WO2016193980A1

WO2016193980A1 - Methods and kits for detection and quantification of large-scale post translational modifications of proteins

Info

Publication number: WO2016193980A1
Application number: PCT/IL2016/050570
Authority: WO
Inventors: Amit TZUR; Doron Gerber
Original assignee: Bar Ilan University
Priority date: 2015-06-03
Filing date: 2016-06-02
Publication date: 2016-12-08

Abstract

The present invention provides a powerful tool for analyzing post-translational modifications (PTMs) in proteins. The invention provides methods and kits enabling accurate determination of PTMs in a biological sample.

Description

METHODS AND KITS FOR DETECTION AND QUANTIFICATION OF LARGE- SCALE POST TRANSLATIONAL MODIFICATIONS OF PROTEINS

FIELD OF THE INVENTION

The invention relates to identification and quantitative detection of post-translational modification (PTM) of proteins. The system provided by the instant invention and methods and kits ensuing therefrom are applicable for identification, classification, characterization and quantification of PTMs using purified components and/or quasicellular environments (cell or tissue extracts), and for diagnosis of biological conditions pertaining to PTMs.

BACKGOUND REFERENCES

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Jacobson. 2012. Enhanced multiplex genome engineering through co-operative oligonucleotide co-selection. Nucleic acids research. 40:el32.

Ciechanover, A., and A.L. Schwartz. 2004. The ubiquitin system: pathogenesis of human diseases and drug targeting. Biochimica et biophysica acta. 1695:3-17.

Ganoth, D., G. Bornstein, T.K. Ko, B. Larsen, M. Tyers, M. Pagano, and A. Hershko. 2001.

The cell-cycle regulatory protein Cksl is required for SCF(Skp2)-mediated ubiquitinylation of p27. Nature cell biology. 3:321-324.

Gerber, D., S.J. Maerkl, and S.R. Quake. 2009. An in vitro microfluidic approach to generating protein-interaction networks. Nature methods. 6:71-74.

Glick, Y., D. Avrahami, E. Michaely, and D. Gerber. 2012. High-throughput protein expression generator using a microfluidic platform. Journal of visualized experiments : JoVE:e3&49.

Harel, M., P. Oren-Giladi, O. Kaidar-Person, Y. Shaked, and T. Geiger. 2015. PROMIS-

Quan: a novel proteomic method for plasma biomarker quantification. Molecular & cellular proteomics : MCP.

Meier, M., R.V. Sit, and S.R. Quake. 2013. Proteome-wide protein interaction measurements of bacterial proteins of unknown function. Proceedings of the National Academy of

Sciences of the United States of America. 110:477-482.

Merbl, Y., and M.W. Kirschner. 2009. Large-scale detection of ubiquitination substrates using cell extracts and protein microarrays. Proceedings of the National Academy of

Sciences of the United States of America. 106:2543-2548. Merbl, Y., P. Refour, H. Patel, M. Springer, and M.W. Kirschner. 2013. Profiling of ubiquitin-like modifications reveals features of mitotic control. Cell. 152:1160-1172.

Neveu, G., R. Barouch-Bentov, A. Ziv-Av, D. Gerber, Y. Jacob, and S. Einav. 2012.

Identification and targeting of an interaction between a tyrosine motif within hepatitis C virus core protein and AP2M1 essential for viral assembly. PLoS pathogens. 8:el002845.

Ozlu, N., M.H. Qureshi, Y. Toyoda, B.Y. Renard, G. Mollaoglu, N.E. Ozkan, S. Bulbul, I.

Poser, W. Timm, A.A. Hyman, T.J. Mitchison, and J.A. Steen. 2015. Quantitative comparison of a human cancer cell surface proteome between interphase and mitosis.

The EMBO journal. 34:251-265.

Singh, S.A., D. Winter, M. Kirchner, R. Chauhan, S. Ahmed, N. Ozlu, A. Tzur, J.A. Steen, and H. Steen. 2014. Co-regulation proteomics reveals substrates and mechanisms of

APC/C-dependent degradation. The EMBO journal. 33:385-399.

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Zur, A., and M. Brandeis. 2001. Securin degradation is mediated by fzy and fzr, and is required for complete chromatid separation but not for cytokinesis. The EMBO journal. 20:792-801.

BACKGROUND OF THE INVENTION

Protein post-translational modifications (PTMs) vastly diversify eukaryotic proteomes and are integrated in essentially all cellular processes. Proteomic approaches, such as mass spectrometry (MS), have been instrumental in monitoring global molecular dynamics for research and clinical applications (Harel et al., 2015; Ozlu et al., 2015; Singh et al., 2014). However, even in this modern era, large-scale analyses of PTMs by MS is challenging due to the limited number of modified peptides derived from proteins that, by themselves, may not be abundant. Moreover, comprehensive PTM analysis by MS often requires significant amounts of biological material that may not be available.

PTM analysis using pre-designed protein arrays was anticipated to overcome these limitations because of the equimolar amount of the arrayed proteins. Large-scale protein arrays have been successfully integrated into PTM research (Merbl and Kirschner, 2009; Merbl et al., 2013). However, this technology relies on pre -purified proteins that are arrayed on a surface and thus, is incompatible with biochemically challenging proteins, let alone insoluble proteins. Moreover, the production of recombinant protein arrays is impractical in-house. Therefore, such arrays cannot be used fresh, and they are inherently limited to certain designs, protein compositions, and model organisms of commercial value.

WO2010/044892 to Merbl Y. and Kirschner MW entitled "Diagnostic method based on large scale identification of post-translational modification of proteins" described methods for PTM analysis of functional extracts from cells and tissues. These methods however are limited to purified proteins stored in an un-physiological environment and thus, limited by protein composition and functionality. These methods are also limited by their ability to provide an easy and accurate quantitation and their ability to confer industrial applicability thereof in clinical or other practice.

There is need to establish modular and versatile systems and methods that rely on minute amounts of biological material and provide a cost-effective technology for accurate determination and analysis of PTMs of diverse and functional proteins and are thus applicable for a broad range of basic, biomedical and biomarker research.

SUMMARY OF THE INVENTION

In a first aspect, the invention relates to a method for analyzing post translational modifications (PTMs) of proteins in a biological sample. More specifically, the method of the invention comprises in its first step (a), providing a plurality of tagged proteins or any peptides thereof. In some embodiments, the tagged protein provided may be further or alternatively labeled with a detectable label or moiety. The second step (b) involves measuring the signal intensity of a detectable tag, and/or in case applicable the detectable label or moiety, for each protein of said plurality of tagged proteins. The next step (c) requires contacting each protein of the plurality of tagged and/or labeled proteins with the sample or any functional extract thereof under conditions that allow PTM reactions. In the next step (d), the signal intensity of a detectable label or moiety specific for PTM generated by each individual PTM reaction of step (c) is measured and determined. The next step (e), involves normalizing for each protein the signal intensity measured in step (d) with the signal intensity measured in step (b), to obtain the PTM value for each protein of said plurality of labeled and/or tagged proteins. The method of the invention thereby provides analysis of the PTMs of proteins by the examined sample. In certain embodiments, the tagged proteins of the invention may be provided as freshly expressed proteins and therefore, in certain embodiments, the method of the invention may involved the step of providing a plurality of expression templates encoding said tagged proteins.

In a second aspect, the invention relates to a diagnostic method for detecting a pathological condition associated with altered PTM state of at least one protein in a subject. In more specific embodiments, such method may comprise in a first step (a) the provision of a plurality of labeled and/or tagged proteins or any peptides thereof. In the next step (b) measuring the signal intensity of a detectable tag for each protein of the plurality of labeled and/or tagged protein. In step (c), contacting a biological sample of the subject or any functional extract of the sample with each protein of said plurality of labeled and/or tagged proteins under conditions that allow PTM reactions. In step (d), measuring the signal intensity of a detectable label or moiety specific for PTM generated by each individual PTM reaction of step (c). The next step (e) involves normalizing for each protein the signal intensity measured in step (d) with the signal intensity measured in step (b), to obtain the PTM value for each protein of said plurality of labeled and/or tagged proteins. Finally, in step (f), determining if the PTM value for each protein obtained in step (e) is any one of positive or negative with respect to a predetermined standard PTM value of the specific protein in at least one of a population of subjects suffering from said condition and in a population of subjects that do not suffer from said condition or alternatively, to a PTM value of the specific protein in at least one control sample. It should be appreciated that in certain embodiments the plurality of tagged proteins provided by the invention may be further labeled with a detectable label or moiety to facilitate quantification thereof as will be detailed herein after.

In a third aspect, the invention provides a kit for use in analyzing PTMs of proteins in a biological sample, the kit comprising:

(a) at least one of a plurality of cDNA molecules encoding tagged and/or labeled proteins or any peptides thereof and a plurality of tagged proteins or any peptides thereof; and

(b) at least one of reagent/s, enzyme/s, substrate/s, co-factor/s and inhibitor/s required for a PTM reaction. BRIEF DESCRIPTION OF THE FIGURES

Figures 1A-1C: Integrated Microfludic platform for PTM Analysis (IMPA) concept Figure 1A shows a typical microfluidic device comprising unit cells with DNA and protein chambers separated by micromechanical valves. Arrayed proteins deposited either as open reading frames (ORFs) spotted and expressed in the DNA chambers and diffused and immobilized in the protein chambers via a protein tag (black triangle), or deposited directly in the protein chambers, are quantified by a fluorescent- or immunodetectable-tag (grey dotted triangle). Surface chemistry is illustrated on the right.

Figure IB shows the step of introducing cell extracts supplemented with quantifiable modifiers into the protein chambers.

Figure 1C shows determination of PTM-to-protein 'bait' ratio (black and grey dots, respectively) in each unit cell.

Figures 2A-2B: Quantification of signal in protein chambers

Figure 2A shows two protein chambers and a pneumatically-regulated 'button' enabling mechanical washing of an unbound material.

Figure 2B shows quantification of net fluorescent signal for each dot on the chip by calculation of a median fluorescent intensity of pixels in the area under 'button' corresponding to the inner circle (80 μπι diameter) and subtraction of a median fluorescent intensity in the area between the inner and outer (160 μπι diameter) circles corresponding to local background noise, using Genepix and Microsoft XI software.

Figures 3A-3C: Compatibility demonstration for membrane, nuclear and cytoplasmic proteins

Figure shows linear Myc/His-double-tagged ORFs encoding for nuclear, membrane and cytoplasmic proteins generated by assembly-PCR using Open Biosystems' full-length human ORF library, deposited on chip via His-Tag and quantified by Cy3-coupled anti-Myc antibodies. The 3D histogram shows an average expression signals (quadruplicates) of the proteins on protein arrays.

Figure 3A shows expression signals of about 1000 nuclear proteins on protein arrays

Figure 3B shows expression signals of about 1000 membrane proteins on protein arrays. The membrane proteins were expressed in the present of a reticulocyte lysate supplemented with canine microsomal membranes.

Figure 3C shows expression signals of about 1000 cytoplasmic proteins on protein arrays. Figures 4A-4G: A proof-of-concept on-chip Tyrosine (Tyr) phosphorylation assay

Figure 4A is a graphic representation of on-chip Tyr phosphorylation of a target protein detected by fluorescently labeled anti-P-Tyr antibodies (Cy5).

Figure 4B shows Btk-mediated on-chip phosphorylation of Btk target peptide, a peptide core sequence VALYDYM (SEQ ID NO. 1), deposited in the protein chamber and incubated with recombinant Btk. P-Tyr levels were quantified using Cy5-coupled P-Tyr-100 antibodies. Figure 4C shows Btk-mediated on-chip phosphorylation of Plcg2 (positive control) and Securin (negative control) deposited as double-tagged In- Vitro Translation (IVT) products. P-Tyr reaction was performed and quantified as above, protein levels were quantified using Cy3-coupled anti-Myc antibodies. Average P-Tyr levels and P-Tyr-to-protein ratios were calculated from 15 < n < 30 dots.

Figure 4D shows on-chip phosphorylation of arrayed proteins mediated by Abl kinase, including four known Abl targets (*) and four negative controls. P-Tyr and protein levels were quantified as above. The threshold value (dotted line) corresponds to 2 SD above the P- Tyr-to-protein ratio of Btk (negative control).

Figure 4E shows on-chip phosphorylation of Btk target peptides incubated with HEK293 extracts per se and supplemented with Tyr phosphatase inhibitor, Sodium Orthovanadate (SOV) and/ or recombinant Btk.

Figure 4F shows on-chip phosphorylation of Plcg2 and Securin in the presence of HEK293extracts under conditions as above.

Figure 4G shows on-chip dephosphorylation analysis of Btk target peptide in a dephosphorylated form, which was deposited on the chip and incubated with buffer (bar) or HEK293 cell extracts supplemented with increasing amounts of SOV. P-Tyr and protein levels were quantified as above. Data were normalized to maximum activity.

Figures 5A-5C: On-chip large-scale Tyr phosphorylation analysis

Figure 5A shows fractions of the array of Figure 3A after incubation with HEK293 extracts supplemented withlO mM SOV. P-Tyr signal for Rad9 and Hck are indicated. Selected signals are magnified in a framed area (white arrowheads). Bars represent average quadruplicates for each protein. A graphic representation of on-chip Tyr phosphorylation of a target protein detected by fluorescently labeled anti-P-Tyr antibodies (Cy5) illustrates the experiment (left) that is also shown as raw data (right).

Figure 5B shows fractions of the same array without SOV supplementation. A graphic representation of on-chip Tyr phosphorylation of a target protein detected by fluorescently labeled anti-P-Tyr antibodies (Cy5) illustrates the experiment (left) that is also shown as raw data (right).

Figure 5C shows top- 10 nuclear proteins identified in the above experiment as having P-Tyr modification, seven of which are known P-Tyr targets (+).

Figure 6A-6C: On-chip large-scale analysis of Tyr Autophosphorylation

Figure shows a newly generated protein array (Fig. 6A) with no HEK293 incubation, specifically areas identified with Hck (Fig. 6B) and Rad9 (Fig. 6C) proteins. P-Tyr signals at Hck are result of auto-phosphorylation.

Figure 7: Proof of concept for PTM analysis on membrane proteins using SRC kinase

The Figure shows a bar graph demonstrating a comparison between Tyrosine phosphorylation analysis of three know membrane or soluble protein targets of SRC kinase (e.g., Anxa6, Trip6, Bmx), a soluble target and negative control membrane proteins (e.g., Pnmt, IL-21 RECEPTOR, ucn2) that are not phosphorylated by SRC kinase. Phosphorylation signal is normalized to target protein level. Kinase activity is expressed as the ratio in phosphor- tyrosine signal between extracts over expressing active or dead SRC kinase.

Figures 8A-8D: On-chip ubiquitination assay

Figure 8A shows a schematic representation of on-chip ubiquitination using fluorescently labeled ubiquitin Ub (circles) and tagged (grey dotted triangle) substrates.

Figure 8B shows off-chip time-dependent degradation of ³⁵S-labeled Securin-eGFP (Sec- eGFP), its stable undegradable mutant (A64Sec-eGFP) and p27 IVT products by incubation with extracts of HeLa S3 cells synchronized to Gl phase (Gl extracts) supplemented with Anaphase-Promoting Complex/Cyclosome (APC/C) activating enzyme UbcHIO or its dominant negative mutant UBcH10^DN, or GST-tagged APC/C inhibitor Emil. Reactions analyzed by SDS-PAGE and autoradiography.

Figure 8C shows reactions as above further supplemented with concentrations of non-labeled or rhodamine-labeled Ub. Data are normalized to maximum signal at time t=0.

Figure 8D shows on-chip analysis of IVT products of Sec-eGFP, A64Sec-eGFP, p27 and Kifcl-eGFP incubated with Gl extracts supplemented with the indicated reagents and Rd-Ub. The histogram shows average (n=20) ratios of Rd-Ub/protein as detected by the GFP tag or Cy2-labeled anti-Myc antibodies, representative raw data are shown below. Figures 9A-9C: Degradation assays of eGFP, p27 and Securin

Figure 9A shows time -dependent degradation of ³⁵S-labeled eGFP IVT product incubated with Gl extracts supplemented, Ub and either UbcHIO or UbcH10^DN. Data are normalized to maximum signal at time i=0.

Figure 9B shows time-dependent degradation of ³⁵S-labeled p27 IVT product incubated with cell extracts generated from asynchronous (unsync) or S-phase arrested HeLa S3 cells.

Figure 9C shows quantification of degradation assays in Figure 8B.

Figure 10: On-chip ubiquitination ofeGFP

Figure shows average ratios («=20) of Rd-Ub/protein (as detected by GFP signal) of GFP IVT product deposited on chip via anti-GFP antibodies and incubated with Gl extracts supplemented with Rd-Ub and either wt- or dominant negative UbcHIO.

Figure 11: On-chip ubiquitination of Geminin

Figure shows an analogous ubiqitination analysis of Geminin-eGFP IVT product deposited on chip (via anti-GFP antibodies) and incubated with Gl extracts supplemented with Rd-Ub and either wt- or dominant negative UbcHIO, or APC/C inhibitor Emil .

Figures 12A-12I: On-chip analysis Ub-chain preference

Figure 12A shows a schematic representation of on-chip Kl l-specific ubiquitination using K48R- mutant Ub to form K-l 1-Ub chains.

Figure 12B shows on-chip analysis of Sec-eGFP ubiquitination with Gl extracts containing Cy5-labeled K48R-Ub or Kl lR-Ub. The histogram shows average ratios (n=20) of Cy5- mutant-Ub/protein, representative raw data are shown on the right.

Figure 12C shows degradation assays of ³⁵S-Sec-eGFP with Gl extracts supplemented with excess of unlabeled wild type (wt) Ub or mutant Ub, wherein Lysin (K) 11 or 48 were substituted with Arginine (R), K48R-Ub or Kl lR-Ub.

Figure 12D shows a schematic representation of on-chip Ub-chain preference of Securin, wherein wt Rd-Ub (dotted gray circle) is displaced by unlabeled K48R-Ub.

Figure 12E shows on-chip ubiquitination of Sec-eGFP with Gl extracts in the presence of wt Rd-Ub and an excess of unlabeled K48R-Ub or Kl lR-Ub. The histogram shows average ratios (n=20) of Rd-Ub/protein (as detected by GFP tag), representative raw data are shown on the right.

Figure 12F and 12G show on-chip ubiquitination of Kifcl-eGFP in the above conditions. Figure 12H and 121 show degradation assays of ³⁵S-Kifc-eGFP with Gl extracts in the presence of wt or mutant Ub (n=3). Kl l/K48-only-Ub mutants carry a single K residue.

Figure 13: On-chip analysis of Ub-chain preference for Geminin

Figure shows on-chip ubiquitination of Geminin-eGFP IVT product incubated with Gl in the presence of wt Rd-Ub and an excess of unlabeled K48R-Ub or Kl lR-Ub. The histogram shows average ratios (n=20) of Rd-Ub/protein (as detected by GFP tag).

DETAILED DESCRIPTION OF THE INVENTION

The present invention makes part of a rapidly growing field of proteomics, a discipline of molecular biology that is concerned with the systematic, high-throughput approach to protein expression analysis of a cell or an organism. The term "proteome" refers to the entire complement of proteins, including modifications of a particular set of proteins, produced by an organism or a cellular system, and further its dynamics (or variation) with time and distinct requirements and stresses that a cell or organism undergoes. The term "proteomics" refers to a large-scale comprehensive analysis of a specific proteome, including information on protein abundances, their variations and modifications, in other words, qualitative and quantitative features of said proteome, along with their interacting partners and networks. The present invention further pertains to the field of "clinical proteomics", a sub-discipline of proteomics that involves application of proteomic technologies on clinical specimens such as blood or tissue samples to identify unique bio-signatures and biomarkers responsible for the diagnosis, prognosis and therapeutic prediction of a disease.

