Bars are for Brigatinib. Error! Reference source not found, shows PC-Mass-Tags, kinases and drug binding constants. Sources for IC₅₀ were from Katayama, R. et al. as well as measurements by Carna Biosciences. * Some IC50 data values were the average of these two sources. Δ Represents IC50 values which were provided by Carna Biosciences only. The remaining IC₅₀ values without any labels were from measurements reported by Katayama, R. et al. only. K_d were from the Drug2Gene database. See main text for full references.

Error! Reference source not found, shows key parameters and results of the 50-kinase and 2-drug Bead-GPS screening experiment. Key parameters and results are shown for the experiment in Example 2.

DETAILED DESCRIPTON OF THE INVENTION

Preparation of photocleavable (PC) biotin labeled peptide Mass-Tags (Mass-Tags). Peptides of unique mass were chemically modified on their N-terminus with photocleavable biotin (PC-Biotin) using N-hydroxysuccinimide (NHS)-activated PC-Biotin labeling reagent [Olejnik, Sonar et al. (1995) Proceedings of the National Academy of Science (USA) 92: 7590-7594] (AmberGen Inc., Watertown, MA). The peptide was prepared at 5 mg/mL in 100 mM sodium bicarbonate and reacted overnight (with mixing) with equimolar amounts of the reagent. The resultant PC-Biotin labeled peptide mass-tags (PC-Mass-Tags) were used without further purification. Because the NHS-activated labeling reagents react only with primary amines, selective labeling of the N-terminus is achieved [Lim, Liu, Braunschweiger, Awad and Rothschild (2014) Rapid Commun Mass Spectrom 28: 49-62]. Preparation of PC-Mass-Tag streptavidin agarose beads.

Preparation of non -fluorescent and fluorescent "Sync Beads" and loading PC-Mass-Tags to the beads has been previously described [Lim, Liu et al. (2014) Rapid Commun Mass Spectrom 28: 49-62]. Briefly, the fluorescent "Sync Beads" were prepared by attaching fluorescent streptavidin to HS-activated 34 μιη agarose beads (NHS HP SpinTrap, GE Healthcare Life Sciences). Fluorescent streptavidin was produced from the reaction of Streptavidin (Thermo Fisher Scientific) and 2 molar equivalents of Alexa Fluor® 647 carboxylic acid, succinimidyl ester (Invitrogen, Carlsbad, CA, USA). The product was purified using Illustra™ NAP™-5 G-25 Sephadex™ columns (GE Healthcare Life Sciences) according to the manufacturer's instructions. For non-fluorescent streptavidin agarose beads, commercially available 34 μπι beads were used (Streptavidin Sepharose High Performance, GE Healthcare Life Sciences, Piscataway, NJ, USA). Peptides (PC-Mass-Tags) listed in Error! Reference source not found, were used. The biotin labeled PC-Mass-Tags were mixed with the streptavidin beads for 30 min in TBS-T, followed by washing steps using TBS-T. Beads were stored at 4oC in TBS or used immediately for subsequent steps. In the case of double PC-Mass-Tags, two PC-Mass-Tags well resolved (mass difference > 2.5 Da) in mass spectrum were incubated with the beads simultaneously at the same concentration as the amount that was used to generate the single-tag beads.

Construction of PC-Mass-Tag encoded protein-beads.

Site specific biotinylated (BTN) protein kinases (Carna Biosciences, Framingham, MA, USA) were loaded to the PC-Mass-Tag beads through biotin-streptavidin interaction. The enzymatic reaction generated BTN-kinase has a single biotinylation at N-terminus region to avoid interference of compound binding and to maintain the kinase activity and an overall functional structure. Each mass tag with unique m/z in mass spectrum serves as the code indicating each individual kinase bead species. In this study, 50 bead species were constructed. Typically, 10 μg of BTN-kinase was incubated with 1 μΙ_, of PC-Mass-Tag streptavidin beads in TBS-T, 1% (w/v) BSA for 30 min and washed with TBS-T.

Incubation of compound library with protein-bead library.