Specifically, the present invention combines structural and functional analyses to identify genuine and significant PTM signatures of a biological sample across multiple pre-defined conditions, and thereby to produce a comprehensive functional map of this sample, as being representative of certain cells and tissues and/or biological conditions, in terms of potential capability in inducing enzyme-substrate and protein-protein interaction as markers of its phenotypic attributes. In this sense, the present invention, unlike genomic-based or simple protein arrays, pertains not only to content but also with functionality of a certain proteome to make inferences about its most probable phenotype. To which end, the present invention focuses on PTMs as being important determinants of functionality of a protein with proven impact and direct bearing on normal and abnormal phenotypes, and further achieves enhancement of PTM analysis to enable bona fide comparison between samples and prediction of phenotypic outcomes. The present invention combines high throughput protein microarrays as a powerful tool for capturing and measuring proteome content and functional analyses represented by many specific directed functional bioassays. This integrated approach is facilitated by use microfluidic platform technology comprising a modular set of microfluidic unit operations containing various cDNA and/or protein molecules, which allow coordinated implementation of multiple biochemical assays including a number of labeling tags and/or detection methods to enable inter- and intra-unit assay standardization. Feasibility and advantageousness of this approach was presently exemplified using modular Integrated Microfluidic Platform for PTM Analysis (IMPA) for systematic and comprehensive on-chip analysis of simple and complex PTMs in cells extracts of various sources. IMPA has proven herein to be compatible with qualitative and quantitative analysis of ubiquitination and large-scale analysis of phosphorylation and dephosphorylation, as well as further preferential analysis of these PTMs relating to autophosphorylation or Ub-chain preference. It is therefore conceivable that IMPA due its unique structural and functional features may represent a modular platform for functional analysis of wide-ranging PTMs of various proteins, specifically, membrane proteins, nuclear protein and cytoplasmic proteins, in biological samples of various origins with reproducibility and analytical validation to enable translation thereof to diagnostic and clinical purposes.

More specifically, the presently introduced inventive concept of Modular Integrated Microfluidic Platform for PTM Analysis (IMPA) contemplates large scale and high throughput qualitative and quantitative analysis of PTMs of proteins and enzymes in biological samples from various sources. The potency, specificity and flexibility of IMPA were exemplified in versatile on-chip assays of simple and complex PTMs, such as tyrosine phosphorylation and ubiquitination, in quasicellular environments employing cell extracts (EXAMPLES 4 to 7). Unlimited by design and protein composition, and further relying only on minute amounts of biological material, IMPA proved to be an improved cost-effective technology with potential applicability to basic and translational research as well as to clinical purposes. IMPA can be utilized for identifying targets of specific enzymes in physiologically relevant contexts, as well as for elucidating PTM fingerprints of particular cells or tissues.

The IMPA concept combines microfluidics with in situ protein expression from a cDNA microarray. More specifically, IMPA uses a microfluidic platform, originally designed for screening protein interactions (Gerber et al., 2009; Glick et al., 2012; Meier et al., 2013; Neveu et al., 2012; Zheng et al., 2012), in which a multilayered microfluidic apparatus is aligned on a spotted cDNA library encoding thousands of double-tagged proteins (DNA chambers) (Figure 1A). Reticulocyte lysate is then applied to the chip for in vitro translation (IVT). Protein expression is nearly unaffected by protein length and compatible with transmembrane- and other insoluble proteins. Each freshly synthesized protein is then (i) captured in a protein chamber controlled by pneumatic valves; (ii) immobilized on the surface via one affinity tag; and (iii) quantified by a fluorescent tag or fluorescently labeled antibodies against the second affinity tag (Figures 2A-2B). After washing, an immunodetectable- or fluorescently labeled-protein modifier, mixed with biologically active cell extracts and/or recombinant enzymes, is applied to all or some protein chambers (Figure IB); about 12 μΐ of reagents are consumed. The use of native cell extracts maintains the stoichiometric environment of a particular cell or tissue at a given physiological/pathological condition during the analysis; bridging the gap between in vitro and in vivo. Moreover, cell extracts are easy to manipulate, and well known for their capacity to recapitulate complex cellular processes and reduce promiscuous enzymatic activities associated with non- physiological environments. Following incubation and washing, PTM signal can then be normalized to the total protein in each unit cell (Figure 1C). This PTM-to-protein normalization is unique.

IMPA can be optimized for any type of PTM that can be fluorescently or isotopically labeled or tagged or alternatively or additionally, immunolabeled. In combination with parallel DNA synthesis (Carr et al., 2012), the microfluidic device, which is placed on a micro arrayed open reading frame (ORF) library, is readily available to study PTM fingerprints of any sequenced animal models in a large spectrum of physiological environments. Automated cherry picking allows the selection of any combination of proteins in a flexible number of repeats. In its current version IMPA enables a maximum of 8 parallel independent experiments per chip. This unique feature enables the simultaneous analysis of multiple PTM signatures in various biochemical settings on a single chip; it also lowers experimental complexity and cost, and simplifies normalization.

Thus, in a first aspect, the invention relates to a method for analyzing post translational modifications (PTMs) of proteins in a biological sample. More specifically, the method of the invention comprises in its first step (a), providing a plurality of tagged proteins or any peptides thereof, or any expression template encoding the same. It should be appreciated that in some embodiments, the tagged proteins may be further labeled by a detectable label or moiety. The second step (b) involves determining the level of each of the tagged protein by measuring the signal intensity of a detectable tag and/or detectable label or moiety for each protein of said plurality of tagged proteins. The next step (c) requires contacting the sample or any functional extract thereof with each protein of the plurality of tagged proteins under conditions that allow individual PTM reactions to occur. In the next step (d), the signal intensity of a detectable label or moiety specific for PTM generated by each individual PTM reaction of step (c) is measured and determined. The next step (e), involves normalizing for each protein the signal intensity measured in step (d) with the signal intensity measured in step (b), to obtain the PTM value for each protein of said plurality of tagged proteins. The method of the invention thereby provides analysis of the PTMs of proteins when these are exposed to an examined sample and thus reflects the ability of the sample to perform the specific PTM reactions.

It should be understood that "individual" PTM reaction is as used herein is meant that each of the tagged protein/s is subjected to at least one PTM reaction, where each of the individual PTM reactions of each of the tagged proteins is determined by measuring the resulting signal intensity of the detectable label or moiety specific for said PTM. Potentially, several PTM different reactions may be performed in parallel, either simultaneously or in either order, for each of the tagged proteins, provided that different detectable label/s or moiety are used for each specific individual PTM reaction.

It should be understood that "analyzing PTMs of proteins in a biological sample" as used herein encompasses in certain embodiments, analyzing PTM of proteins by a biological sample that reflect physiological conditions of the subject or the biological source of the sample. In these embodiments, specific enzymes, factors or co-factors in the sample or any extract or preparation thereof enable PTMs of the tagged proteins provided by the methods of the invention.

It should be appreciated that the plurality of tagged proteins provided by the method of the invention may in some embodiments reflect or represent at least some of the proteins in the biological sample or in the source subject for said sample.

As used herein, the term "post-translational modification" or "PTM", as used herein refers to the covalent and generally enzymatic modification of amino acid side chains, and/or proteins backbone during or after protein biosynthesis. It should be understood that this term refers to reactions wherein a chemical moiety is covalently added to or alternatively removed from a protein, specifically, by enzymatic or non-enzymatic reaction. Many proteins can be post- translationally modified through the covalent addition of a chemical moiety (also referred to herein as a "modifying moiety") after the initial synthesis (i.e., translation) of the polypeptide chain. Such chemical moieties usually are added by an enzyme to an amino acid side chain or to the carboxyl or amino terminal end of the polypeptide chain, and may be cleaved off by another enzyme. Single or multiple chemical moieties, either the same or different chemical moieties, can be added to a single protein molecule. It should be noted however that other forms of protein post-translational modification that include proteolytic cleavage of peptide bonds, removing the initiator methionine residue, as well as the formation of disulfide bonds using linking cysteine residues, and protein splicing are also encompassed by the invention.

PTM of a protein can alter its biological function, such as its enzyme activity, its binding to or activation of other proteins, its cellular localization or its turnover, and is important in cell signaling events, development of an organism, and disease. As will be described in more detail herein after, examples of PTM covered by the method of the invention include, but are not limited to phosphorylation, ubiquitination and ubiquitin-chain preference, as demonstrated herein, as well as to any PTM reaction performed by ubiquitin-like protein, for example, sumoylation, neddylation, pupylation, ISGylation, and the like.

It should be appreciated that in some embodiments, the PTM reaction as defined by the invention further encompass the addition of Hydrophobic groups for membrane localization include myristolation, that involves the attachment of myristate (that is a C14 saturated acid), palmitoylation, attachment of palmitate, a ci6 saturated acid, isoprenylation or prenylation, that involve the addition of an isoprenoid group (e.g. farnesol and geranylgeraniol), farnesylation, geranylgeranylation, glypiation, glycosylphosphatidylinositol (GPI) anchor formation via an amide bond to C-terminal tail, and the like.

Still further, several modifications may enhance the enzymatic activity of a given enzyme. Such PTMs may include for example, lipoylation, that involves the attachment of a lipoate (C₈) functional group, covalent attachment of flavin moiety (FMN or FAD), attachment of heme C via thioether bonds with cysteins, phosphopantetheinylation, that involves the addition of a 4'-phosphopantetheinyl moiety from coenzyme A as well as retinylidene Schiff base formation.

Still further embodiments of PTMs include diphthamide formation, ethanolamine phosphoglycerol attachment and hypusine formation. PTMs involving the attachment or removal of small chemical groups include acylation, e.g. 0-acylation (esters), N-acylation (amides), 5-acylation (thioesters), and crotonylation that involves for example, addition of crotonyl to histons and acetylation, that involves the addition of an acetyl group, either at the N-terminus of the protein or at lysine residues, or alternatively deacetylation involving the removal of said acetyl group and formylation. Still further PTMs relate to alkylation, that involve the addition of an alkyl group, e.g. methyl, ethyl, methylation or demethylation (addition or removal of at least one methyl group at lysine or arginine residues).

Still further modifications include amide bond formation that may encompass amidation at C- terminus and amino acid addition that may include arginylation, a tRNA-mediation addition, polyglutamylation, that involves the covalent linkage of glutamic acid residues and polyglycylation, covalent linkage of at least one glycine residue. Still further, butyrylation, gamma-carboxylation and glycosylation, that involves the addition of a glycosyl group to either arginine, asparagine, cysteine, hydroxy lysine, serine, threonine, tyrosine, or tryptophan. In further embodiments, PTMs may also include polysialylation, malonylation, hydroxylation, iodination nucleotide addition such as ADP-ribosylation, oxidation, phosphate ester (O- linked) or phosphoramidate (N-linked) formation, phosphorylation, the addition of a phosphate group, usually to serine, threonine, and tyrosine (0-linked), or histidine (N-linked), adenylylation, the addition of an adenylyl moiety, usually to tyrosine (O-linked), or histidine and lysine (N-linked), propionylation, pyroglutamate formation, 5-glutathionylation, S- nitrosylation, 5-sulfenylation, succinylation that involves the addition of a succinyl group to lysine, sulfation, the addition of a sulfate group to a tyrosine and the like.

It should be further appreciated that the term PTM as used herein further encompasses non enzymatic modifications, for example, glycation, carbamylation the addition of Isocyanic acid to an N-terminus of either lysine, histidine, taurine, arginine, or cysteine, carbonylation the addition of carbon monoxide to other organic/inorganic compounds.

A state of PTM can be altered, for example, if there is a change in the average number of a given chemical group attached per protein molecule, if there is a change in the type of chemical group or groups attached per protein molecule, or if there is a different mixture of protein molecules having distinct modification patterns in a patient sample. Alteration of a PTM state of a protein includes going from an unmodified protein to a modified one and vice- versa, as well as changes in the number or type of chemical moieties added to the protein. It should be appreciated that the term "analyzing" of PTMs in a sample as referred to herein encompasses quantification and or characterization of the level, nature, extent and/or pattern of PTMs of proteins in the examined sample.

Still further, the method of the invention involves the step of measuring and thereby determining or assessing the signal intensity of the detectable tag or detectable moiety. Reference to "determining", as used herein, includes estimating, quantifying, calculating or otherwise deriving by measuring an end point indication that may be for example, the appearance of a detectable moiety, any detectable change in the levels or any change in the rate of the appearance or alternatively, the disappearance of the detectable moiety. As used herein, "assessing" refers to quantitative and/or qualitative determination of the detectable moiety attached to the tagged protein/s used by the invention, e.g., obtaining an absolute value for the amount or concentration of the tagged protein/s, and also of obtaining an index, ratio, percentage, visual or other value indicative of the level of the tagged protein/s in each individual PTM reaction.

It should be understood that an essential step in the method of the invention involves normalization of each individual PTM reaction by measuring the amount of the tagged protein in each reaction prior, in parallel or after the addition of the sample. The intensity of the PTM reaction is then normalized with the signal measured for the tagged protein. It should be therefore appreciated that in addition to the accuracy and validity of the results obtained, the use of this specific step provide a tool for analyzing reversible-PTMs, more specifically, removal of specific PTM of a protein by the sample. Specific example for such application is the dephosphorylation that is demonstrated in Example 3 and will be discussed in more detail herein after.

As indicated above, in a first step the method of the invention involves providing a plurality of tagged proteins or of any expression templates encoding the same. "Plurality" as used herein is meant one or more, two or more, three or more, four or more, five or more, six or more, seven or more, eight or more, nine or more or ten or more. In more specific embodiments, plurality of tagged proteins may range between about 2 to about 50,000 or more, specifically, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more, specifically, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 550, 600, 650, 700, 750, 800, 850, 900, 950, 1000 or more, specifically, 2000, 3000, 4000, 5000, 6000, 7000, 8000, 9000, 10,000, 20,000, 30,000, 40,000, 50,000 or more. In more specific embodiments, the method of the invention may provide between about 2 to 5,000, tagged proteins. It should be noted that although the range of between about 2 to about 50,000 or more, is preferred, in some embodiments, further tagged proteins may be provided.

In some specific embodiments, each of the plurality of tagged proteins provided by the method of the invention may be immobilized to a solid support, optionally, in a predetermined and recorded position.

In some specific embodiments, the tagged proteins used by the methods and kits of the invention may be freshly expressed proteins that are immobilized to the solid support. More specifically, in some embodiments, the tagged proteins may be freshly translated from expression template/s, specifically, cDNA molecules, specifically, using in vitro translation techniques. Thus, in more specific embodiments, a plurality of tagged proteins according to step (a) of the method of the invention may be provided by (i) providing a plurality of expression template/s, specifically, cDNA molecules encoding tagged proteins or any peptides thereof; (ii) incubating the plurality of expression template/s, specifically, cDNA molecules under conditions allowing in vitro translation of the cDNA molecules into tagged proteins; and (iii) immobilizing each of the tagged proteins obtained in step (ii) to a solid support in a predetermined and optionally recorded position.

It should be noted that the method of the invention is highly modular and can easily be scaled up to thousands of tagged proteins encoded by the expression templates provided herein. This approach only requires an open reading frame (ORF) as starting material, which may be obtained from a variety of sources including yeast, bacterial or eukaryotic cDNA clones. More specifically, this method is based on flow dependent surface deposition of expression templates (e.g., linear expression templates, specifically, cDNA molecules) to be used in in vitro transcription/translation for the in situ generation of fresh protein/s. Expression of the template encoded proteins may be used for highly efficient in vitro protein synthesis. As noted above, using this method it is possible to analyze a vast number of protein PTMs.

As used herein, an "expression template" is a DNA molecule, specifically, cDNA molecule with a protein-encoding sequence (open reading frame) and operably linked sequences required for transcription and translation to produce the protein. These sequences, or elements, are known in the art and include a RNA polymerase start site for transcription, a ribosome binding site and associated regulatory structures, a start codon defining the start of the template, and optionally a stop codon, poly A tail, RNA polymerase stop sequence, sequences that extend the life of the mRNA. The expression template may be linear or have a closed- circular topology (e.g., a template in a plasmid vector). Specifically, the expression template may be a linear or circular double stranded molecule. In certain embodiments, the expression template may include a covalently linked ligand (e.g., biotin) or other molecule that allows the template to be immobilized on a surface of a microfluidic chamber (e.g., a DNA chamber as will be described herein after). Such ligands used for immobilizing the tagged proteins, may be easily introduced during synthesis of the template using PCR amplification methods.

In some non-limiting embodiments, PCR based approach may be used for generating a linear expression vector, which is highly modular and can easily be scaled up to thousands of target genes. Such approach only requires an ORF as starting material as described above. All other components of the system may be commercially procurable oligomers.

The expression templates may be used for microarray spotting or flow deposition as described above, and may be specifically applicable to on-chip synthesis, for example, by introducing primer pairs and their respective template by co-spotting as mentioned above and running the PCR reaction in situ on-chip.

This method may be therefore used for rapid in situ synthesis of protein using in vitro transcription/translation. This allows the generation of large libraries of proteins of any chosen sequenced organism to be tested for PTM reactions.

In some specific embodiments that are also illustrated in the Examples, the tagged proteins used by the methods of the invention are freshly expressed in vitro on-chip by Transcription and Translation (TnT, or TNT), or IVT (in vitro translation). In vitro transcription-translation provides the means to synthesize proteins rapidly from a DNA template sequence. Using polymerase chain reaction, DNA templates can be generated rapidly from any coding sequence and protein produced without the use of time-consuming protein expression systems using living cells. In certain embodiments, different cellular extracts may be added, specifically, human extracts, rabbit reticulocyte, wheat germ, E. coli and the like. Non limiting examples for TnT systems are TnT Procedure with PCR-Generated DNA.

The use of freshly expressed tagged proteins enables analyzing PTMs of any protein. Therefore, in some embodiments, the plurality of tagged proteins may comprise any purified, partially purified, semi-purified or non-purified protein of any size, type or nature or of any cellular localization, including but not limited to soluble proteins, insoluble proteins, cytosolic proteins, secreted proteins, nuclear proteins, membrane proteins, transmembranal proteins, mitochondrial proteins, lysosomal proteins, or any proteins associated with any cellular organelle. It should be further appreciated that the tagged proteins may be any native or any mutated protein, as well as any chimeric or fusion protein.

In some specific embodiments, the tagged proteins used by the method of the invention may be at least one of membrane protein/s, nuclear protein/s and cytoplasmic protein/s. The invention therefore provides in some embodiments thereof specific methods for analyzing PTMs of membrane proteins. The invention further encompasses the provision of freshly prepared and functional membrane protein arrays.

It should be appreciated that membrane proteins, let alone modifications of membrane proteins, are usually in low abundance and thus, more challenging for mass-spectrometry- based analysis. In addition, they are incompatible with high-throughput methods (e.g., yeast two-hybrid) and are particularly difficult to purify in functional form (e.g., protein arrays).

Combining integrated microfluidics with microarrays and in vitro transcription and translation (TNT) systems, as provided by the present invention, overcome all of the above mentioned difficulties. The integrated microfluidic device provided by the invention allows smart liquid management in very low volumes, partitioning, and process integration (i.e., protein expression, immobilization, and PTM reactions). Microarray technology provides the means for programming thousands of different experiments. In vitro TNT expression systems allow protein biosynthesis and are compatible with high throughput.

As shown in Example 2 and Figure 3B, adding microsomal membranes enable the correct folding of membrane proteins and support post translational modifications. In short, the microfluidic platform using the method of the invention facilitates using in vitro TNT systems to produce a reliable membrane protein array from DNA with high sensitivity, low material and protein consumption, and compatibility with membrane proteins. As such, the present disclosure platform is the only available tool for large-scale functional membrane protein arrays.

Thus, in some specific embodiments, the tagged proteins used by the method of the invention may be membrane proteins. The present invention therefore provides functional and freshly expressed membrane protein arrays that in some specific and non-limiting embodiments may be applicable in analyzing PTMs of membrane proteins. As example for on-chip analysis of PTMs of membrane proteins, specifically phosphorylation is provided in Figure 7, that will be further discussed herein after.

It should be note that, "membrane protein/s" as meant herein, refers to a type of protein that is attached to or associated with, a biological membrane (i.e. of a cell or of an organelle, specifically mitochondria). Membrane proteins are a common type of proteins along with soluble globular proteins, fibrous proteins, and disordered proteins. They include integral membrane proteins that are permanently anchored to the membrane and peripheral membrane proteins, which are only temporarily attached to the lipid bilayer or to other integral proteins. In some embodiments, such attachment may be facilitated via a linking element, for example via a fatty acid such as myristic acid, palmitic acid, prenyl groups and the like, that promote attachment to cell membrane.

The integral membrane proteins (also called intrinsic proteins) are classified as transmembrane proteins that span across the membrane and integral monotopic proteins that are attached to only one side of the membrane. More specifically, integral proteins have one or more segments that are permanently embedded within the phospholipid bilayer and have their domains on both sides of the membrane. Most integral proteins contain residues with hydrophobic side chains that interact with fatty acyl groups of the membrane phospholipids, thus anchoring the protein to the membrane. Most integral proteins span the entire phospholipid bilayer.

As noted above, peripheral proteins (or extrinsic proteins) are proteins that have a much weaker interaction with the membrane than integral proteins. They are temporarily bound either to the lipid bilayer or to integral proteins by hydrophobic, electrostatic, and other non- covalent interactions. This type of proteins does not interact with the hydrophobic core of the phospholipid bilayer. They are usually bound to membrane by interactions with integral membrane proteins or directly by interactions with lipid polar head groups.

It should be understood that the methods and arrays provided by the invention are applicable for any of the membrane proteins discussed herein.

Still further, it should be also appreciated that the tagged proteins used by the method of the invention may be of any desired organism or any combination of organisms, specifically, yeast, bacterial or eukaryotic organisms. More specific and non-limiting examples for eukaryotic organisms include but are not limited to Protists, Fungi, plants and animals, including vertebrates (birds, mammals, amphibians, reptiles, fish); molluscs (clams, oysters, octopuses, squid, snails); arthropods (millipedes, centipedes, insects, spiders, scorpions, crabs, lobsters, shrimp); annelids (earthworms, leeches); sponges; and jellyfish. In more specific embodiments the tagged protein or any peptides thereof may be a mammalian protein, more specifically, a human protein. As noted above, the tagged proteins provided by the methods and arrays of the invention reflect at least some of the proteins in the source organism, e.g., human or any mammal, of the examined sample.

In some specific embodiments, each of said expression template/s, specifically, cDNA molecules or tagged proteins may be contained in an individual chamber, slot, dot, well, vessel, tube, bottle, unit, container, capillary and the like.

In further specific embodiments, the tagged proteins of the invention or any expression template/s, specifically cDNA encoding the same, may be localized, embedded, spotted or printed in a pre-recorded position or spot on a solid support.

It should be noted that in certain embodiments each of the tagged proteins of the invention may comprise at least one tag. In some specific embodiments, such tag may be used for immobilization purpose. It should be appreciated that the plurality of tagged protein/s provided by the invention may comprise in certain embodiments the same immobilization tag that facilitates immobilization of all tagged proteins to the solid support. However, the different protein/s provided may comprise different tags used for immobilization purpose. In case the tagged protein/s of the invention comprise only one tag that is used for immobilization to a solid support, an additional label or any detectable moiety may be required to facilitate the quantification of the tagged immobilized protein/s. Such detectable label/s or moiety may be in the form of isotope or fluorescent labeling of the protein molecule/s, for example by way of metabolic labeling that may occur during synthesis of the protein. In still further alternative or additional embodiments, the tagged proteins may comprise at least two different tagging molecules, or even more. More specifically, at least one, at least two, at least three, four, five, six, seven, eight, nine, ten tags or even more. In further embodiments, the proteins used by the methods of the invention may comprise at least 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more different or identical tag molecules or detectable moieties.

In further embodiments, at least one tag may be used to immobilize the tagged protein to a solid support. Still further, in some embodiments one or more tags may be used in determining the level and quantity of each of the tagged protein as performed and measured by step (b) of the method of the invention, specifically for normalization purpose. In some specific embodiments, the protein of the invention may contain only one tag. This tag may be used either for immobilizing the protein on a solid support or alternatively or additionally, to determine the amount of the protein in the reaction. In case the sole tag is used for immobilization, the amount of the protein may be determined using a specific directly or indirectly labeled molecule that may be either a detectable label or moiety incorporated into the protein, or alternatively a directly or indirectly labeled molecule that recognizes and binds the protein. Non-limiting example for such molecule may be an antibody or any other affinity ligand specific for the tagged protein/s of the invention. It should be understood however, that in most cases, a double tagged protein may be used as discussed above. In yet another embodiment, where a sole tag is used to immobilize the tagged protein of the invention to the solid support, detection thereof and determination of the protein amount for the normalization step, may be accomplished and facilitated by incorporation of labeling moiety or any other detectable label, for example, using a metabolically labeled tagged protein that carry a fluorescent or isotope moiety. More specifically, a freshly expressed tagged protein used by the invention may be a fluorescently labeled protein, for example, using labeled Lysine or any other amino acid integrated into the protein during translation.

Determination of the level of the protein and in some embodiments determination of the signal intensity of the PTM reaction may be accomplished using colorimetric methods or alternatively, using mass spectrometry (MS), as will be elaborated herein after.

In certain embodiments, the tags are attached directly or indirectly (via a connecting linker, spacer or tether) to at least one of the N-terminal or the C-terminal end of the protein. The term "spacer" or "tether" as referred to herein, relates to a polypeptide (or any oligonucleotide encoding the same) comprising between about 2-5 amino acid residues, or about 5-10 amino acid residues, or about 10-15 amino acid residues, or about 15-30 amino acid residues, or about 30-50 amino acid residues, or about 50-70 amino acid residues, or about 70-90 amino acid residues, or about 90-150 amino acid residues, or about 150-300 amino acid residues or more.

The invention provides tagged proteins or any peptides thereof. As used herein, the terms "protein" and "polypeptide" are used interchangeably to refer to a chain of amino acids linked together by peptide bonds. In more specific embodiments, the protein/s of the invention may be composed of less than 3000, less than 2000, less than 1750, less than 1500, less than 1250, less than 1000, less than 500, less than 450, less than 400, less than 350, less than 300, less than 250, less than 200, less than 150, less than 100, or less than 95, 90, 85, 80, 75, 70, 65, 60, 55, 50, 45, 40, 35, 30, 25, 20, 15, 10 or 5 amino acids linked together by peptide bonds. In other embodiments, a protein may be composed of between 30 to 1500 or more amino acids linked together by peptide bonds. It should be noted that peptide bond as described herein is a covalent amid bond formed between two amino acid residues.

In certain embodiments, tag molecules applicable for the invention include but are not limited to myc, His, HA, Flag, GFP, ABP, GST, Biotin/avidin, and the like. "Tagged" as used herein may further include fusion or linking of the protein or any fragment or peptide thereof to a tag that in some embodiments may contain several amino acids or a peptide that may be recognized by affinity or immunologically, using specific antibodies.

More specifically, The term ^vtag\ as used herein, refers to a molecule that can be attached to a larger macromolecule, and which can be used to separate that macromolecule from macromolecules that do not have the tag, or facilitate the specific visualization of said macromolecule. More specifically, in some specific embodiments, the tag referred to herein may be protein tags, which consist of specific amino acid sequences that are recognized and bound by known ligands. It is understood that, where appropriate, when adding an N-terminal tag to a polypeptide, the polypeptide initial methionine encoded by the initiation codon (ATG) may be re-located to the N-terminus of said tag, to facilitate translation initiation. In a similar manner, where a C-terminal tag is used, the stop codon (any one of TAA, TGA and TAG) may be re-located to the C-terminus of the aged protein.

More specifically, under protein tags refers to peptide sequences genetically grafted onto a recombinant protein or peptide. Often these tags are removable by chemical agents or by enzymatic means, such as proteolysis or splicing. An important feature of protein tags is that they do not or should not alter functionality of the tagged protein. Tags are attached to proteins for various purposes, of particular relevance to the present invention are protein tags serving as capture or reporter molecules.

Affinity tags are appended to proteins so that they can be purified or immobilized to a matrix. Notable examples thereof include, but not limited to, a poly(His) tag due to its affinity to metal matrices, and Strep-tag, a peptide which binds to streptavidin or the modified streptavidin called streptactin (Strep-tag II: WSHPQFEK, as also denoted by SEQ ID NO. 2). Epitope tags are short peptide sequences with high immunoreactivity and high-affinity to antibodies, these tags are particularly useful for detection and/or visualization of tagged proteins by various molecular technologies, e.g. western blotting, immunofluorescence and immunoprecipitation. Non limiting examples of epitope tags that are applicable to the present invention include:

. AviTag (GLNDIFEAQKIEWHE, as also denoted by SEQ ID NO. 3) allowing biotinylation by the enzyme BirA enabling recognition by streptavidin.

• E-tag (GAPVPYPDPLEPR, as also denoted by SEQ ID NO. 4) recognized by a specific antibody.

. FLAG-tag (DYKDDDDK, as also denoted by SEQ ID NO. 5) recognized by a specific antibody.

• HA-tag (YPYDVPDYA, as also denoted by SEQ ID NO. 6) recognized by a specific antibody.

• Mvc-tag, (EQKLISEEDL, as also denoted by SEQ ID NO. 7) recognized by a specific antibody.

• S-tag (KET A A AKFERQHMDS , as also denoted by SEQ ID NO. 8) a novel fusion peptide tag allowing detection by rapid and sensitive homogeneous assay or by colorimetric reaction

. SBP-tag (MDEKTTGWRGGHVVEGLAGELEQLRARLEHHPQGQREP, as also denoted by SEQ ID NO. 9) binds to streptavidin

• TC tag (CCPGCC, as also denoted by SEQ ID NO. 10) recognized by FlAsH and ReAsH biarsenical compounds

• V5 tag (GKPIPNPLLGLDST, as also denoted by SEQ ID NO. 11) recognized by a specific antibody.

. VSV-tag (YTDIEMNRLGK, as also denoted by SEQ ID NO. 12) recognized by a specific antibody.

• Xpress tag (DLYDDDDK, as also denoted by SEQ ID NO. 13) recognized by a specific antibody.

Fluorescence tags are used to give visual readout on a protein. Green Fluorescent Protein (GFP) and its variants are the most commonly used fluorescence tags. More advanced applications of GFP include using it as a folding reporter (fluorescent if folded, colorless if not). For specific purposes, GFP may be combined with affinity tags such as in His-tagged GFP. Protein tags may also include specific enzymatic modifications (such as biotin ligase tags) and chemical modifications (FlAsH tag) to enable colorimetric or other detection. Other protein tags which may be applicable in the context of present invention include:

. Isopeptag (TDKDMTITFTNKKDAE, as also denoted by SEQ ID NO. 14) a strong affinity tag that covalently binds to the bacterial pilin-C protein.

• SpyTag (AHIVMVDAYKPTK, as also denoted by SEQ ID NO. 15) a strong affinity tag acting via SpyCatcher protein.

• BCCP (Biotin Carboxyl Carrier Protein), a protein domain biotinylated by BirA enabling recognition by streptavidin.

• Glutathione-S-transferase-tag (GST-tag), a protein which binds to immobilized glutathione and is recognized by specific antibodies.

• Halo-tag, a mutated hydrolase that covalently attaches to the HaloLink™ Resin (Promega) a modified haloalkane dehalogenase designed to covalently bind to synthetic ligands (HaloTag ligands).

• Ty tag, amore recent an antibody reactive tag.

• Maltose-Binding Protein (MBP) may be immobilized using amylose resin.

It should be noted that said tag molecules may be labeled by a detectable label or moiety directly or indirectly. In some other specific embodiments an indirect label may be measured using an immunoassay or any other affinity assay.

The term "label moiety" or "label" as referred to herein, relates to a specific group of atoms within a molecule having the ability to either bind other known specific molecules including those which have associated with them a molecule that emits a detectable signal or emit a detectable signal itself. The label either emits or produces a detectable signal directly or through interaction with one or more additional members of a signal producing system. Any type of label can be used consistent with the invention, with conventional labeling methods known in the art being suitable.

In some specific embodiments, conditions that allow PTM reactions to occur may include but are not limited to supplementing the sample or any functional extract thereof with at least one of reagent/s, enzyme/s, substrate/s, co-factor/s and inhibitor/s, prior to or during the specific PTM reaction.

It should be appreciated that in certain embodiments, the method of the invention may be applicable for analyzing any PTM reactions. Non-limiting examples include at least one of phosphorylation, ubiquitination, sumoylation (using sumo 1-4), neddylation, isgylation (using ISG15), fatylation (using FAT10), pupylation, and any other eukaryotic or prokaryotic ubiquitin like modifier. It should be appreciated that the method of the invention encompasses any of the PTM reactions indicated herein before. To name but few, the method of the invention may be further applicable for myristolation, palmitoylation, isoprenylation or prenylation, farnesylation, geranylgeranylation, glypiation, covalent attachment of flavin moiety, attachment of heme C, phosphopantetheinylation, retinylidene Schiff base formation, diphthamide formation, ethanolamine phosphoglycerol attachment, hypusine formation, acylation, [e.g. 0-acylation (esters), N-acylation (amides), 5-acylation (thioesters), and crotonylation], acetylation, deacetylation, formylation, alkylation, methylation or demethylation, amide bond formation, arginylation, polyglutamylation, polyglycylation, butyrylation, gamma-carboxylation and glycosylation, polysialylation, malonylation, hydroxylation, iodination, ADP-ribosylation, oxidation, phosphate ester (0-linked) or phosphoramidate (N-linked) formation, phosphorylation, adenylylation, propionylation, pyroglutamate formation, 5-glutathionylation, 5-nitrosylation, 5-sulfenylation, succinylation, sulfation, glycation, carbamylation, carbonylation, as well as proteolytic cleavage of peptide bonds, and formation of disulfide bonds by linking cysteine residues.

In certain embodiments, the present invention pertains to PTMs by Ubiquitin-Like proteins or modifiers (UBLs). The term UBLs as meant herein encompasses the phylogenetically distinct classes of UBLs, including SUMO, NEDD8, ISG15, FUB l, FAT10, Atg8, Atgl2, Urml, and Ufml, and further a putative recently identified modifier known as DWNN. UBLs confer diverse functions on their target proteins, depending on the cellular role of their targets, UBLs have been implicated in DNA replication, signal transduction, cell cycle control, embryogenesis, cytoskeletal regulation, metabolism, stress response, homeostasis and mRNA processing. The most common feature of UBLs, rather than sharing primary sequence similarity, is their three-dimensional fold.

Thus in more specific embodiments, the present invention pertains to PTMs by Small Ubiquitin-Related Modifier (SUMO). Although SUMO shares low amino acid identity with Ubiquitin (approximately 18%), but their three-dimensional structures are virtually super- imposable. However, it differs from Ubiquitin in overall charge topology indicating differences in the nature of their interacting partners. It now seems that SUMO affects the widest range of proteins when compared to other UBLs. Under SOMO is meant the four functional SUMO genes in the human genome producing four isoforms (SUMO- 1/2/3 and 4). Specifically, the SUMO-3 gene is derived from SUMO-2 and the encoded proteins share 86% sequence identity; SUMO-1 shares only 44% sequence identity with SUMO-2 and -3; SUMO-4 is encoded by a separate gene that shares 85% identity with SUMO-2 and is expressed mainly in the kidney.

Further, the term 'SUMOylation' refers to a series of reactions catalyzed by SUMO-specific enzymes, wherein SUMO is conjugated via the C-terminal glycine to a ε-amino group on an internal lysine residue of the target protein in a stepwise mechanism that is analogous to modification by Ubiquitin. The SUMOylation pathway begins with the ATP-dependent activation of SUMO at the C-terminus by a heterodimeric SUMO activation enzyme consisting of SAEI (Aosl)/SAE2 (Uba2). The activated SUMO is then transferred through a transesterification reaction to Ubc9, the only known SUMO-conjugating enzyme (E2), forming a SUMO-Ubc9 thioester intermediate. The specificity for the substrate is determined by both Ubc9 and the E3 ligase. SUMOylation was found to be involved in both negative and positive regulation of gene transcription, wherein SUMO does not bind directly to DNA but appears to influence transcriptional activity indirectly by interacting with transcription factors. SUMOylation confers several functions on target proteins namely; protein stability, subcellular localization, transcription activation, DNA repair, and other cellular events.

In other specific embodiments, the present invention pertains to PTMs by Neural Precursor Cell-Expressed Developmentally Down-Regulated (NEDD8), UBL that shares the highest sequence identity (approximately 60%) with Ubiquitin. NEDD8 modifies target proteins in a manner analogous to ubiquitin in a series of reactions involving NEDD8 activating El-like enzyme (APP-BPl/Uba3), NEDD8-specific E2 enzymes (Ube2F or Ubcl2) and a more recent gene (RING)-finger protein ROC1 (NEDD8 E3 ligase). The nature of chain formation by NEDD8 was found to have only subtle differences to polyubiquitination. Neddylation was found to be required for regulation of the multifunctional transcription factor, NF-κΒ, which is crucial in immune response and apoptotic pathways.

In yet other specific embodiments, the present invention pertains to PTMs by Human HLA-F Adjacent Transcript 10 (FAT10), an 18 kDa UBL that shares moderate sequence similarity with Ubiquitin, (29% and 36% at the N- and C- termini respectively). FAT10 was found to be constitutively expressed in lymphoblastoid cells and in dendritic cells and to be induced in certain other cells by pro-inflammatory stimuli. The unique feature of this modification is that it is found in vertebrates only and is expressed by specific stimuli in a tissue-specific manner. FAT10 modification, also known as diubiquitin (owing to the two Ubiquitin-like domains in tandem, head- to-tail) relies on its C- terminal diglycine motif for modification of its substrates. FAT10 is mediated through the Uba6 (El) and USE1 (E2) enzymes, which are specific to both FAT10 and Ubiquitin. FAT10 was implicated in regulating the cell cycle, tumorigenesis, inhibition of cell proliferation and in survival. It also plays a significant role in immune response. For example, studies have shown that FAT10 expression is induced by interferon-γ and tissue necrosis factor a (TNFa), and that deletion of FAT 10 results in lymphocytes that are prone to spontaneous apoptotic death and sensitive to endotoxin exposure and more recently that FAT 10 appears to mediate the activation of NFKB, a key mediator of innate immunity.