A pool of 50 kinase-beads and one control PC-Mass-Tag bead species without any kinases was generated by combining ~ 5000 beads/species together in TBS-T, 1% (w/v) BSA. The beads were washed and re-suspended in 800 μΙ_, TBS-T, 1% (w/v) BSA. The compounds— Dasatinib (LC Labs, Woburn, MA, USA) and Brigatinib (AP26113) (Selleckchem, Houston, TX, USA) were dissolved in DMSO (10 mM) and further diluted with the incubation solution before use. The drugs were mixed and incubated with the protein-bead library at a final concentration of 0.25 μΜ for each compound for 1.5 hours at room temperature, followed by TBST-T wash.

Forming random bead-arrays.

Custom fiber optic bead-array micro-well plates were manufactured for AmberGen, Inc. by INCOM USA, Inc. (Charlton, MA, USA). The 1 75 25 mm plates contained ~1 million hexagonally packed fiber optic wells created from 50 μπι fibers etched to 40 μπι depth. The fiber cladding yields wells of approximately 45 μπι i.d. For optimal performance in mass spectrometric imaging, plates were coated by sputtering on indium tin oxide (ITO) with sheet resistance (Rs) of 50 ohms/sq or less (ThinFilms, Inc., Hillsborough, NJ, USA). The plates were pre-hydrated and assembled into an AHC1X16 Microarray Hybridization Cassette (Arraylt® Corporation, Sunnyvale, CA, USA) which subdivided the plate into 16 square sub-array zones, each zone measuring 7.5 mm x 7.5 mm.

Prior to loading the beads onto the plate, a wash step with MS grade water was added to remove the salts from the beads. Typically, 100 μΐ. of the bead suspension (~ 2.8 X 10⁴ beads) were loaded to each chamber of the Microarray Hybridization Cassette. Centrifugation at 1430 g was performed when the beads were settled into the micro-wells. The fluid supernatant was discarded. The plate was then washed using water. The plate was allowed to dry overnight at room temperature and covered from light. When fluorescent dye was used, fluorescence image of the bead-arrays was acquired using a GenePix 4200 microarray scanner (Molecular Devices LLC, Sunnyvale, CA, USA).

Photocleavage of the PC-Mass-Tags from the beads was performed by irradiating the microtiter plate from above for 5 min with 365 nm UV light using a Blak-Ray lamp (model XX- 15; UVP, Upland, CA, USA), at a 5 cm distance (the power output under these conditions was approximately 2.6 mW/cm² at 360 nm, 1.0 mW/cm² at 310 nm and 0.16 mW/cm² at 250 nm).

Matrix-assisted laser desorption/ionization (MALDI) mass spectrometric imaging (MSI) and data analysis.

A mixture of 2,5-Dihydroxybenzoic acid (DHB) (10 mg/mL) and a-cyano-4-hydroxycinnamic acid (CHCA) (5 mg/mL) (Sigma-Aldrich, St. Louis, MO, USA) in 70% methanol (0.1% trifluoro acetic acid and 0.1% phosphoric acid) was sprayed to provide a thin and uniform film of matrix using the HTX TM- Sprayer™ (HTXImaging by HTX Technologies, LLC, Carrboro, NC, USA). The spray parameters were: nozzle XY velocity of 800 mm/min, 18 passes, 0.1 mL/min matrix flow rate, nozzle temperature at 30 °C and track spacing of 3 mm.

MALDI-MSI was performed using an AB Sciex 4800 Plus MALDI-TOF/TOF mass spectrometer with a Nd/YAG pulsed laser (355 nm) (AB Sciex, Foster City, CA) and SimulTOF 200 Combo MALDI TOF mass spectrometer (SimulTOF Systems, Sudbury, MA) in the positive ion operating in reflector mode. For AB Sciex 4800 MS, images were acquired with a scan raster of 40 μπι steps and 100 laser shots per pixel. For SimulTOF 200 Combo MS, all imaged areas were collected using a laser repetition rate of 1 kHz, 10 μΐ laser power, 100 laser shots/spectrum, and step size of 25 μπι between raster lines.

Acquisition of the MALDI-MSI using AB Sciex 4800 Plus was achieved using the public domain software 4000 Series Imaging (Novartis & Applied Biosystems, Markus Stoeckli), which works with the native software on the mass spectrometer, and the images analyzed using the software TissueView™ (AB Sciex). SimulTOF 200 MALDI-MSI was acquired using SimulTOF softwares: Wizard, Controller and Plate Editor. The data analysis was performed using SimulTOF Viewer. For peak detection, the signal-to-noise threshold was set to three and the mass tolerance was set to ±0.5 Da.