In further specific embodiments, the present invention pertains to PTMs by Interferon Stimulated Gene 15 (ISG15), a 17 kDa Ubiquitin-like I protein that is understood to be primarily an anti-viral response gene whose expression is induced by type I IFN (interferon), LPS (lipopolysaccharide), and pI:pC, a synthetic inosine polymer that resembles the RNA of infectious viruses. More specifically, the upstream enhancer element of ISG15 was found to be responsive to IFN and p53. Over 150 to 300 proteins have been identified as ISGylation targets. Conjugation of substrate proteins by ISG15 (i.e. ISGylation) is similar to ubiquitination in that it follows a three-step enzymatic cascade using an El activating enzyme (UbelL), an E2 conjugating enzyme (UbcH6 and UbcH8), and several E3 ligases, such as EFP (estrogen-responsive finger protein) and the HECT (homologous to E6-AP C-terminus)- type E3 ligase Herc5, both of which are inducible by interferon. The main ISG15 characteristics are pointing to its vital role in the innate immune response to viral infections.

In yet further specific embodiments, the present invention pertains to PTMs by Autophagy- Related (ATG) Genes, more specifically to the two Atg proteins, Atg8 and Atgl2 that possess an Ubiquitin-like fold and adopt an Ubiquitin-like mechanism to modify substrate proteins. By the virtue of being associated with autophagy, ATGs have potentially important bearing on processes and diseases wherein autophagy has an important role, such as neurodegeneration and cancer.

In specific embodiments, the present invention further pertains to PTMs by Ubiquitin-Fold Modifier 1 (Ufml), a 9.1 kDa protein sharing 16% sequence identity with Ubiquitin, but unlike other UBLs it has only a single glycine at its C-terminus. Although information about Ufml biological significance is still patchy, Ufmlconjugation was found to be abundant in the liver and lungs and further found essential for erythroid differentiation.

In further specific embodiments, the present invention pertains to PTMs by Domain with no Name (DWNN), a recently discovered putative UBL sharing about 28% identity with Ubiquitin but having an almost superimposable three-dimensional structure.

In yet other specific embodiments, the present invention pertains to proteins with an Ubiquitin domain, multi-domain proteins with UBLs that are often arranged together with the RING- finger motif. These proteins are encoded by a single transcript and consist of the Ubiquitin- like domain usually at the N-terminus and a RING-finger motif along the length of the protein. As for now, this groups includes Homocysteine-Inducible Endoplasmic Reticulum Stress-Inducible Ubiquitin-Like Domain Member 1 (Herpudl), Parkin, Retinoblastoma Binding Protein 6 (RBBP6) and Ubiquilin.

Crotonylation, as used herein refers to acetylation, for examples of histones at DNA regulatory elements by addition of crotonyl, specifically to lysine residues ((Kcr). Crotonylation is a newly discovered histone PTM that is enriched at active gene promoters and potential enhancers in mammalian cell genomes. However, the cellular enzymes that regulate the addition and removal of Kcr are unknown although several studies indicate the involvement of crotonyl-CoA.

Glycosylation as meant herein encompasses a diverse selection of sugar-moiety additions to proteins that ranges from simple monosaccharide modifications, e.g. characteristic of nuclear transcription factors, to highly complex branched polysaccharide, e.g. changes of cell surface receptors. Carbohydrates in the form of aspargine-linked (N-linked) or serine/threonine- linked (O-linked) oligosaccharides are major structural components of many cell surface and secreted proteins. Protein glycosylation is acknowledged as one of the major PTMs, with significant effects on protein folding, conformation, distribution, stability and activity.

N-acetylation refers to a transfer of an acetyl group to nitrogen, which occurs in almost all eukaryotic proteins through both irreversible and reversible mechanisms. This term, as meant herein, includes the N-terminal acetylation which requires the cleavage of the N-terminal methionine by methionine aminopeptidase (MAP) before replacing the amino acid with an acetyl group from acetyl-CoA by N-acetyltransferase (NAT) enzymes. This type of acetylation is co-translational, in that N-terminus is acetylated on growing polypeptide chains that are still attached to the ribosome. While 80-90% of eukaryotic proteins are acetylated in this manner, the exact biological significance of this type of acetylation is still unclear.

Under N-acetylation is further meant acetylation at the ε-ΝΗ2 of lysine (termed lysine acetylation), which in the case of histones, constitutes an important epigenetic mechanism. Histone acetylation is a reversible event that reduces chromosomal condensation to promote transcription, and the acetylation of these lysine residues is regulated by transcription factors that contain histone acetyletransferase (HAT) activity. While transcription factors with HAT activity act as transcription co-activators, histone deacetylase (HDAC) enzymes are compressors that reverse the effects of acetylation by reducing the level of lysine acetylation and increasing chromosomal condensation. Sirtuins (silent information regulator) are a group of NAD-dependent deacetylases that target histones, they maintain gene silencing by hypoacetylating histones and have been reported to aid in maintaining genomic stability. While acetylation was first detected in histones, cytoplasmic proteins have been reported to also be acetylated, and therefore acetylation seems to play a greater role in cell biology than simply transcriptional regulation.

Further, the term "acylation" refers to herein to PTM proteins via the attachment of functional groups through acyl linkages. One prominent type is fatty acylation, the addition of fatty acids to particular amino acids, such as myristoylation or palmitoylation.

N-myristoylation is facilitated specifically by N-myristoyltransferase (NMT) and uses myristoyl-CoA as the substrate to attach the myristoyl group to the N-terminal glycine. Because methionine is the N-terminal amino acid of all eukaryotic proteins, this PTM requires methionine cleavage by the above-mentioned MAP prior to addition of the myristoyl group. The myristoyl group is a 14-carbon saturated fatty acid (C14), which gives the protein sufficient hydrophobicity and affinity for membranes, but not enough to permanently anchor the protein in the membrane. N-myristoylation can therefore act as a conformational localization switch, in which protein conformational changes influence the availability of the handle for membrane attachment, such in Src-family kinases.

S-palmitoylation adds a CI 6 palmitoyl group from palmitoyl-CoA to the thiolate side chain of cysteine residues via palmitoyl acyl transferases (PATs). Because of the longer hydrophobic group, this anchor can permanently anchor the protein to the membrane. This localization can be reversed, though, by thioesterases that break the link between the protein and the anchor; thus, S-palmitoylation is used as an on/off switch to regulate membrane localization. S- palmitoylation is often used to strengthen other types of lipidation, such as myristoylation or farnesylation.

Acylation as used herein is meant the process of adding an acyl group to a protein. Protein acylation is the post-translational modification of proteins via the attachment of functional groups through acyl linkages. One prominent type is fatty acylation, the addition of fatty acids to particular amino acids (e.g. myristoylation or palmitoylation). Protein acylation has been observed as a mechanism of biological signaling.

Still further, ADP-ribosylation as used herein is the addition of one or more ADP-ribose moieties to a protein. It is a reversible post-translational modification that is involved in many cellular processes, including cell signaling, DNA repair, gene regulation and apoptosis.

It should be noted that ubiquitination and phosphorylation that are specific embodiments exemplified by the invention, are described in more detail herein after.

Apart from the above mentioned most ubiquitous PTMs, the present invention is further relevant for the analysis of other PTM including, although not limited to, the ones detailed below.

S-nitrosylation refers to formation of S-nitrothiols (SNOs) by the reaction of nitric oxide (NO) with free cysteine residues on a target protein. S-nitrosylation is not random and only specific cysteine residues are S-nitrosylated. S-nitrosylation is reversible due to SNOs' short half life in the cytoplasm because of reducing enzymes, such as glutathione and thioredoxin, which denitrosylate proteins. Therefore, SNOs are often stored in membranes, vesicles, the interstitial space and lipophilic protein folds to protect them from denitrosylation, e.g. caspases that mediate apoptosis are stored as SNOs in the mitochondrial inter-membrane space. S-nitrosylation was proved to be critical PTM for the stabilization of proteins, regulation of gene expression and provision of NO donors within a cell, therefore generation, localization, activation and catabolism of SNOs are tightly regulated.

Methylation refers to the transfer of one-carbon methyl groups to nitrogen or oxygen (N- and O-methylation, respectively) to amino acid side chains increases the hydrophobicity of the protein and can neutralize a negative amino acid charge when bound to carboxylic acids. Methylation is mediated by methyltransferases, and S-adenosyl methionine (SAM) is the primary methyl group donor. While N-methylation is irreversible, O-methylation is potentially reversible. Methylation is a well-known mechanism of epigenetic regulation, as histone methylation (e.g., by histone methyl transferase, HMTs) and demethylation influences the availability of DNA for transcription. Amino acid residues such as lysine and arginine can be conjugated to a single methyl group or multiple methyl groups to increase the effects of modification. As noted above, in certain embodiments the invention further pertains to demethylation, e.g., by protein demethylases.

Lipidation refers to types of PTMs that increase the hydrophobicity of a protein and thus its affinity for membranes. Under this definition are included C-terminal glycosyl phosphatidylinositol (GPI) anchor, N-terminal myristoylation, S-myristoylation and S- prenylation. The different types of lipidation are not mutually exclusive, in that two or more lipids can be attached to a given protein.

GPI-anchored proteins are often localized to cholesterol- and sphingolipid-rich lipid rafts, which act as signaling platforms on the plasma membrane. This type of modification is reversible, as the GPI anchor can be released from the protein by phosphoinositol-specific phospholipase C.

S-prenylation covalently adds a farnesyl (CI 5) or geranylgeranyl (C20) group to specific cysteine residues within 5 amino acids from the C-terminus via farnesyl transferase (FT) or geranylgeranyl transferases (GGT I and II). Unlike S-palmitoylation, S-prenylation is hydrolytically stable. Approximately 2% of all proteins are prenylated, including all members of the Ras superfamily. This group of molecular switches is farnesylated, geranylgeranylated or a combination of both. Additionally, these proteins have specific 4-amino acid motifs at the C-terminus that determine the type of prenylation at single or dual cysteines. Prenylation occurs in the endoplasmic reticulum (ER) and is often part of a stepwise process of PTMs that is followed by proteolytic cleavage by Reel and methylation by isoprenyl cysteine me thyltransf erase (ICMT).

In some specific embodiments, the method of the invention may be used for analyzing ubiquitination.

Ubiquitination as meant herein refers to an enzymatic PTM process wherein the carboxylic acid of the terminal glycine from the di-glycine motif in the activated ubiquitin forms an amide bond to the epsilon amine of the lysine in the modified protein. Ubiquitin is an 8-kDa polypeptide consisting of 76 amino acids that is appended to the NH2 of lysine in target proteins via the C-terminal glycine of ubiquitin. Following an initial monoubiquitination event, the formation of an ubiquitin polymer may occur, and polyubiquitinated proteins are then recognized by the 26S proteasome that catalyzes the degradation of the ubiquitinated protein and the recycling of ubiquitin. Ubiquitin possesses a total of 7 lysine residues. Historically the original type of ubiquitin chains identified were those linked via lysine 48. More recent work has uncovered a wide variety of linkages involving all possible lysine residues and in addition chains assembled on the N-terminus of a ubiquitin molecule ("linear chains"), and also branched ubiquitin chains containing multiple "atypical" (non-lysine 48- linked) linkage types.

Ubiquitination is carried out by a set of three enzymes, El, E2 and E3. Ubiquitin is first activated by ubiquitin-activating enzyme El, before being transferred to its active site, the amino acid cystein. This transfer requires ATP, making the process energy-dependent. The ubiquitin molecule is then passed on to the second enzyme of the complex, E2 (ubiquitin- conjugating enzyme), before reaching the final enzyme, E3, the ubiquitin protein ligase, which recognizes, binds the target substrate and labels it with the ubiquitin. The process can be repeated until a short chain is formed, with three or more ubiquitin molecules usually targeting the protein to the proteasome.

Substrate specificity is mainly defined by the multiple E2 and E3 combinations possible. E2 and E3 belong to large protein families, but while E2 share many well-conserved catalytic domains, E3 ligases only share a few conserved motifs and are, therefore, very specific. As the three-steps process advances, specificity increases: El interacts with all E2s, which interact with a more limited subset of E3s, which in turn target a limited array of protein substrates, based on shared recognition motif within the proteins to be labeled. This enables the ubiquitination-proteasome pathway to be highly specific in the selection of proteins to be labeled. The anaphase-promoting complex (APC) and the SCF complex (for Skpl-Cullin-F- box protein complex) are two examples of multi-subunit E3s involved in recognition and ubiquitination of specific target proteins for degradation by the proteasome.

Ubiquitin labeling is however not always fatal for the protein, with several non-proteolytic functions associated with the addition of a single ubiquitin molecule (mono-ubiquitination) or specific cases of polyubiquitination. Mono-ubiquitination can alter the fate of the protein in a less terminal fashion, potentially affecting its cellular sub-location, function or its degradation though lysosomes. The process is also reversible with enzymes (deubiquitinases) able to cleave ubiquitin from its target. Ubiquitin is the founding member of a family of ubiquitin- like proteins (such as the SUMO protein). Modification of proteins with SUMO (or Small Ubiquitin-related Modifier), known as SUMOylation, often increases the protein lifespan and stability. It is also linked to nuclear-cytosolic transport, regulation and transcription.

The ubiquitination system functions in a wide variety of cellular processes, including antigen processing, apoptosis , biogenesis of organelles, cell cycle and division, DNA transcription and repair, differentiation and development, immune response and inflammation, neural and muscular degeneration, morphogenesis of neural networks, modulation of cell surface receptors, ion channels and the secretory pathway, and further response to stress and extracellular modulators , ribosome biogenesis, and viral infection.

In some specific embodiments, conditions that allow a ubiquitination reaction according to the method of the invention may include supplementing the sample prior to or during the PTM reaction, with at least one of ubiquitin ligase/s, mutated ubiquitin ligase/s, ubiquitin ligase inhibitor/s, wild type ubiquitin, mutated ubiquitin, wild type ubiquitin comprising a detectable label or moiety and mutated ubiquitin comprising a detectable label or moiety. In specific embodiments of the present invention, for performing ubiquitination assay, more than one enzyme may be necessary to carry out the reaction, and while one or more enzyme is supplied by the extract or fluid sample, one or more other enzymes required for optimal activity may be limited or missing. In such cases, the missing or limited enzyme or enzymes can be added to the extract or fluid to establish an optimal PTM reaction. A further useful strategy is to add to the extract an inhibitor of an enzyme that inhibits a particular type of PTM. Examples include methyl-ubiquitin and dominant-negative E2 enzymes for ubiquitination or sumoylation.

In further embodiments of the invention, particularly those implementing ubiquitination, a PTM reaction may be supplemented with Ubiquitin-Like Modifiers (ULMs), which may include, but not limited to, Interferon-Stimulated Gene (ISG15) also Ubiquitin Cross- Reacting Protein (UCRP); Fau Ubiquitin-like protein (Fubi, FUB 1) also Monoclonal Nonspecific Suppressor Factor β (MNSF ); Neural precursor cell-Expressed Developmentally Downregulated-8 (NEDD8) also Related to Ubiquitin 1 (Rubl) in yeast; F-Adjacent Transcript-10 (FAT 10); Small Ubiquitin-Related Modifiers 1, 2 and 3 (SUMO-1/-2/-3) also ubiquitin-like protein SMT3A, B and C, Ubiquitin-like protein (UBLl), Global Mobility Professional protein (GMP1); Autophagy ubiquitin-like enzymes Apg8 and Apg 12; and more recently discovered ULMs such as Ubiquitin-related modifier Urml ; Ubiquitin-Like protein 5 (UBL5) also homologous to unbiquitin-1 in Saccharomyces cerevisiae (hubl); and Ubiquitin- fold modifier 1 (Ufml).

In certain particular embodiments, the method of the invention may use as a ligase, the anaphase-promoting complex/cyclosome APC/C, that mediates ubiquitination during the mitosis and Gl phase. In certain embodiments, a mutated ligase may be also added to the reaction. In more specific embodiments a, dominant negative APC/C activating E2 ligase applicable in the method of the invention may be UbcHlO. Still further, in certain embodiments ligase inhibitors may be added to the reaction. As a specific APC/C inhibitor, Emil may be used.

In yet some other embodiments, as an ubiquitin ligase, Ub ligase SCF^Skp2, mediating ubiquitination during the S phase, may be used in the methods of the invention.

In some embodiments, the method of the invention involves supplementing the sample with ubiquitin that comprises a detectable label or moiety. In such case, the intensity of the detectable label in the ubiquitin is measured and is considered as a detectable label specific for PTM generated by each individual PTM reaction.

The terms "detectable label" or "moiety" (also "reporter molecule") refers herein to any atom, molecule or a portion thereof, the presence, absence or level of which is directly or indirectly monitorable. One example includes radioactive isotopes. Other examples include (i) enzymes which can catalyze color or light emitting (luminescence) reactions and (ii) fluorophores. The detection of the detectable moiety can be direct provided that the detectable moiety is itself detectable, such as, for example, in the case of fluorophores. Alternatively, the detection of the detectable moiety can be indirect. In the latter case, a second moiety reacts with the detectable moiety, itself being directly detectable is preferably employed. The detectable moiety may be inherent to the antibody or any other affinity molecule. For example, the constant region of an antibody can serve as an indirect detectable moiety to which a second antibody having a direct detectable moiety can specifically bind.

The detection can be accomplished by colorimetric methods, which employ a chromogenic substrate for the enzyme. Detection may also be accomplished by visual comparison of the extent of enzymatic reaction of a substrate in comparison with similarly prepared standards.

Detection may be further accomplished with a specific antibody that can be detectably labeled is by linking the same to an enzyme. This enzyme, in turn, when later exposed to an appropriate substrate, will react with the substrate in such a manner as to produce a chemical moiety which can be detected, for example, by spectrophotometric, fluorometric or by visual means. Enzymes which can be used to detectably label the antibody include, but are not limited to, malate dehydrogenase, staphylococcal nuclease, delta-5-steroid isomerase, yeast alcohol dehydrogenase, alpha-glycerophosphate dehydrogenase, triose phosphate isomerase, horseradish peroxidase, alkaline phosphatase, asparaginase, glucose oxidase, beta- galactosidase, ribonuclease, urease, catalase, glucose-6-phosphate dehydrogenase, glucoamylase and acetylcholinesterase.

Alternative affinity reagents such as aptamers have recently shown great promise as an adjunct to antibodies. These nucleic acid-based molecules possess protein-binding specificity, similar to antibodies that make them useful as protein capture and detection reagents.

In yet some further alternative embodiments, detection methods applicable for the purpose of the invention may involve mass-spectrometry (MS) analysis of tagged or untagged target proteins and modifiers. More specifically, in such scenario, a desired set of proteins (the plurality of proteins provided by the invention) may be expressed and modified using the IMPA of the invention. The modified or unmodified target proteins may be eluted from the chip and processed for MS or liquid chromatography (LC)-MS analysis for monitoring the possible mass change of the peptides corresponding to one or more modifications.

Alternatively, the arrayed target proteins undergo proteolysis on chip, and the peptides may be collected and analyzed by MS or LC-MS. As with fluorescence detection, the ratio between modified and unmodified peptides provides the mean of normalization for each of the examined target proteins.

PTM detection by MS may indicate the modified site. Information about the modified site is particularly relevant in situations where certain proteins are modified by one or more types of PTMs at different positions depending on the specific physiological conditions (e.g., normal vs. pathological).

As shown in Example 7, the method of the invention may be also used for determining Ub- chain preference of a protein. It should be noted that Ub may form eight different chain types, through each of its 7 lysine residues and via N terminal (linear ubiquitination), that vary in length and linkage type. The various types of Ub modification are linked to distinct physiological functions in cells. Ub-chains include but are not limited to K6, Kl l, K27, K29, K33, K48, K63 and the like. The type of chain impacts protein turnover and fate. Thus, in some specific embodiments, the method of the invention may be applicable for determining Ub-chain preference of a protein. The invention therefore contemplates the two approaches disclosed in the Examples for analyzing Ub-chain preference by the examined sample.

For determining the Ub-chain preference of a protein, in some specific embodiments the sample may be supplemented with at least one of wild type ubiquitin, mutated ubiquitin, wild type ubiquitin comprising a detectable label or moiety and mutated ubiquitin comprising a detectable label or moiety. Non limiting examples for such mutated ubiquitin may include the lysine (K) to arginine (R) mutants, for example, UbKl lR, and UbK48R.

In some alternative embodiments, Ub-chain preference may be detected and analyzed by immuno-assay employing specific antibodies that recognize and bind a specific Ub-chain.

In yet some alternative embodiments, the method of the invention may be applicable for analyzing phosphorylation in a sample.

Phosphorylation as meant herein refers to a reversible phosphorylation of proteins, principally on serine, threonine or tyrosine residues and to lesser extent on histidine and arginine. It should be appreciated that the use of the phrase "analyzing phosphorylation by the sample" as used herein encompasses in addition to phosphorylation, also, dephosphorylation and autophosphorylation. Phosphorylation plays critical roles in the regulation of many cellular processes including cell cycle, growth, apoptosis and signal transduction pathways.

More specifically, for a large subset of proteins, phosphorylation is tightly associated with protein activity and is a key point of protein function regulation. Phosphorylation regulates protein function and cell signaling by causing conformational changes in the phosphorylated protein. These changes can affect the protein in two ways. First, conformational changes regulate the catalytic activity of the protein. Thus, a protein can be either activated or inactivated by phosphorylation. Second, phosphorylated proteins recruit neighboring proteins that have structurally conserved domains that recognize and bind to phosphomotifs. These domains show specificity for distinct amino acids. For example, Src homology 2 (SH2) and phosphotyrosine binding (PTB) domains show specificity for phosphotyrosine (pY), although distinctions in these two structures give each domain specificity for distinct phosphotyrosine motifs. Phosphoserine (pS) recognition domains include MH2 and the WW domain, while phosphothreonine (pT) is recognized by forkhead-associated (FHA) domains. The ability of phosphoproteins to recruit other proteins is critical for signal transduction, in which downstream effector proteins are recruited to phosphorylated signaling proteins. Phosphorylation only occurs at the side chains of three amino acids, serine, threonine and tyrosine, in eukaryotic cells. These amino acids have a nucleophilic (-OH) group that attacks the terminal phosphate group (γ-Ρ032-) on the universal phosphoryl donor adenosine triphosphate (ATP), resulting in the transfer of the phosphate group to the amino acid side chain. This transfer is facilitated by magnesium (Mg2+), which chelates the γ- and β- phosphate groups to lower the threshold for phosphoryl transfer to the nucleophilic (-OH) group. This reaction is unidirectional because of the large amount of free energy that is released when the phosphate -phosphate bond in ATP is broken to form adenosine diphosphate (ADP).

Protein phosphorylation, as being reversible PTM, is mediated by kinases and phosphatases that phosphorylate and dephosphorylate substrates, respectively. These two families of enzymes facilitate the dynamic nature of phosphorylated proteins in a cell. Indeed, the size of the phosphoproteome in a given cell is dependent upon the temporal and spatial balance of kinase and phosphatase concentrations in the cell and the catalytic efficiency of a particular phosphorylation site.

In this connection it should be understood that kinases and phosphatases and their essential co-factors or co-substrates are optional components/ additives to the methods and kit of the present invention, applicability of which has been demonstrated in the present examples.

More specifically, in some embodiments, to provide conditions that allow phosphorylation, the sample may be supplemented prior to or during the PTM reaction with at least one of kinase/s, mutated kinase/s, kinase inhibitor/s, phosphatase inhibitor/s, kinase substrate/s, and ATP.

Kinases are enzymes that facilitate phosphate group transfer to substrates. Greater than 500 kinases have been predicted in the human proteome; this subset of proteins comprises the human kinome. Protein kinases are categorized into subfamilies that show specificity for distinct catalytic domains and include tyrosine kinases or serine/threonine kinases. Approximately 80% of the mammalian kinome comprises serine/threonine kinases, and >90% of the phosphoproteome consists of pS and pT. Studies have shown that the relative abundance ratio of pS:pT:pY in a cell is 1800:200: 1. Although pY is not as prevalent as pS and pT, global tyrosine phosphorylation is at the forefront of biomedical research because of its relation to human disease via the dysregulation of receptor tyrosine kinases (RTKs). ATP is the co-substrate for almost all protein kinases, although guanosine triphosphate is used by a small number of kinases. ATP is the ideal structure for the transfer of α-, β- or γ- phosphate groups for nucleotidyl-, pyrophosphoryl- or phosphoryltransfer, respectively. While the substrate specificity of kinases varies, the ATP-binding site is generally conserved.

Other optional component on methods and kits of the invention, especially for purposes of refined and differential analyses, are protein kinase and/ or phosphatase inhibitors that block the action of one or more protein kinases and/ or phosphatases. Some of the kinase inhibitors may be specific inhibitors of tyrosine kinases. Numerous compounds have been identified and used to inactivate or block these enzymes.

In some specific embodiments, the sample may be supplemented with a tyrosine kinase, for example, Btk. In yet another embodiment, the sample may be further supplemented with phosphatase inhibitor/s, for example, the tyrosine phosphatase inhibitor sodium orthovanadate (SOV), as shown in Examples 3, 4 and 5.

It should be appreciated that in some embodiments, a detectable label specific for phosphorylation may be a label attached to an antibody (either to a primary or a secondary antibody), that specifically recognizes and binds phospho-Tyrosine, phospho-Serine or phospho-Threonine. In some specific and non-limiting examples, the phosphorylation may be therefore measured and identified by an immuno-assay. In yet some further alternative embodiments, the PTM, e.g., phosphorylation may be assessed using mass spectrometry (MS) assay.

As shown by Example 4 and Figure 6, the normalization step of the method of the invention enables one to distinguish between autophosphorylation and phosphorylation of a specific protein. Therefore, according to some embodiments, the method of the invention may be applicable for detecting autophosphorylation of a protein in a sample.

More specifically, in this embodiment, a further normalization step has been applied on each protein. Specifically, the PTM reactions for each protein were performed in the presence or in the absence of the sample, e.g., cell extract or lysate. Normalizing the PTM measured levels of each protein on-chip, in the presence as compared to the absence of the sample, reveals auto-phosphorylated proteins, namely, proteins that are phosphorylated in the absence of any external kinase. This method further provides more accurate information that relates to the kinases present in the examined sample and may further reduce irrelevant background. Still further, as shown in Figure 7 (Example 5), the invention demonstrates phosphorylation of membrane proteins by Src kinase. In this assay, phosphorylation of plurality of tagged proteins (freshly expressed membrane or soluble proteins) by Src kinase was compared and normalized to their phosphorylation upon incubation with an inactive mutated Src. This experiment further exemplifies the use of at least one further additional normalization step to the method of the invention. More specifically, two normalization steps were used in this embodiment. The first was normalization of the signal reflecting the amount of each individual protein as assessed by measuring the tag of each protein, or alternatively, the label or label moiety (e.g., flurorecent or isotope label). The second normalization step includes normalizing the PTM (e.g., phosphorylation) results of each protein by active Src with the results of the same protein in the presence of inactive Src. This approach provides information indicating auto-phosphorylation of specific proteins.

It should be appreciated that similar analysis may be performed using other kinases (comparing active wild type form to an inactive mutated form), or any other proteins involved with any PTM reactions.

The invention further provides in alternative specific embodiments thereof, an additional approach for evaluating phosphorylation and autophosphorylation of proteins by a biological sample. More specifically, plurality of cDNA molecules encoding tagged kinases is provided as the tagged proteins. In some embodiments, the invention additionally provides plurality of cDNA molecules encoding the same tagged kinases, but in inactive mutated form (e.g., mutations in their active site). These mutated kinases lack ability of autophosphorylation. PTM reactions performed in the presence of the examined sample reflect the phosphorylation of said kinases by other kinases present in the sample. This embodiment also uses a further normalization step in addition to the normalization of the PTM reaction results with the protein levels of each protein. More specifically, the PTM levels (phosphorylation) in the presence of the examined sample are normalized for the mutated kinases as compared to the non-mutated kinases in the array.

Thus, the invention provides variety of approaches for accurate determination of PTMs, specifically, phosphorylation as well as for auto-phosphorylation in a sample.

It should be further understood that these examples demonstrate specific embodiments of the methods of the invention that involve more than a single normalization step. Thus, in some embodiments, the method of the invention may comprise at least one, at least two, at least three, at least four, at least five, at least six at least seven, at least eight, at least none, at least ten or more additional normalization steps.

In yet another embodiment, the normalization step allows further flexibility of the method of the invention enabling the analysis and detection of reversible PTMs. A non limiting example for reversible PTM is dephosphorylation as demonstrated in Example 3. Thus, in certain embodiments, the methods and kits of the invention may be used for analyzing dephosphorylation.

It should be appreciated that in some embodiments, the method of the invention may be applicable for screening and identification of protein targets for specific PTM enzyme/s. According to more specific embodiments the method may comprise; first in step (a), providing a plurality of candidate tagged target proteins or any peptides thereof. In some embodiments, the tagged protein/s may be further labeled by a detectable label or moiety. The next step (b) involves measuring the signal intensity of a detectable tag or detectable label or moiety for each protein of the plurality of candidate target tagged proteins. In the next step (c) a sample or any functional extract thereof supplemented with said PTM enzyme/s is contacted with each protein of said candidate tagged proteins under conditions that allow PTM reactions to occur. In the next step (d), the signal intensity of a detectable label or moiety specific for the PTM generated by each individual PTM reaction of step (c), is measured and determined. The next step (e) involves normalizing for each protein the signal intensity measured/determined in step (d) with the signal intensity measured in step (b), to obtain the PTM value for each protein of the plurality of candidate target tagged proteins. The final step (f) involves determining if the PTM value for each candidate protein obtained in step (e) is any one of positive or negative with respect to a PTM value of at least one of positive control protein/s (known as a target for the specific PTM reaction) and negative control protein/s (known as proteins that are not a target for the specific PTM reaction analyzed).

It should be further appreciated that in certain embodiments, the methods and systems provided by the invention may be useful for screening and identification of inhibitors of specific PTM enzyme/s. In such case, the above PTM reactions are performed in the presence of candidate inhibitors or modulators and any change in the PTM value of the protein in the presence of the candidate may indicate that the examined candidate may be used as an inhibitor or modulator for the specific PTM enzyme examined. Still further, the method of the invention may be applicable for determining PTM state of at least one protein in a subject. In more specific embodiments, the method of the invention may comprise: (a) providing a plurality of tagged proteins or any peptides thereof; In some embodiments, the tagged proteins may be further labeled by at least one detectable label or moiety, (b) measuring the signal intensity of a detectable tag or detectable label for each protein of the plurality of tagged proteins; (c) contacting a biological sample of the examined subject or any functional extract of the sample with each protein of said plurality of tagged proteins under conditions that allow individual PTM reactions to occur; (d) measuring and determining the signal intensity of a detectable label or moiety specific for PTM generated by each individual PTM reaction of step (c). In the next essential step (e), normalizing for each protein the signal intensity measured in step (d) with the signal intensity measured in step (b), to obtain the PTM value for each protein of said plurality of tagged proteins.

In more specific embodiments, the method of the invention described above may be applicable for diagnosing a pathologic condition associated with alteration of PTM of at least one protein. In such specific embodiments, the method may further comprise the step of (f), determining if the PTM value for each candidate protein obtained in step (e) is any one of positive or negative with respect to a predetermined standard PTM value of said protein in at least one of a population of subjects suffering from said condition and in a population of subjects that do not suffer from said condition or to a PTM value of said protein in at least one control sample.

As demonstrated by Example 1 and Figures 1 and 2, the invention provides a powerful platform for analyzing PTM reactions in a sample, based on a microfluidic device. Thus, in certain embodiments, the methods of the invention may be performed using the microfluidic device of the invention. Certain embodiments of the invention therefore provide methods for analyzing PTMs, implemented using a microfluidic device. In more specific embodiments, such device may comprise at least one chip comprising plurality of single assay units. More specifically, each of the units comprise at least one DNA chamber and at least one protein chamber, said chambers are isolated from one another by micromechanical valve/s.

In some embodiments, the assay units further include a reaction chamber where the PTM reaction is performed. In yet some specific and non-limiting embodiments, at least one of the protein chambers may be used as reaction chambers, specifically, as isolated PTM reaction chambers. It should be appreciated that in some embodiments, in each reaction chamber that comprise at least one copy of one tagged protein, a single PTM reaction may be performed in one immobilized tagged and/or labeled protein. In yet some other embodiments, more than one PTM reaction may be performed, either simultaneously or in either order in each single reaction chamber, specifically in each single tagged and/or labeled protein. In such case, different detectable labels or moieties must be used for each PTM reaction.

In some embodiments, the use of the microfluidic device by any of the methods of the invention may involve further specific steps that may include applying a cell-free transcription-translation system into the DNA chambers that comprise an expression template, maintaining the reaction under conditions in which protein synthesis occurs thereby producing said tagged proteins by the expression templates.

As noted above, the IMPA platform of the invention is based on a microfluidic chip of between about 2 to 50,000 or more assay units. Specifically, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60, 61, 62, 63, 64, 65, 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, 100 or more, specifically, 110, 120, 130, 140, 150, 160, 170, 180, 190, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900, 1000, 2000, 3,000, 4,000, 5,000, 6,000, 7,000, 8,000, 9,000 and 10,000 or more. In further embodiments, 20,000, 25,000, 30,000, 35,000, 40,000, 45,000, 50,000 and more assay units may be used. Each assay unit contains at least two spotting chambers (e.g., DNA chamber and protein chamber) that encapsulate the sample. Assay units are isolated from one another during incubation steps with isolation valves to eliminate cross-contaminations. Samples to be analyzed may be automatically picked from an appropriate microtiter plate with a microarray robot, and precisely spotted on the reaction chambers.

As used herein, the term "microfluidic" device refers to a device with structures (channels, chambers, valves and the like, forming the assay unit/s or reaction unit/s) at least some of which have at least one dimension on the order of tens or hundreds of microns. In general, at least one structure of the device has dimension(s) below 1000 microns.

As used herein, "assay unit/s" or "reaction unit" refers to a combination of microftuidic structural elements that is repeated many times (e.g., 2 to 10,000 times or more, 100 to 5,000 times, or 250-2500 times) in a microfluidic device, where assay or reaction units can operate simultaneously to carry out a function in a highly parallel manner. Common polymers that may be used for preparing the device used by the method of the invention may include perfluoropoly[beta]thers, polyisoprene, polybutadiene, polychloroprene, polyisobutylene, poly(styrene-butadiene-styrene), polyurethanes, and silicones, for example, or poly(bis(fluoroalkoxy)phosphazene) (PNF, Eypel-F), poly(carborane-siloxanes) (Dexsil), poly(acrylonitrile-butadiene) (nitrile rubber), poly(l- butene), poly(chlorotrifluoroethylene- vinylidene fluoride) copolymers (Kel-F), poly(ethyl vinyl ether), poly(vinylidene fluoride), poly(vinylidene fluoride-hexafluoropropylene) copolymer (Viton), elastomeric compositions of polyvinyl chloride (PVC), polysulfone, polycarbonate, polymethylmethacrylate (PMMA), and polytertrafluoroethylene (Teflon), polydimethylsiloxane, polydimethylsiloxane copolymer, and aliphatic urethane diacrylate.

As used herein, the term "flow channel" that may be included in the assay unit/s, refers to a microfluidic channel through which a solution can flow. The dimensions of flow channels can vary widely but typically include at least one cross-sectional dimension (e.g., height, width, or diameter) less than 1 mm, preferably less than 0.5 mm, and often less than 0.3 mm. Flow channels often have at least one cross- sectional dimension in the range of 0.01 to 1000 microns.

An "isolated reaction chamber" generally refers to a reaction chamber that is not in fluid communication with other reactions chambers present on the device (i.e., one or more valves are closed to isolate the site). In some specific embodiments, the PTM reactions are performed in the reaction chambers. For illustration and not limitation, reaction chambers may often have volumes of about 0.1 - 100 nL. Exemplary reaction chambers may have a generally circular, squared, triangle, or hexagonal (or any appropriate shape) footprint and have dimensions including a diameter of about 10 to about 1000 microns, e.g., from about 200-300 microns, e.g., about 250 microns, and heights of from about 1 to about 200 microns, e.g., about 5 to about 20 microns, e.g., about 10 microns. Chambers having a non-circular shape may have similar volumes.

It should be understood that the invention further provides functional arrays, chips and devices of membrane, nuclear, cytoplasmic, secreted, wild type, mutated, chimeric and fusion proteins useful for analyzing PTM thereof in a sample.

As noted above, alterations in PTMs have been shown as associated with different pathological conditions including neurodegenerative disorders, proliferative disorders and metabolic disorders, therefore, the method of the invention that provides a powerful tool for analyzing PTMs in a sample, may be also applicable in diagnosing such disorders.

Thus, in a second aspect, the invention relates to diagnostic method for detecting a pathological condition associated with altered PTM state of at least one protein in a subject. In more specific embodiments such method may comprise in a first step (a) providing a plurality of tagged proteins or any peptides thereof. In the next step (b) measuring the signal intensity of a detectable tag for each protein of the plurality of tagged proteins. In step (c) contacting a biological sample of the subject or any functional extract of said sample with each protein of said plurality of tagged proteins under conditions that allow individual PTM reactions to occur. In step (d) measuring/determining the signal intensity of a detectable label or moiety specific for PTM generated by each individual PTM reaction of step (c). The next step (e) involves normalizing for each protein the signal intensity measured/determined in step (d) with the signal intensity measured in step (b), to obtain the PTM value for each protein of said plurality of tagged proteins. Finally, in step (f) determining if the PTM value for each protein obtained in step (e) is any one of positive or negative with respect to a predetermined standard PTM value of said protein at least one of a population of subjects suffering from said condition and in a population of subjects that do not suffer from said condition or to a PTM value of said protein in at least one control sample.

It must be appreciated that detecting a pathological condition associated with altered PTM state of at least one protein in a subject according to the invention is achieved using a sample obtained from the subject. The physiological conditions that are present in the subject as mimicked by providing the sample from the subject, reflects specific pathological states or specific tissue type. It should be further appreciated that the ability of the sample or any extract or preparation thereof to perform PTMs of the tagged proteins, indicates the ability of the subject in a specific physiological condition to modify the same proteins.