Although the 100 (AB Sciex 4800) or 50 (SimulTOF) μιη diameter laser beam of the MALDI instrument is larger than the actual diameter of wells and individual beads, it is still possible to resolve individual beads by performing imaging in incremental steps that are smaller than the beam diameter. This oversampling approach has been proved to be ideal for MSI experiments due to the higher spatial resolution it provides [16].

Fluorescence imaging of the plates, as detailed earlier [17], could also be performed after MALDI-MSI. This is particularly useful as the matrix provides some auto-fluorescence when excited with the 488-nm laser (fluorescein channel), thereby allowing visualization of the region scanned by MALDI-MSI, observed as the zone of matrix depletion.

EXAMPLES

The following examples are provided in order to demonstrate and further illustrate certain preferred embodiments and aspects of the present invention and are not to be construed as limiting the scope thereof.

EXAMPLE 1

CONSTRUCTION OF A MODEL RECOMBINANT KTNASE-AGAROSE-BEAD LIBRARY

In this Example a test was performed to demonstrate feasibility of the Bead-GPS approach, a model 50-member recombinant kinase-bead library encoded with PC-Mass-Tags was prepared and validated. To prepare the library, 50 recombinant kinases which had been site-specifically biotinylated at the N-terminus (Carna Biosciences) were attached to 34 μπι streptavidin agarose beads. Peptides (7 to 15-mer) labeled at the N-terminus with photocleavable biotin (PC-Biotin) [Olejnik, Sonar, Krzymanska-Olejnik and Rothschild (1995) Proceedings of the National Academy of Science (USA) 92: 7590-7594] were used as PC-Mass-Tags. Each kinase-bead species was encoded with a single unique PC-Mass-Tag (see Error! Reference source not found, for all kinases and PC-Mass-Tags). Higher capacity coding for a larger bead library is possible using two or more PC -Mass-Tags on each bead as demonstrated in Figure 2A-C.

Synchronization of MALDI-MS and fluorescence images as described previously [Lim, Liu et al. (2014) Rapid Communications in Mass Spectrometery 28: 49-62] was used to validate the PC-Mass-Tag coding of individual kinase-beads in the library. Since all 50 kinases contained a common FLAG epitope tag, the bead library was probed with a fluorescently labeled (DyLight650®) anti-FLAG antibody. A specific kinase (LCK) chosen in the library was also simultaneously probed with an LCK-specific antibody that was fluorescently labeled with a different fluorophore (Phycoerythrin). After antibody binding, the 50-member kinase-bead library was randomly incorporated into a micro-well plate to form the array (micro-well plates contain approximately 1 million wells in the footprint of a standard microscope slide, each well sized large enough to hold only a single 34 μπι bead [Lim, Liu et al. (2014) Rapid Communications in Mass Spectrometery 28: 49-62]).

Results:

Figure 3A shows a 2-color fluorescence image of the resulting array, whereby red indicates anti-FLAG antibody detection (all kinase-beads) and yellow is the anti-LCK antibody detection (specific kinase-beads). This same array was also imaged by MALDI-MS to detect the PC-Mass-Tag residing on each bead. The blue in Figure 3A is the overlaid (synchronized) MALDI-MS image of the PC-Mass-Tag species used to code for the LCK kinase beads. As seen, it correctly aligns with the yellow fluorescence of the anti-LCK antibody. Note that MALDI-MSI detected approximately 50% of the beads based on visual comparison to the anti-LCK and anti-FLAG fluorescence images, which may be the result of uneven MALDI-MS matrix coating or incomplete matrix penetration into all wells.

Figure 3B shows a colorized MALDI-MS image of the same random bead-array for 9 more (excluding LCK) selected PC-Mass-Tags corresponding to 9 different kinase-bead species from the entire 50-member library. The MALDI-MS image was again overlaid onto the common anti-FLAG fluorescence image (with the fluorescence now shown in white). Each discrete MALDI-MS spot aligns with a bead detected by the anti-FLAG antibody. Figure 3C shows color-coded overlaid MALDI-MS spectra from 10 representative individual beads selected from the 10 kinase-bead species (see circled beads in Figure 3 A &B). This data confirms that single-bead resolution was obtained and that no bead-to-bead cross-contamination of PC-Mass-Tags occurs, since only a single PC-Mass-Tag species appears in each spectral trace.

EXAMPLE 2

DEMONSTRATION OF DRUG-PROTEIN INTERACTION SCREENING USING BEAD-GPS TECHNOLOGY

This example validated the ability of Bead-GPS to detect drug-protein interactions and also to simultaneously screen multiple drugs against the entire kinase-bead library. Two model drugs were chosen for this purpose, Dasatinib and Brigatinib. Dasatinib (BMS-354825) is a BCR-ABL and SRC family tyrosine kinase inhibitor used to treat chronic myelogenous leukemia (CML) [9]. Brigatinib (AP26113) is a dual inhibitor of anaplastic lymphoma kinase (ALK) and mutant epidermal growth factor receptor (mEGFR), developed to be used as a second-line drug for non-small cell lung cancer patients that exhibit a drug -resistant mutation in ALK (mALK with L1196M) [10].

The kinase-bead library was simultaneously treated with both drugs, extensively washed and then incorporated into the micro-well plate to form the random bead-array. MALDI-MSI scanning was then performed on a 5.8 x 6 mm region of the array (in this case, fluorescence imaging is not required). The readout was performed using a SimulTOF 200 Combo MALDI-MS instrument (SimulTOF Systems, Sudbury, MA) which provided high mass resolution (m/Am = 20,000), a high repetition rate Nd:YLF laser (5 kHz), high digitizer acquisition rate (50-100 pixel s/second) and continuous laser raster scanning. The MALDI laser acted to simultaneously desorb/ionize the PC-Mass-Tag and the drugs which non-covalently bind to the proteins attached to each bead. Key parameters for the experiment are listed in Error! Reference source not found.. Briefly, the MALDI-MSI scan was performed at 50 μιη pixel size over a period of 40 minutes which covered 16,800 micro-wells. By manually enumerating discrete beads (spots) in representative areas of the MALDI-MSI image and comparing to the pixel count for that area, it was determined that on average each bead comprised 1 pixel (which agrees with the 45 μιη diameter of the micro-wells [18] compared to the 50 μιη pixel size). Therefore, based on pixel counts, an estimated 9,235 beads were detected by MALDI-MSI in the scanned region (average bead redundancy per kinase of 181). This corresponded to beads detected in 55% of the total micro-wells. At this detected bead density (264/mm²) it is estimated that 495,000 total beads would be detected on a single chip the size of a standard 75 x 25 mm microscope slide (note that either the entire array can be used for an experiment or the array can be sectored into sub-arrays using silicone gaskets).

To process the data, the SimulTOF software was first used to detect PC-Mass-Tag and drug peaks in all scanned spectra and calculate the monoisotopic peak area. All pixels containing none or more than one detected PC-Mass-Tag were then removed from the dataset, leaving only those pixels with a single PC-Mass-Tag species. For each kinase species (each PC-Mass-Tag), monoisotopic peak area for each drug was averaged for all PC-Mass-Tag-positive pixels for that kinase (average of 181 such pixels, i.e. beads, per kinase). To reduce noise, on a per kinase basis, pixels where the monoisotopic peak area for the drug fell outside of one standard deviation of the mean were rejected. Furthermore, kinases which had pixel (bead) counts <10% of the average were eliminated due their low number of replicate data points, which resulted in the loss of 3 kinases out of 50 for a 94% success rate.

Results:

Figure 4A-C displays the Bead-GPS results for Dasatinib and Brigatinib. For many but not all of the kinases in the library, the K_d or IC₅₀ values for the drugs are known (see Error! Reference source not found.). Therefore, to validate the Bead-GPS results, drug-kinase interactions were categorized following an earlier study [10] as weak for K_d or IC₅₀ >100 nM; medium for <100 nM and >10 nM; and strong for <10 nM. Since Bead-GPS measures binding, K_d values were used instead of IC₅₀ when available. For Dasatinib, K_d values were available from the Drug2Gene database[19] for 42 kinases. Bead-GPS results for Dasatinib for these kinases are shown in Figure 4A. The known weak interactions give consistently zero or negligible MALDI-MS Dasatinib signals, while the known strong interactions give a range of strong positive MALDI-MS signals (note there were no known interactions falling into the medium category for Dasatinib). If a cutoff to score hits was set at three standard deviations above the mean for the known weak interactions (black dotted lines in Figure 4A-C), 100% sensitivity and specificity was achieved for the known strong interactions.