More specifically, abnormalities in the ubiquitin-dependent proteolysis pathway, also referred to as Ubiquitin Proteasome System (UPS) have been implicated in a number of human disorders, including Parkinson and Alzheimer diseases, prion infection, amyotrophic lateral sclerosis, polyglutamine expansion disorders, cystic fibrosis, multiple myeloma and other cancers. In some disorders, a direct pathogenetic linkage between an UPS aberration and the resulting disease has been identified, for example, Angelman syndrome, Autosomal recessive juvenile parkinsonism, Liddle syndrome, Von Hippel-Lindau protein associated malignant conditions, Colon cancer, Fanconi anemia, Uterine cervical carcinoma, Various malignant conditions, Breast and ovarian cancer and Familial cylindromatosis.

SUMOylation is important for the normal functions of proteins in the cell. However, in the last several years a number of studies have suggested that sumoylation also plays a role in human disease pathogenesis. Indeed, proteins that play key roles in a number of human disease states, including huntingtin, ataxin-1, tau, a-synuclein, DJ-1 (also called PARK7 (Parkinson's disease 7) and superoxide dismutase 1 (SOD1), are targets of SUMO modification.

Several lines of evidence point to a role for the SUMO modification pathway in tumorigenesis and reduced survival. In addition, sumoylation can regulate the activities of important tumor suppressor proteins, including p53, pRB (retinoblastoma protein), p63, p73, and Mdm2 (murine double minute 2). A number of proteins that play important roles in neurodegenerative diseases are known to be sumoylated. These include proteins involved in Huntington's disease (huntingtin), spinocerebellar ataxia type 1 (ataxin-1), Parkinson's disease (tau, a-synuclein, DJ-1), amyotrophic lateral sclerosis (SOD1), and Alzheimer's disease (tau, APP).

Alteration in glycosylation has been also indicated for neurodegenerative disorders. More specifically, glycosylation of microtubule- associated protein tau related to AD have been shown. Acetylation, deacetylation and methylation were shown as involved in neurologic and psychiatric disorders, including Huntington's Disease (HD), PD, anxiety and mood disorders, Rubinstein-Taybi syndrome and Rett syndrome: Epigenetic targets of HDAC inhibition in neurodegenerative and psychiatric disorders.

Thus, it should be appreciated that the diagnostic method of the invention encompasses any of the pathologic conditions associated with or linked to alteration in PTMs, as indicated above.

It is understood that the interchangeably used terms "associated", "linked" and "related", when referring to pathologies herein, mean diseases, disorders, conditions, or any pathologies which at least one of: share causalities, co-exist at a higher than coincidental frequency, or where at least one disease, disorder condition or pathology causes the second disease, disorder, condition or pathology. More specifically, as used herein, "disease", "disorder", "condition", "pathology" and the like, as they relate to a subject's health, are used interchangeably and have meanings ascribed to each and all of such terms. In some embodiments, the plurality of tagged proteins provided by the methods of the invention may be immobilized to a solid support in a predetermined position.

In more specific embodiments, a plurality of tagged proteins according to step (a) of the method of the invention may be provided by (i) providing a plurality of expression templates, specifically, cDNA molecules encoding tagged proteins or any peptides thereof; (ii) incubating the plurality of expression templates, specifically, cDNA molecules under conditions allowing in vitro translation of the expression templates, specifically, cDNA molecules into tagged proteins; and (iii) immobilizing each of the tagged proteins obtained in step (ii) to a solid support in a predetermined and optionally recorded position.

In some specific embodiments, each of said cDNA molecules or tagged proteins may be contained in an individual chamber.

In certain embodiments, the tagged proteins may comprise at least one tag. In such case, the tagged protein may be optionally labeled by isotope or fluorescent detectable label. In further embodiments, the tagged protein of the invention may comprise at least two different tagging molecules, or even more. More specifically, at least one, two, three, four, five, six, seven, eight, nine, ten tags or even more.

In some specific embodiments, conditions that allow PTM reactions to occur may include but are not limited to supplementing the sample or any functional extract thereof with at least one of reagent/s, enzyme/s, substrate/s, co-factor/s and inhibitor/s, prior to or during said PTM reaction.

It should be appreciated that in certain embodiments, the method of the invention may be applicable for analyzing any PTM reactions. Non-limiting examples include at least one of phosphorylation, ubiquitination, sumoylation, neddylation, ISGylation (using ISG15), fatylation (using FAT10), fatylation, pupylation and any other eukaryotic or prokaryotic ubiquitin like modifier. It should be appreciated that the invention encompasses any PTM reaction, and specifically, any of those disclosed herein before.

In such specific embodiments, conditions that allow a ubiquitination reaction may include supplementing the sample prior to or during the PTM reaction, with at least one of ubiquitin ligase/s, mutated ubiquitin ligase/s, ubiquitin ligase inhibitor/s, wild type ubiquitin, mutated ubiquitin, wild type ubiquitin comprising a detectable label or moiety and mutated ubiquitin comprising a detectable label or moiety.

In some embodiments, the method of the invention involves supplementing the sample with ubiquitin that comprises a detectable label or moiety. In such case, the intensity of the detectable label in the ubiquitin is measured and is considered as a detectable label specific for PTM generated by each individual PTM reaction. Determination of the detectable label that reflects the extent of the ubiqutination of the analyzed protein may be performed by any colorimetric method described above. However, in some further embodiments, it must be understood that the MS may be successfully employed as discussed above.

In some specific embodiments, the method of the invention determines Ub-chain preference of a protein in a subject.

For determining the Ub-chain preference of a protein, in some specific embodiments the sample may be supplemented with at least one of wild type ubiquitin, mutated ubiquitin, wild type ubiquitin comprising a detectable label or moiety and mutated ubiquitin comprising a detectable label or moiety. Non limiting examples for such mutated ubiquitin may include the lysine (K) to arginine (R) mutants, for example, UbKl lR, and UbK48R. Alternatively, Ub- chain preference may be detected using antibodies or any other affinity molecules specific for different Ub-chains. In yet some further embodiments, detection and analysis of Ub-chain preference of a protein may be performed using MS assays as described herein before.

In some embodiments, the method of the invention involves analyzing phosphorylation in the subject.

In some embodiments, to provide conditions that allow phosphorylation, the sample may be supplemented prior to or during the PTM reaction with at least one of kinase/s, mutated kinase/s, kinase inhibitor/s, phosphatase inhibitor/s, kinase substrate/s, and ATP. In some embodiments, the sample may be supplemented with a tyrosine kinase, for example, Btk. In yet another embodiment, the sample may be further supplemented with phosphatase inhibitor/s, for example, the tyrosine phosphatase inhibitor sodium orthovanadate (SOV), as shown in Examples 3 and 4.

It should be appreciated that in some embodiments, a detectable label specific for phosphorylation may be a label attached to an affinity molecule, for example, an antibody (either to a primary or a secondary antibody), that specifically recognizes and binds phospho- Tyrosine, phospho-Serine or phospho-Threonine. The phosphorylation may be therefore measured and identified by an immuno-assay, or alternatively, by MS analysis.

In some specific embodiments, the method of the invention may be used for detecting at least one of phosphorylation, autophosphorylation and dephosphorylation of a protein in a sample.

As mentioned above, the methods and kits (described herein after) of the invention may use for detection of the tagged proteins, detection of the PTM reactions (e.g., phosphate group and the like) or for immobilization purpose, antibodies. The term "antibody" as used in this invention includes whole antibody molecules as well as functional fragments thereof, such as Fab, F(ab')2, and Fv that are capable of binding with antigenic portions of the tagged polypeptides. The antibody is preferably monospecific, e.g., a monoclonal antibody, or antigen-binding fragment thereof. The term "monospecific antibody" refers to an antibody that displays a single binding specificity and affinity for a particular target, e.g., epitope. This term includes a "monoclonal antibody" or "monoclonal antibody composition", which as used herein refer to a preparation of antibodies or fragments thereof of single molecular composition.

As noted above, the term "antibody" also encompasses antigen-binding fragments of an antibody. The term "antigen-binding fragment" of an antibody (or simply "antibody portion," or "fragment"), as used herein, may be defined as follows:

(1) Fab, the fragment which contains a monovalent antigen- binding fragment of an antibody molecule, can be produced by digestion of whole antibody with the enzyme papain to yield an intact light chain and a portion of one heavy chain;

(2) Fab', the fragment of an antibody molecule that can be obtained by treating whole antibody with pepsin, followed by reduction, to yield an intact light chain and a portion of the heavy chain; two Fab' fragments are obtained per antibody molecule;

(3) (Fab')2, the fragment of the antibody that can be obtained by treating whole antibody with the enzyme pepsin without subsequent reduction; F(ab')2 is a dimer of two Fab' fragments held together by two disulfide bonds;

(4) Fv, defined as a genetically engineered fragment containing the variable region of the light chain and the variable region of the heavy chain expressed as two chains; and (5) Single chain antibody ("SCA", or ScFv), a genetically engineered molecule containing the variable region of the light chain and the variable region of the heavy chain, linked by a suitable polypeptide linker as a genetically fused single chain molecule.

Methods of generating such antibody fragments are well known in the art (See for example, Harlow and Lane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, New York, 1988, incorporated herein by reference).

Antibodies used by the present invention may optionally be covalently or non-covalently linked to a detectable label. The term "labeled" can refer to direct labeling of the antibody via, e.g., coupling (i.e., physically linking) a detectable substance to the antibody, and can also refer to indirect labeling of the antibody by reactivity with another reagent that is directly labeled. Examples of indirect labeling include detection of a primary antibody using a fluorescently labeled secondary antibody. More specifically, detectable labels suitable for such use include any composition detectable by spectroscopic, photochemical, biochemical, immunochemical, electrical, optical or chemical means. Useful labels in the present invention include magnetic beads, fluorescent dyes (e.g., fluorescein isothiocyanate, Texas red, rhodamine, green fluorescent protein, and the like), radiolabels (e.g., ³H, ¹²⁵1, ³⁵S, ¹⁴C, or ³²P), enzymes (e.g., horseradish peroxidase, alkaline phosphatase) and colorimetric labels such as colloidal gold or colored glass or plastic (e.g. polystyrene, polypropylene, latex, etc.) beads.

As noted above, the methods of the invention are performed using biological samples. The term "biological sample" in the present specification and claims is meant to include any biological samples. Biological samples may be obtained from mammal, specifically, a human subject, include fluid, solid or tissues. The term "sample" may also include body fluids such as whole blood sample, blood cells, bone marrow, lymph fluid, serum, plasma, urine, sputum, saliva, faeces, semen, spinal fluid or CSF, the external secretions of the skin, respiratory, intestinal, and genitourinary tracts, tears, milk, any human organ or tissue, any biopsy, for example, lymph node or spleen biopsies, any sample taken from any tissue, tissue extract, cell or cell culture any sample obtained by lavage optionally of the breast ductal system, plural effusion, samples of in vitro or ex vivo cell culture and cell culture constituents. Some samples that are a priori not liquid may be contacted with a liquid buffers which are then used according to the methods of the invention.

Biological samples may be obtained from all of the various families of domestic animals, as well as feral or wild animals, including, but not limited to, such animals as ungulates, bear, fish, lagamorphs, rodents, etc. Specifically, the sample is liquid, specifically, a body fluid sample, more specifically, a sample of mammalian origin, specifically, human.

Still further, the methods of the invention involve the step of contacting the sample or any extract or preparation thereof with the tagged proteins. The term "contacting" means to bring, put, incubate or mix together. As such, a first item is contacted with a second item when the two items are brought or put together, e.g., by touching them to each other or combining them. In the context of the present invention, the term "contacting" includes all measures or steps which allow interaction between each of the tagged proteins and the tested sample or any extract or preparation thereof. The contacting is performed in a manner so that at least one of the PTM enzymes, factors and co-factors in the sample can interact with the tagged protein and perform a PTM reaction.

Still further, as noted before, an essential step in the method of the invention is the normalization step. It should be noted that any assayed sample may contain more or less biological material than is intended, due to human error and equipment failures. Importantly, the same error or deviation applies to both the tagged proteins and the sample. Thus, division of the PTM value measured in a specific reaction by the signal measured for each tagged protein before sample has been added yields a quotient which is essentially free from any technical failures or inaccuracies (except for major errors which destroy the sample for testing purposes) and constitutes a normalized PTM value. More specifically, the essential normalization step in certain embodiments, involves dividing the PTM reaction value with the values measured for the protein amount of each tagged protein (e.g., quantifying the tag). As noted herein before, the methods of the invention may comprise further normalization steps that are performed for each and every tagged and/or labeled protein of the invention that is normalized to itself. Such further normalization steps may include dividing the normalized PTM results for each protein with PTM results in the absence of a sample (as exemplified in Figure 6), dividing the normalized PTM results achieved using the active kinase with normalized PTM results performed using the inactive mutated kinase (as shown in Figure 7).

The present invention relates to the diagnosis of subjects, or patients. By "patient" or "subject" it is meant any organism who may be affected by the above-mentioned conditions, and to whom the diagnosis methods herein described is desired, including humans, domestic and non-domestic mammals such as canine and feline subjects, bovine, simian, equine and murine subjects, rodents, domestic birds, aquaculture, fish and exotic aquarium fish. It should be appreciated that the treated subject may be also any reptile or zoo animal. More specifically, the methods of the invention are applicable for mammals. By "mammalian subject" is meant any mammal which the proposed diagnostic methods are desired, including human, equine, canine, and feline subjects, most specifically humans.

In certain embodiments, the method of the invention may be implemented using a microfluidic device comprising at least one chip comprising plurality of single assay units, wherein each of said units comprise at least one DNA chamber and at least one protein chamber, said chambers are isolated from one another by micromechanical valve/s.

(a) at least one of a plurality of cDNA molecules encoding tagged proteins or any peptides thereof; and a plurality of tagged and/or labeled proteins or any peptides thereof; and(b) at least one of reagent/s, enzyme/s, substrate/s, co-factor/s and inhibitor/s required for at least one PTM reaction.

(b) In some specific embodiments, each of the plurality of tagged proteins provided by the method of the invention may be immobilized to a solid support in a predetermined and recorded position.

As indicated above, the PTM assays described herein may involve attaching, immobilizing or spotting of the plurality of proteins and/or cDNA molecules in a solid support. As used herein, the terms "attaching" and "immobilizing" refer to a process of depositing, binding or linking the plurality of proteins and/or cDNA molecules onto a substrate or solid support.

As used herein, "immobilized" or "stably bound" when used in connection with the tagged proteins or the cDNA molecules of the invention, refer to a plurality of proteins and/or cDNA molecules that are stably bound to a solid substrate or support via covalent bonds, hydrogen bonds or ionic interactions such that the plurality of proteins and/or cDNA molecules retain the unique pre-selected position relative to all other proteins and/or cDNA molecules that are stably associated with the support, or to all other pre-selected regions or compartments on the solid substrate under conditions in which PTM reactions are typically analyzed (i.e., during one or more steps of washes and/or , incubation etc.).

As used herein, "substrate" or "support" or "solid support", when referring to the methods and kits of the invention, refers to a material having a rigid or semi-rigid surface. The support may be biological, non-biological, organic, inorganic, or a combination of any of these, existing as particles, strands, precipitates, gels, sheets, tubing, chambers, spheres, beads, containers, capillaries, pads, slices, films, plates, slides, chips, etc. Often, the substrate is a silicon or glass surface, (poly)tetrafluoroethylene, (poly) vinylidendifmoride, polystyrene, polycarbonate, a charged membrane, such as nylon or nitrocellulose, or combinations thereof. Preferably, at least one surface of the substrate may be substantially flat. The support may optionally contain reactive groups, including, but not limited to, carboxyl, amino, hydroxyl, thiol, and the like. In one embodiment, the support may be optically transparent. As noted above, the solid support may include polymers, such as polystyrene, agarose, sepharose, cellulose, glass, glass beads and magnetizable particles of cellulose or other polymers. The solid-support can be in the form of large or small beads, chips or particles, tubes, plates, or other forms.

In some specific embodiments, each of said cDNA molecules or tagged proteins may be contained in an individual chamber or any tube, vessel, well and the like, allowing the performance of a separated individual PTM reaction.

In certain embodiments, the tagged proteins may comprise at least two different tagging molecules, or even more. More specifically, at least one, at least two, at least three, four, five, six, seven, eight, nine, ten tags or even more. In some specific embodiments, the tagged proteins used by the methods and kits of the invention are doubly tagged proteins, where one tag being employed for attachment and the second tag being used for quantification.

In certain embodiments, where each of the proteins in the plurality of proteins comprises only one tag, specifically, a tag used for on-chip immobilization, the tagged proteins may be optionally further labeled by at least one fluorescent or isotope label. In some embodiments, the kit of the invention may be used for detection of at least one PTM, e.g., phosphorylation and ubiquitination. It should be noted however that any PTM reaction may be applicable for the kit of the invention. In some specific and non limiting examples the PTM reaction may be at least one of sumoylation, fatylation, neddylation, pupylation and ISGylation, myristolation, palmitoylation, isoprenylation or prenylation, farnesylation, geranylgeranylation, glypiation, covalent attachment of flavin moiety, attachment of heme C, phosphopantetheinylation, retinylidene Schiff base formation, diphthamide formation, ethanolamine phosphoglycerol attachment, hypusine formation, acylation, [e.g. 0-acylation (esters), N-acylation (amides), S- acylation (thioesters), and crotonylation], acetylation, deacetylation, formylation, alkylation, methylation or demethylation, amide bond formation, arginylation, polyglutamylation, polyglycylation, butyrylation, gamma-carboxylation and glycosylation, polysialylation, malonylation, hydroxylation, iodination, ADP-ribosylation, oxidation, phosphate ester (O- linked) or phosphoramidate (N-linked) formation, phosphorylation, adenylylation, propionylation, pyroglutamate formation, 5-glutathionylation, 5-nitrosylation, 5-sulfenylation, succinylation, sulfation, glycation, carbamylation, carbonylation, as well as proteolytic cleavage of peptide bonds, and formation of disulfide bonds by linking cysteine residues.

In some embodiments PTM reaction is ubiquitination.

In some embodiments the kit of the invention may comprise at least one of ubiquitin ligase/s, mutated ubiquitin ligase/s, ubiquitin ligase inhibitor/s, wild type ubiquitin, mutated ubiquitin, wild type ubiquitin comprising a detectable label or moiety and mutated ubiquitin comprising a detectable label or moiety.

In some particular embodiments, the kit of the invention may be used for analyzing ubiquitination in a sample. Non-limiting example concerns a kit that may comprise the following components: (a) a plurality of expression template/s may be provided in certain embodiments as cDNA library of interest (e.g., such library may be obtained from different organisms and may encode different protein composition, for example, membrane proteins, or specific enzymes or mutants and the like); (b) Lysates required for protein expression (e.g., human, mouse or lysates of any mammalian subjects, reticulocyte, wheat germ, E. coli lysate); (c) Energy regeneration mix comprising for example ATP, Creatine phosphate, and creatine phopho kinase); (d) Detection reagents that may include fluorescently labeled Ubiquitin (Rodamin, fluorescein, etc.). Alternatively, the kit may comprise unlabeled ubiquitin, in case the ubiqutination is indirectly labeled using for example, a specific antibody. In such case, the kit of the invention may further comprise detection reagents for unlabeled ubiquitin that may include fluorescently labeled or immunodetectable anti-ubiqutin antibodies; (e) the kit of the invention may further comprise reagents and buffers, for example, wash buffer. It should be noted that in certain cases several buffers, and wash buffers may be required. In certain embodiments, such kit may further comprise antibodies or any other affinity molecule specific for Ub-chain. Such antibodies may be used for analyzing Ub-chain preference in the examined sample.