Although the second drug, Brigatinib, is not nearly as well characterized as Dasatinib (and K_d values not available), IC₅₀ values for 21 of the kinases have been published [10]. Figure 4b shows the Bead-GPS results for these kinases. Using the same cutoff and scoring method as above, 100% sensitivity for the known strong interactions was observed. In the case of medium interactions, an 83% sensitivity and 100% specificity was obtained. The full Bead-GPS binding profile for all kinases and both drugs is shown in Figure 4c. Importantly, as expected Dasatinib and Brigatinib show completely different binding profiles with no overlap. Based on the data in Figure 4A-C, the weakest interaction detected by Bead-GPS of the drug-protein pairs expected to bind was -80 nM (IC₅₀). However, future optimizations such as washing steps and studies of more interactions of various strengths will be necessary to more accurately determine the detection limits. It should be emphasized that the magnitude of the Bead-GPS signals among different kinases is not expected to quantitatively correlate with the magnitude of the corresponding K_d or IC₅₀. For example, if the Bead-GPS signals are plotted against K_d or IC₅₀ for all kinases, a linear correlation is not found. Factors which would tend to produce MALDI-MS intensities which do not directly correlate with binding strength include different amounts of target protein on the different bead species and, differential extraction/elution of drugs from the various bead species (by matrix compound and laser energy). We propose that this approach would be most useful as a method to initially screen for potential off-target interactions (hits). Once these hits are determined, they would be further validated and analyzed with existing quantitative and kinetic methods such as FRET and SPR assays which are better suited for more in-depth analysis of smaller numbers of protein targets.

Thus, specific compositions and methods of proteome-wide drug screening using mass spectrometric imaging (MSI) of bead-arrays have been disclosed. It should be apparent, however, to those skilled in the art that many more modifications besides those already described are possible without departing from the inventive concepts herein. Moreover, in interpreting the disclosure, all terms should be interpreted in the broadest possible manner consistent with the context. In particular, the terms "comprises" and "comprising" should be interpreted as referring to elements, components, or steps in a non-exclusive manner, indicating that the referenced elements, components, or steps may be present, or utilized, or combined with other elements, components, or steps that are not expressly referenced.

Although the invention has been described with reference to these preferred embodiments, other embodiments can achieve the same results. Variations and modifications of the present invention will be obvious to those skilled in the art and it is intended to cover in the appended claims all such modifications and equivalents. The entire disclosures of all applications, patents, and publications cited above, and of the corresponding application are hereby incorporated by reference.

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Photocleavable Peptide-Coded Random Bead- Arrays," Rapid Communications in Mass Spectrometery 25(1), 49-62.

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TABLE 1

TABLE 2

Claims

CLAIMS: We claim:

1. A label -free method to measure the binding of one or more prey molecules to a bead library consisting of bait molecules immobilized on beads comprising:

a) Making a mass-tag encoded bait-bead library whereby each bead carries a unique bait molecule and a corresponding mass-tag;

b) Incubating said prey molecules with the bead library;

c) Making an array with said bead library;

d) Measuring the mass spectrum from at least one bead from each member of the bait-bead library carrying a unique bait molecule in said array; and

e) Determining from the mass spectral measurements in step (d) the relative binding of each prey molecule with each member of the bait-bead library.

2. The method of claim 1, wherein said bait molecules are selected from the group consisting of proteins, polypeptides, peptides, glycoproteins, proteoglycans, carbohydrates, lipids, nucleic acids, DNA, RNA and macromolecular structures comprised of a combination of biomolecules such as ribosomes which contain proteins and nucleic acid.

3. The method of claim 1, wherein said prey molecules are drug compounds, candidate drug compounds or lead drug compounds which are selected from the group consisting of small molecule compounds, organic chemical compounds, inorganic chemical compounds, proteins, glycoproteins, proteoglycans, antibodies, polypeptides, peptides, nucleic acids, lipids, carbohydrates, linear polymers, and branched polymers as well as biomimetics such as peptoids.