Still further embodiments relate to kits for analyzing any other ubiquitin like modifiers (UBL) such as Nedd8, Sumol, Sumo2, Sumo3, Sumo4, Fubl , FatlO, ISG15, Atg8, Atgl2, Urml and Ufml. According to some embodiments, such kit per one or more modifiers may comprise: (a) cDNA library of interest (as indicated above, for different organisms and different desired protein compositions); (b) lysate/s for protein expression (human, mouse reticulocyte, wheat germ, E. coli lysate); (c) Energy regeneration mix containing ATP, Creatine phosphate, and creatine phopho kinase; (d) detection reagents may include fluorescently labeled UBL (Rodamin, fluorescein, etc.) or unlabeled UBL. In case unlabeled UBL are used detection reagents may include fluorescently labeled or immunodetectable anti-UBL antibodies; and (e) any appropriate buffers and reagents, specifically, wash buffer.

In other embodiments, the kit of the invention may be used for analyzing phosphorylation.

In more specific embodiments, such kit may comprise at least one of kinase/s, mutated kinase/s, kinase inhibitor/s, phosphatase inhibitor/s, kinase substrate/s, and ATP.

In some specific and noon-limiting embodiments, the invention may provide kits for analyzing tyrosine phosphorylation. Such kit may comprise (a) a plurality of expression templates that may be provided according to certain embodiments, as a cDNA library of interest (different organisms and protein composition). As noted herein before, such library comprise cDNA molecules encoding for different tagged proteins, thereby providing a plurality of tagged proteins for allowing in vitro translation of the fresh tagged protein/s from the cDNA templates; the kit may further comprise (b) lysate/s for protein expression (e.g., human, mouse reticulocyte, wheat germ, E. coli lysate); a further component of the kit of the invention may include (c) Energy regeneration mix that may comprise ATP, Creatine phosphate, and creatine phopho kinase; (d) Detection reagents required for such kit may include fluorescently labeled or immunodetectable anti-phospho tyrosine antibodies; and (e) Wash buffer/s and any further buffers or reagents for every step of the analytic method performed using this kit.

It should be noted that the microfluidic platform provided by the present invention allows flexibility and versatility and provides a powerful tool for analyzing specific PTMs in specific protein groups. Non limiting examples for such application may provide a specific kit for analyzing tyrosine phosphorylation of tyrosine kinases. Such specific kit may be further designed for example to distinguish between kinases that are auto-phosphorylated and kinases that are phosphorylated by other kinases present in the examined sample. A specific example for such kit may require the provision of plurality of kinases as well as plurality of mutants of each one of said kinases. The mutated kinases (mutated for example in their active site) cannot undergo auto-phosphorylation. Normalization of the phosphorylation of plurality of wild type kinases by the examined sample with the phosphorylation of the identical inactive mutants of said kinases, reflects therefore the phosphorylation performed by kinases in the sample. Thus, in some specific embodiments, such particular kit may comprise: (a) plurality of expression templates, specifically cDNA molecules provided for example as a cDNA library of wild type and library of the corresponding mutated kinases that carry at least one mutation in the active site. It should be noted that any organism may be used as the origin for said kinases (for example, human, to pathogens, plants, fungi and the like); (b) Lysate for protein expression (e.g., human or mouse reticulocyte, wheat germ, E. coli lysate); (c) Energy regeneration mix containing ATP, Creatine phosphate, and creatine phopho kinase; (d) Detection reagents may include fluorescently labeled or immunodetectable anti-phospho tyrosine antibodies; and (e) wash buffer and/or any appropriate reagents and buffers.

As noted above, the powerful microfluidic platform provided by the invention enables analyzing the PTM performed by a sample on any particular type or subset of proteins, for example, nuclear, secreted, cytoplasmic, membrane, mitochondrial, lysosomal, enzymes, kinases, or any wild type, chimeric, or mutated protein and the like.

A particular example provided by the invention is a kit specific for membrane proteins. It should be noted that in certain embodiments, such membrane protein kits may be further adapted for particular PTM reaction, by employing the components described above for phosphorylation, ubiquitination or for any other PTM reactions disclosed in the present invention herein before. It should be of course understood that the kit/s of the invention may be designed to provide information for any combination of PTM reactions. Thus, in certain specific and non-limiting embodiments, kits for analyzing PTMs of membrane proteins may comprise (a) plurality of tagged membrane proteins or any expression templates encoding the same, specifically, cDNA molecules, that may be provided as cDNA library of membrane- and membrane-associated proteins of any organism of interest; (b) any lysate and reagents required for in vitro protein expression (e.g., human, mouse reticulocyte, wheat germ, E. coli lysate); (c) an important component that in some specific embodiments may be an essential component of a kit designed for membrane proteins, may be microsomal membranes; (d) the kit may further comprise energy regeneration mix containing ATP, Creatine phosphate, and creatine phopho kinase; (e) labeled or immunodetactable modifier of interest; (f) any appropriated detection reagents, that may include reagents for fluorescently labeled or immunodetectable anti- modifier (e.g., phopho-tyrosine, Ubiquirin, Sumo, Acetylation, and the like); and any buffers, for example wash buffer and further reagents. Overall, it should be noted that the microfluidic platform of the invention, as well as any method or kit of the invention may be adapted for analyzing any PTMs. It should be also noted that PTMs may include modification of the proteins and alternatively, addition or deletion of small molecules. In cases involving modifications of proteins, these may be labeled directly (as exemplified by the rhodamin ubiquitin used in the present invention, specifically in Examples 6 and 7) or indirectly using antibodies (as exemplified for tyrosine phosphorylation). PTM reactions involving small molecules (e.g., phosphate group) may be labeled by antibodies or radioactively. It should be noted that the kits of the invention may be adapted for any of these PTM reactions.

Still further, it should be appreciated that the kits of the invention may be applicable and therefore may comprise appropriate reagents, such as at least one of (a) chemically-Labeled modifiers (fluorescence, radioactive; e.g., Rhodamine-coupled ubiqutin); (b) chemically- labeled primary antibodies (e.g., florescent anti-PTM antibodies) as well as suitable reagents for different assays such as chemiluminescence immunoassay or for any other colorimetric methods, gold (metal) -coupled antibodies, etc. It should be further appreciated that any chemically labeled secondary antibodies may be used, for example chemically labeled (florescence, radioactive, horseradish peroxidase or any other colorimetric assay) secondary antibodies.

The invention further provides a system comprising a plurality of tagged proteins or cDNA molecules immobilized, preferably, in predetermined and recorded position/s, to a solid surface adapted for separate individual PTM reactions. This system may comprise at least one chip and/or device forming a solid support for the plurality of tagged proteins and/or cDNA adapted for separate individual PTM reactions. Non-limiting examples for such system is provided by the invention in the IMPA system described in Example 1, and in some specific embodiments, the device illustrated in Figures 1 and 2. In some embodiments, the predetermined and recorded position/s of the tagged proteins used by the methods of the invention may be also defined by the compartmentalization through the microfluidic device. It is understood that the invention encompass any of the described devices, systems and chips described herein as well as any version thereof. The invention also encompasses any use of the systems and chips described herein in analyzing PTMs of proteins.

Thus, in more specific embodiments, the kit of the invention as described above may further comprise at least one microfluidic device. In more specific embodiments, such device may comprise at least one chip comprising plurality of single assay units. It should be noted that each of these units may comprise at least one DNA chamber and at least one protein chamber. It should be noted that these chambers may be isolated from one another for example by valve/s, specifically micromechanical valve/s.

In still further embodiments, the invention provides the use of any of the kits of the invention for performing any of the methods for analyzing PTMs of proteins in a biological sample as defined by the invention and disclosed herein before. More specifically, any of the kits of the invention may be adapted to perform any of the methods provided and disclosed by the present invention.

All scientific and technical terms used herein have meanings commonly used in the art unless otherwise specified. The definitions provided herein are to facilitate understanding of certain terms used frequently herein and are not meant to limit the scope of the present disclosure.

The terms "comprises", "comprising", "includes", "including", "having" and their conjugates mean "including but not limited to". The term "consisting essentially of" means that the composition, method or structure may include additional ingredients, steps and/or parts, but only if the additional ingredients, steps and/or parts do not materially alter the basic and novel characteristics of the claimed composition, method or structure.

The terms "comprises", "comprising", "includes", "including", "having" and their conjugates mean "including but not limited to". This term encompasses the terms "consisting of" and "consisting essentially of". The phrase "consisting essentially of" means that the composition or method may include additional ingredients and/or steps, but only if the additional ingredients and/or steps do not materially alter the basic and novel characteristics of the claimed composition or method. Throughout this specification and the Examples and claims which follow, unless the context requires otherwise, the word "comprise", and variations such as "comprises" and "comprising", will be understood to imply the inclusion of a stated integer or step or group of integers or steps but not the exclusion of any other integer or step or group of integers or steps.

It should be noted that various embodiments of this invention may be presented in a range format. It should be understood that the description in range format is merely for convenience and brevity and should not be construed as an inflexible limitation on the scope of the invention. Accordingly, the description of a range should be considered to have specifically disclosed all the possible sub ranges as well as individual numerical values within that range. For example, description of a range such as from 1 to 6 should be considered to have specifically disclosed sub ranges such as from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as individual numbers within that range, for example, 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range. Whenever a numerical range is indicated herein, it is meant to include any cited numeral (fractional or integral) within the indicated range. The phrases "ranging/ranges between" a first indicate number and a second indicate number and "ranging/ranges from" a first indicate number "to" a second indicate number are used herein interchangeably and are meant to include the first and second indicated numbers and all the fractional and integral numerals there between.

As used herein the term "method" refers to manners, means, techniques and procedures for accomplishing a given task including, but not limited to, those manners, means, techniques and procedures either known to, or readily developed from known manners, means, techniques and procedures by practitioners of the chemical, pharmacological, biological, biochemical and medical arts.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub combination or as suitable in any other described embodiment of the invention. Certain features described in the context of various embodiments are not to be considered essential features of those embodiments, unless the embodiment is inoperative without those elements.

Various embodiments and aspects of the present invention as delineated hereinabove and as claimed in the claims section below find experimental support in the following examples.

Disclosed and described, it is to be understood that this invention is not limited to the particular examples, methods steps, and compositions disclosed herein as such methods steps and compositions may vary somewhat. It is also to be understood that the terminology used herein is used for the purpose of describing particular embodiments only and not intended to be limiting since the scope of the present invention will be limited only by the appended claims and equivalents thereof.

It must be noted that, as used in this specification and the appended claims, the singular forms "a", "an" and "the" include plural referents unless the content clearly dictates otherwise. EXAMPLES

Materials and methods

Plasmids

Human Securin open reading frame (ORF) was amplified using the plasmid pCS2-FA- Securin as a template, and Xhol (5') and BamHI (3') flanked primers, and cloned into pEGFP-Nl vector (Clontech). Securin-eGFP ORF was then amplified using Fsel flanked primer (5') also carrying six repeats of His tag and AscI flanked primer (3'), and cloned into the pCS2-FA vector. For generating the Δ64 Securin mutant, Securin ORF encoding for amino acids 65-202 was amplified, using Securin-eGFP as a template and Fsel (5')/AscI (3') flanked primers. p27 was amplified from the human full-length clone (Open Biosystems), using Fsel (5')/AscI (3') flanked primers, and cloned into pCS2-FA vector. Geminin cDNA was amplified from U20S cDNA using Fsel (5')/AgeI (3') primers. PCR products were cloned in-frame upstream to eGFP in pCS-FA vector. Plasmids, pCS2-FA-Securin, pCS2-FA, pCS2-FA-Kifcl-eGFP, pCS2-GFP, and Emil C terminus (amino acids 299-477) in pGEX, were obtained from previous studies. Myc-p27-His was generated by a two-step assembly PCR using human p27 ORF, a first primer set containing a c-Myc tag (5') and a His tag (3'), and a second primer set containing a T7 promoter (5') and a T7 terminator sequence (3'). A similar two-step assembly PCR was used to generate all other synthetic genes.

Cell culture and synchronization

HeLa S3 and HEK293 cells were maintained in tissue culture plates (Nunc) containing Dulbecco's Modified Eagles Medium (DMEM) (Gibco) supplemented with 10% fetal bovine serum (FBS) (Biological Industries, Israel), 2 mM L-Glutamine (Gibco) and penicillin (100 u)/streptomycin (0.1 mg/ml) (Gibco). Cells were maintained at 37 °C in a humidified 5% CO2 environment. For Gl extract preparation, HeLa S3 cells were grown in 1 L-spinner flask. At a concentration of ~5xl0⁵ cells/ml, cells were treated with 2 mM thymidine (Sigma, Israel) for 22 h, washed twice, released into fresh prewarmed media for 3 h and incubated with 50 ng/ml nocodazole (Sigma, Israel) for 11 to 12 h. Cells were washed twice and released into fresh prewarmed media for 3.5 h to generate a Gl synchronous population. In order to generate S- phase extracts, HeLa S3 cells, ~5xl0⁵ cells/ml, were incubated 22 h with thymidine, washed twice, released into fresh pre-warmed media for 3 h and harvested. Preparation of cell extracts

Extracts of HeLa S3 and HEK293 cells were prepared as follows: cells were lysed in a swelling buffer [20 mM Hepes pH 7.5, 2 mM MgC12, 5 mM KCl, 1 mM DTT, 1 tablet of complete protease inhibitor cocktail (Roche)] supplemented with energy-regeneration mix (1 mM ATP, 7.5 mM creatine phosphate, 70 mg/ml creatine phosphokinase, 0.1 mM EGTA) and homogenized by freeze-thawing and passage through 21G needle successively. Extracts were cleared by subsequent centrifugations and stored at -80 °C.

Degradation assay

Target proteins were expressed in vitro in rabbit reticulocyte-coupled transcription and translation reaction (TNT-coupled reticulocyte system, Promega) supplemented with ³⁵S- Methionine (IsoLabel). Unless otherwise indicated, degradation assays were performed in a final volume of 25 μΐ containing 20 μΐ human cell, 1 μΐ of X20 energy-regeneration mix, 0.4 mg/ml Ub, 0.3 mg/ml His-tagged UbcHIO or UbcH10^DN, 0.48 mg/ml C-terminus-GST Emil and 1 μΐ radiolabeled IVT product. Samples were incubated at 23 to 30 °C. Aliquots were taken every 15 or 20 min, denatured and quick-frozen in liquid nitrogen. Samples were resolved by SDS-PAGE and visualized by autoradiography using GE phosphorimager, Typhoon-9500. Ub, rhodamine N-terminus-labeled Ub (Rd-Ub) and Ub mutants (Kl l-only, K48-only, K11R, and K48R), purchased from Boston Biochem, were supplemented to the reaction at a final concentration of 0.04 or 0.4 mg/ml.

Molds fabrication

The device was designed in AutoCAD2011 (Autodesk, Inc.) and each layer reproduced as a chrome mask at 40,000 dpi (Fineline-Imaging). Flow molds were fabricated on 4" silicon wafers (Silicon Quest International) coated with hexamethyldisilazane (HMDS) in a vapor bath for 10 min. Subsequently, wafers were spin-coated with SPR 220-7 (Shipley) 1500 rpm for 60 sec yielding a substrate height of around 12-14 μπι. Molds were baked at 105 °C for 6 min, followed by a 60-sec I-line exposure on a MJB-4 contact mask aligner (Karl Suss). Molds were then incubated for 2 h in RT, baked at 110 °C for 10 min, incubated for an additional 45 min at RT and developed with MF319 developer (ROHM and HAAS ) followed by H2O wash. Molds were annealed at ramping temp (70-200C, 10C\h) for 15 h. Control molds were fabricated on 4" silicon wafers by spin coating SU-8 2025 (MicroChem) initially at 500 rpm for 10 sec, followed by 3000 rpm for 60 sec, yielding a substrate height of around 16-20 μπι. Subsequently, molds were baked at 65 °C for 2 min and 95 °C for 5 min. Wafers were exposed for 6 sec on the mask aligner, followed by a post-exposure bake series of 65 °C for 1 min and 95 °C for 7 min. Wafers were developed in PGMEA developer (KMG) for 4.5 min followed by isopropanol wash.

Device fabrication

The microfluidic devices were fabricated on silicone molds casting silicone elastomer polydimethylsiloxane (PDMS, SYLGARD 184^®, Dow Corning). Each device consisted of two aligned PDMS layers, the flow and control layers. The molds were first exposed to chlorotrimethylsilane (Sigma- Aldrich) vapor for 10 min to promote elastomer release after the baking steps. A mixture of silicone-based elastomer and curing agent was prepared in two different ratios, 5: 1 and 20: 1, for the control and flow molds, respectively. The control layer was degassed and baked for 30 min at 80 °C. The flow layer was initially spin-coated (Laurell Technologies) at 2000 rpm for 60 sec, and baked at 80 °C for 30 min. The control layer was separated from its mold, and control-channel-access-holes were punched. Flow and control layers were aligned manually under a stereoscope and baked for 1.5 h at 80 °C. The two-layer device (chip) was peeled from the flow mold, and flow-channels-access-holes were punched.

Surface chemistry

In order to prevent non-specific adsorption and achieve suitable binding orientation of expressed proteins, the entire accessible surface area within the microfluidic device was chemically modified. This surface chemical modification also facilitates the self-assembly of a protein array on the surface. To which end, biotinylated-BSA (1 μg/μl, Thermo) was flowed for 30 min through the device, binding BSA to the epoxy surface. On top of the biotinylated- BSA, 0.5 μg/μl of Stepavidin (Neutravidin, Pierce) was added for 30 min. The 'button' valve was closed and biotinylated-PEG (1 μg/μl, Nanocs) was flowed over for 30 min, thus passivating the rest of the device. Following passivation, the 'button' valve was released and a flow of 0.2 μg/μl penta-His (Qiagen; #34440) or GFP (abeam; #ab6658) biotinylated antibodies was applied. The antibodies bound to the exposed Streptavidin, specifically to the area under the 'button', thereby creating an array of anti-His- or anti-GFP tags. Hepes buffer (50 mM) was used for washing between each surface chemistry step.

Production of human synthetic sene library

Synthetic linear genes were generated by using two-step assembly PCRs. A library of human nuclear ORFs was cherry-picked from an Open Biosystem library of 15,500 full-length human ORFs, and used as a template for the first PCR step. Two epitope tags were added by using a 5' primer carrying a c-Myc tag and a 3' primer carrying a His tag. A PCR reaction mix (20 μΐ) contained 0.8 units of high-fidelity hot-start DNA polymerase (Phusion II, Finnzymes) per reaction. Second-step PCRs was performed using the first PCR products as a template and a primer set containing T7 promoter (5') and T7 terminator (3')· The reaction mixture (50 μΐ) contained 1.5 units of DNA polymerase. PCR products were filtered in multi- well 10 K filter plates (AcroPrep™, Pall Corporation), and eluted with 40 μΐ DDW. PCR products were distributed into UV-transparent 384-well dishes, and their concentration was determined by the Synergy™ 4 Hybrid Microplate Reader (BioTek). All PCRs were performed in 96-well plates.