The method of claim 1, wherein said array is formed in micro-well plates, each well sized to hold only a single bead.

The method of claim 4, wherein said micro-well plates used to form the array comprise an indium tin oxide surface coating.

A label-free method to screen drug compounds for on and off -target interactions comprising: a) Making a mass-tag encoded bait-bead library whereby each bead carries a unique target molecule and a corresponding mass-tag;

b) Incubating said compound molecules with the bead library;

c) Making an array with said bead library;

d) Measuring the mass spectrum from at least one bead from each member of the target-bead library carrying a unique target molecule in said array; and

e) Determining from the mass spectral measurements in step (d) the relative binding of each compound molecule with each member of the target -bead library.

The method of claim 6, wherein said target molecules are selected from the group consisting of proteins, polypeptides, peptides, glycoproteins, proteoglycans, carbohydrates, lipids, nucleic acids, DNA, RNA and macromolecular structures comprised of a combination of biomolecules such as ribosomes which contain proteins and nucleic acid.

8. The method of claim 6, wherein said compound molecules are drug compounds, candidate drug compounds or lead drug compounds which are selected from the group consisting of small molecule compounds, organic chemical compounds, inorganic chemical compounds, proteins, glycoproteins, proteoglycans, antibodies, polypeptides, peptides, nucleic acids, lipids, carbohydrates, linear polymers, and branched polymers as well as biomimetics such as peptoids.

9. The method of claim 6, wherein said array is formed in micro-well plates, each well sized to hold only a single bead.

10. The method of claim 9, wherein said micro-well plates used to form the array comprise an indium tin oxide surface coating.

11. A label -free method to measure the binding of one or more prey molecules to a bead library consisting of bait molecules immobilized on beads comprising:

b) Making an array with said bead library;

c) Incubating said prey molecules with the bead library;

12. The method of claim 11, wherein said bait molecules are selected from the group consisting of proteins, polypeptides, peptides, glycoproteins, proteoglycans, carbohydrates, lipids, nucleic acids, DNA, RNA and macromolecular structures comprised of a combination of biomolecules such as ribosomes which contain proteins and nucleic acid.

13. The method of claim 11, wherein said prey molecules are drug compounds, candidate drug compounds or lead drug compounds which are selected from the group consisting of small molecule compounds, organic chemical compounds, inorganic chemical compounds, proteins, glycoproteins, proteoglycans, antibodies, polypeptides, peptides, nucleic acids, lipids, carbohydrates, linear polymers, and branched polymers as well as biomimetics such as peptoids.

14. The method of claim 11, wherein said array is formed in micro-well plates, each well sized to hold only a single bead.

15. The method of claim 14, wherein said micro-well plates used to form the array comprise an indium tin oxide surface coating.

16. A label-free method to screen drug compounds for on and off-target interactions comprising: a) Making a mass-tag encoded bait-bead library whereby each bead carries a unique target molecule and a corresponding mass-tag;

b) Making an array with said bead library;

c) Incubating said compound molecules with the bead library;

d) Measuring the mass spectrum from at least one bead from each member of the target-bead library carrying a unique target molecule in said array; and e) Determining from the mass spectral measurements in step (d) the relative binding of each compound molecule with each member of the target -bead library.

17. The method of claim 16, wherein said target molecules are selected from the group consisting of proteins, polypeptides, peptides, glycoproteins, proteoglycans, carbohydrates, lipids, nucleic acids, DNA, RNA and macromolecular structures comprised of a combination of biomolecules such as ribosomes which contain proteins and nucleic acid.

18. The method of claim 16, wherein said compound molecules are drug compounds, candidate drug compounds or lead drug compounds which are selected from the group consisting of small molecule compounds, organic chemical compounds, inorganic chemical compounds, proteins, glycoproteins, proteoglycans, antibodies, polypeptides, peptides, nucleic acids, lipids, carbohydrates, linear polymers, and branched polymers as well as biomimetics such as peptoids.

19. The method of claim 16, wherein said array is formed in micro-well plates, each well sized to hold only a single bead.

20. The method of claim 19, wherein said micro-well plates used to form the array comprise an indium tin oxide surface coating.