DNA arraying and device alignment

A subset of 8 synthetic genes or 1024 nuclear synthetic genes was mixed with 0.125% polyethylene glycole and 12.5 mg/ml D-trehalose dihydrate (Sigma- Aldrich) in 384-well plates (Greiner Bio-One). PCR products were spotted in quadruplicates or in 20 copies onto epoxy-coated glass slides (CEL Associates) using a MicroGrid 610 microarrayer (Bio Robotics) equipped with SMT-S75 silicone pins (Parallel Synthesis). The device contained 64 columns and either 64 or 16 rows, with a pitch of 281.25 μπι (columns) by 562.5 μπι (rows). The array was manually aligned to the microfluidic device under a microscope and bonded for 4 h on heated plate at 80 °C.

Protein expression on-chip

A pre-mixed reticulocyte lysate supporting protein expression by T7 promoter (12.5 μΐ), was flowed into the DNA chambers, following surface chemistry. Next, the valves separating each unit cell were closed and the device was incubated on a hot plate for 2.5 h at 32 °C. The IVT product in each unit cell was then diffused from the DNA chamber to the protein chamber. Proteins were immobilized on the anti-His antibodies under the 'button' through their C- terminus His tag (C-terminal tagging ensures an array of full-length proteins). Unbound proteins, protein fragments, and reticulocyte lysate were mechanically washed and then discarded from the chip by 50 mM Hepes buffer. The immobilized proteins were detected by immunofluorescence as follows: Cy3-coupled anti-Myc antibodies (1/100 dilution; Sigma C6594) were flowed into the device, and incubated with the immobilized proteins under the 'button' for 30 min at RT. Unbound antibodies were mechanically washed and then discarded from the chip (50 mM Hepes buffer). Protein expression levels were determined with a microarray scanner (LS Reloaded, Tecan) using a 532 nm laser and 575/50 nm filter. Membrane Protein Expression

Proteins were expressed on the device by using rabbit reticulocyte quick-coupled TNT reaction (Promega). Microsomal membranes (Promega) were added to the extract to express membrane proteins (including L-HDAg). The expression of the proteins from the spotted synthetic genes on the device created an array of proteins ready for use in a PTM reaction. By opening the "neck" valves, 12.5 μΐ. of the expression mix was flowed through the device into the gene chamber. Next, the "sandwich" valves were closed, leaving each unit cell separated from its environment, and the device was incubated on a hot plate for 2.5 h at 32 °C. Expressed proteins were then diffused through the DNA chamber to the protein reaction chamber, binding their C terminus His tag to the anti-His antibody or their N terminus c-Myc tag to the c-Myc antibody under the button valve, immobilizing the protein. Proteins were labeled with a c-Myc (SigmaAldrich) or penta-His (Qiagen) Cy3 antibody, which bound to its corresponding epitope and labeled it.

Mutant Ub labeling

K11R and K48R Ub mutants (Boston Biochem) were fluorescently labeled using DyLight™ 650 (Thermo; #62266). The dye-to-protein ratio was calculated by measuring optical absorption of the dyed proteins with NanoDrop™ (Thermo) at 280- and 655 nm wavelengths.

On-chip ubiquitination and Ub-preference assays

Unlabeled IVT products flowed into the chip and immobilized on the surface under the 'button' at the protein chamber through their C-terminus His or GFP tags (see 'Surface chemistry'). Next, the 'button' valves opened and Gl extract mixtures were flowed for 10 min (RT). Extracts contained 6 μΜ MG132 (Sigma- Aldrich), and either 0.04 mg/ml Rd-labeled Ub or Cy5-labeled K11R-Ub/K48R-Ub. Cell extract mixtures used for the ubiquitination assays further included 0.2 mg/ml unlabeled Ub-Kl lR or Ub-K48R. Unbound material was washed by Hepes buffer (50 mM). In the case of relatively high background, an additional washing step with Hepes buffer containing 0.5 M NaCl was performed. On-chip ubiquitination reactions containing Rd-Ub or Cy5-Ub-mutants were determined by 535- or 633 nm excitation, respectively (emission filters: 575/50 and 692/40). Ub signal was normalized to protein level, as measured by 488 nm-excited eGFP or immunofluorescence with Cy2-coupled anti-Myc antibodies (Abeam; #ab69258) (emission filter: 535/25). On-chip Tyr kinase assay

For monitoring Btk-mediated phosphorylation, Btk target peptide VALYDYM (200 nM; Cell Signaling; #1330) was immobilized under the 'button', through Biotin-Sterptavidin interaction. Double-tagged target proteins (IVT products) were immobilized on the protein chamber through their C-terminus His-tag. Immobilized target peptides or proteins were incubated (30- 60 min, 37 °C) with 300 nM recombinant human Btk (Abeam) in either Btk buffer [Hepes 50 mM, MgCl₂ 3 mM, MnCb 3 mM, ATP 10 mM, sodium orthovanadate (SOV) 3 mM] or HEK293 cell extracts supplemented with mock or 300 nM Btk. Subsequently, immobilized peptides/proteins were mechanically washed and incubated for 30 min with anti-P-Tyr antibodies coupled to Cy5 (Cell Signaling; p-Tyr- 100 - #9415). Levels of target proteins under the 'button' were measured by a second immunofluorescence using Cy3-coupled anti- Myc antibodies. To determine the P-Tyr-to-protein ratio were used 633- and 535 nm lasers and matching 575/50 and 692/40 filters. P-Tyr background signal (normalized to protein level) was determined on the same chip by an equivalent reaction with no kinase. These values were subtracted from the gross phosphorylation signal to calculate average net signals. Background signal was negligible.

1. For monitoring Abl-mediated phosphorylation, eight double-tagged proteins were expressed on-chip, immobilized on the protein chamber through their C-terminus His tag, and incubated (45 min, 37 °C) with 200 nM human recombinant Abl (Abeam) in Abl buffer (Hepes 25 mM, SOV 10 mM, MgCl₂ 25 mM, EGTA 5 mM, EDTA 2 mM, ATP 10 mM). An equivalent reaction with no kinase was performed on the same chip. P-Tyr-to-protein ratio was determined as described for the Btk assay. Background signal was negligible.

2. For Tyr phosphorylation of nuclear proteins, an array of 1024 nuclear proteins was incubated (30 min, 37 °C) with HEK293 cell extracts in the presence or absence of 10 mM SOV and P-Tyr-to-protein ratios were determined and calculated as the average P-Tyr-to- protein ratio of a single nuclear protein (n=4). P-Tyr-to-protein ratios were measured immediately after protein expression.

On-chip dephosphorylation assay

Phosphorylated Btk target peptide VALY*DYM (200 nM, Cell Signaling; #1460) was immobilized on the chip (details are mentioned above), incubated (30 min, 37 °C) with HEK293 cell extracts supplemented with increasing doses of SOV and P-Tyr signal was determined. Imase and data analysis

PTM and protein net signal were measured under the 'button' and PTM-to-protein ratio was determined, using LS Reloaded microarray scanner (Tecan) and GenePix7.0 (Molecular Devices) image analysis software for all experiments.

EXAMPLE 1

IMP A concept and device

To establish an effective and accurate assay overcoming the limitations of previous methods for PTM analysis, the inventors designed a modular system, Integrated Microfluidic platform for PTM Analysis (IMP A), illustrated in Figures 1A-1C and 2A-2B. This system is based on multilayer microfluidics regulated by pneumatic valves and thus combines on-chip in vitro protein synthesis with microarray and in situ PTM assays of freshly synthesized tagged proteins.

Figure 1A shows a typical microfluidic device spotted with a DNA microarray encoding a target protein library (left). Each unit cell on the microfluidic device comprises a DNA chamber and protein chamber controlled by three micromechanical valves, which enable first, to isolate each unit cell, second, to separate the DNA and protein chambers, and third, to establish a surface chemistry for immobilization of a protein to the protein chamber surface (middle). This design enables versatility of applications, whereby a protein can be produced in the DNA chamber by incubation with reticulocyte lysate to enable in vitro translation (IVT), diffused and immobilized via a first protein tag (black triangle) in the protein chamber, and further quantified via a second fluorescent or immunodetectable tag (grey dotted triangle), or directly if the proteins are translated in the presence of a labeled amino acid. Surface chemistry is illustrated on the right. In an alternative embodiment, externally expressed proteins can be applied to the chip through common channels and deposited directly into the protein chamber and quantified. Figure IB illustrates the step of application of cell extracts, used in small volumes (5 - 10 μΐ) and supplemented with recombinant enzyme cocktail for initiation of PTM reaction. Following intensive washing, covalently bound PTMs can be quantified either directly (using fluorescent modifiers) or by immunofluorescence. Figure 1C is a schematic representation of a target protein, 'bait (grey dot), which is covalently modified by fluorescently labeled PTM (black dot) to enable normalization of PTM signal to total protein in each unit cell. Figures 2A-2B are enlarged images of signal detection in two protein chambers. Figure 2A shows the pneumatic -regulated 'button' enabling mechanical washing of unbound material in the protein chamber. Figure 2B shows signal detection using Genepix and Microsoft XI software for data analysis, whereby the 1^st inner 80 μm-diameter- circle is formed around the 'button' and the 2^nd outer 160 μm-diameter-circle is formed around the inner circle. The median fluorescent intensity at the area between the two circles defines the local background signal, which is subtracted from the median fluorescent intensity of pixels of the inner circle to provide the net signal for each dot on the chip.

EXAMPLE 2

Membrane protein expression

The human genome contains about 21,000 distinct protein-coding genes, out of which about 5,360 encode membrane proteins. Membrane proteins are critical for many cellular processes, such as normal and malignant signaling, transport, cell-cell communication, and also interaction with pathogens associated with many human diseases. It is not surprising that 60% of drugs currently in the market target proteins at the cell surface. Mapping protein interactions and post-translational modifications of membrane proteins is, therefore, of utmost importance.

Figure 3 shows linear Myc/His-double-tagged ORFs encoding for nuclear, membrane and cytoplasmic (Figs 3A, 3B and 3C, respectively) proteins generated by assembly-PCR using Open Biosystems' full-length human ORF library as a template. More specifically, linear PCR products were printed on epoxy-coated glass slides in quadruplicates. Matched microfluidic device was aligned accordingly to generate 4096 unit cells optimized for protein expression by reticulocyte lysate. Expressed proteins were deposited on chip via His-tag and quantified by Cy3-coupled anti-Myc antibodies. In the plot each bar averages quadruplicates of a single protein. The expression signals of nuclear protein (Figure 3A), membrane proteins (Figure 3B) and cytoplasmic proteins (Figure 3C) are expressed in a 3D bar graph. Figure 3B demonstrates the compatibility of the platform of the invention to membrane protein expression. Over 1000 different human membrane or membrane-associated proteins (according to the UNIPROT database) were expressed in quadruplicates in the present of a reticulocyte lysate supplemented with canine microsomal membranes. The latter ensure that the membrane proteins express into membrane properly. It should be noted that no obvious difference in expression levels was observed between membrane proteins and nuclear or cytoplasmic proteins. There was no bias due to spotted DNA concentrations or in protein length. The number of transmembrane domains in a protein also did not affect expression levels.

The inventors thus established the feasibility of applying the platform of the invention for generating functional membrane proteins arrays, as well as functional nuclear and cytoplasmic protein arrays.

EXAMPLE 3

IMPA a proof-of-concept

Validity and operability of IMPA system for on-chip detection and quantification of PTM in biological samples was exemplified in a model assay of Tyrosine Phosphorylation (P-Tyr), as being an intensively studied PTM and also due availability of fluorescently labeled P-Tyr antibodies (P-Tyr- 100) by which it can be visualized. A series of experiments using this tool are illustrated in Figures 4A-4G.

Figure 4A is a graphic representation of a tagged target protein covalently modified by P-Tyr, and detected by the Cy5 labeled P-Tyr-100 antibody. Figures 4B-4C illustrate on-chip P-Tyr using Bruton's tyrosine kinase (Btk) and various Btk targets. Figure 4B shows P-Tyr of the biotinylated Btk target peptide (VALYDYM, as also denoted by SEQ ID NO. 1) deposited on chip (via streptavidin binding), incubated with buffer or recombinant Btk (300 nM, 30 min, 37 °C) and immunolabeled with Cy5-coupled P-Tyr-100 antibodies. P-Tyr signals were evident in chambers exposed to Btk (raw data below). The histogram shows average signals from 27 dots normalized to maximum activity. Figure 4C shows on-chip P-Tyr of Plcg2 (known Btk target) and Securin (negative control) deposited on chip as Myc/His double- tagged IVT products (via anti-His Antibodies), incubated with Btk (as above) and immunolabeled with Cy5-coupled P-Tyr-100 and Cy3-coupled anti-Myc antibodies for quantification of relative P-Tyr-to-total protein levels. Raw data are shown below. The histogram shows average net signals for the two proteins calculated from gross phosphorylation signals normalized to total protein («= 15 dots) minus average background Cy5 signals from equivalent reactions with no kinase. These data clearly indicate P-Tyr signals for Plcg2 but not for Securin, thus proving specificity of IMPA.

Figure 4D shows on-chip P-Tyr for Tyr proto-oncogenic kinase Abl, using an array of IVT proteins, including known Abl targets. To which end, a chip comprising eight Myc/His- double-tagged IVT proteins, including Abl targets (Crkl, Caveolin-1 [Cavl], Rinl, and Annexin Al [Anxal]) and negative controls (Btk, PLC-gamma 2 [Plcg2], Securin, and Frs2) was incubated with recombinant Abl kinase (200 nM, 30 min, 37 °C) and subjected to P-Tyr analysis as above. The histogram shows net P-Tyr levels («=30 dots) with respect to a threshold (dotted line) corresponding to two standard deviations (2 SD) above average Cy5/Cy3 for Btk (negative control). All together, these results suggest IMPA compatibility with the detection and quantification of P-Tyr on various protein targets.

Figures 4E to 4G further illustrate compatibility of IMPA with the analysis of P-Tyr in quasi- cellular environment (i.e. cell extracts). To which end, various Btk targets were deposited on chip and incubated with HEK293 extracts (37 °C, 30 min), per se or supplemented with Tyr phosphatase inhibitor, sodium orthovanadate (SOV) or recombinant Btk, or both. Figure 4E shows on chip P-Tyr of Btk target peptides incubated with either buffer or HEK293 extracts. The histogram shows net P-Tyr signals quantified and normalized as above («=20 dots). Unambiguous P-Tyr signals were observed in reactions supplemented with SOV, and increased signals - with recombinant Btk (lower panel). Figure 4F shows results of an analogous experiment using Plcg2 and Securin and Cy5-coupled P-Tyr-100 and Cy3-coupled anti-Myc antibodies («=20 dots). Figure shows that HEK293 extracts supplemented with SOV and Btk induced P-Tyr of Plcg2, but not Securin, thus pointing to authenticity of IMPA for the detection and quantification of P-Tyr in these conditions.

Figure 4G further illustrates compatibility of IMPA with the detection of on-chip Tyr dephosphorylation. To which end, biotinylated Btk target peptides were deposited on chip in a phosphorylated form, the chip was incubated with buffer (bar) or HEK293 cell extracts supplemented with increasing SOV concentrations (37 °C, 30 min) (plot) and immunolabeled with Cy5-coupled P-Tyr-100 antibodies. Overall, Tyr dephosphorylation was tested on the same chip under eight distinct conditions. The histogram represents average signals from 20 dots normalized to maximum activity. Data show a typical inhibition curve (EC50 ~ 2.5mM), pointing to quantitative attributes of IMPA in these conditions. As demonstrated herein, the method of the invention may be used to analyze both, phosphorylation and dephosphorylation, reflecting its ability to analyze different reversible modifications.

Altogether, these results demonstrate competence and performance of IMPA for analyzing PTM on Tyr and potentially other PTMs in cell extracts and further underscore quantitative aspects of this analysis. EXAMPLE 4

On-chip large scale P-Tyr analysis

Performance and capability of IMPA in a large scale analysis of samples was further investigated. To which end, the inventors used the 64 x 64 unit-cell array capturing quadruplicates of 1024 nuclear proteins shown in Figure 3A, for analyzing P-Tyr modifications induced by HEK293 extracts. This line of experiments is illustrated in Figures 5A-5C.

Figure 5A shows signals in chambers incubated with HEK293 cell extracts (37 °C, 30 min) supplemented with 10 mM SOV and Figure 5B - in chambers non-supplemented with SOV. The right panel (raw data) shows fractions of microarray with signals for two nuclear proteins, Tyr protein kinase Hck P and Rad9 (cell cycle checkpoint protein). Significant and uniform P- Tyr signals obtained in all four Rad9 and Hck chambers emphasize the importance of multiple repeats in minimizing false hits, as well as the overall selectivity and accuracy of IMGA. Selected signals are magnified in a framed area (5A, top panel, white arrowheads). Figure 5C shows top-10 nuclear proteins detected in this experiment, seven of which are known P-Tyr targets (+), suggesting low false -positive rate. Interestingly, two of the top-10 were non-receptor Tyr kinases (Hck and Frk).

The inventors next demonstrated the feasibility of using the platform of the invention to distinguish between PTMs resulting from autophosphorylation and phosphorylation by kinases present in the examined sample (cell extract), as shown in Figure 6.

Figure 6A is a schematic representation of on-chip phosphorylation showing labeled phosphate (dark) and tagged (grey triangle) substrates. The protein array was analyzed with no incubation with HEK293 extracts. P-Tyr signals were evident for Hck as shown in Figure 6B but not for Rad9 as shown in Figure 6C (raw data) pointing to Hck signals being a result of autophosphorylation, thus supporting functionality of arrayed IVT products.

EXAMPLE 5

On-chip Phosphorylation of membrane proteins

Demonstrating the on-chip phosphorylation of nuclear proteins, the inventors next examined the functionality of the membrane protein array presented in Figure 3B. Thus, a proof of concept for PTM analysis on membrane proteins was exemplified using SRC kinase as illustrated in Figure 7. Library of positive and negative controls of the SRC kinase were expressed on chip from DNA using reticulocyte lysate with microsomal membranes. The proteins were then incubated (30°C for 60 min) with cell extract generated from HEK293 over-expressing Y527F SRC (constitutively active SRC mutant) or a K297M SRC (inactive SRC mutant). All experiments were performed with Sodium ortho vanadate (a tyrosine phosphates inhibitor). Phosphorylation was detected using anti-phosphotyrosine antibody labeled with Cy5. The inventors normalized the phosphorylation level to protein. Then in a further normalization step, the phosphorylation level of each protein was normalized under active SRC to phosphorylation level when incubate with inactive SRC. The data was normalized to protein level. Kinase activity was expressed as the difference in phosphor- tyrosine signal between extracts over expressing active or dead SRC kinase. There was a significant difference between active and dead for SRC specific targets, with no obvious difference between membrane or soluble targets. Proteins chosen as negative controls demonstrate little difference between active and dead SRC signals.

These results clearly demonstrate the potential of this unique platform for analyzing PTM of membrane proteins by showing a specific Src-mediated phosphorylation of membrane proteins.

EXAMPLE 6

On-chip ubiquitination assay

After showing capability of IMPA in detecting a simple PTM, as P-Tyr, the inventors pursued testing IMPA in a more complex PTM, such as ubiquitination. Unlike phosphorylation, ubiquitination is a multi -enzymatic process that covalently binds one or more Ubiquitins (Ub) to a target protein via Lysine (K) residues. Sequential binding of Ub via one of its seven K residues produces distinct Ub-chains that differentially regulate proteins' fate, most notably, their degradation by proteasome. Figure 8A is a schematic representation of on-chip ubiquitination using fluorescently labeled Ub (circle) and tagged (grey dotted triangle) substrates. For proof-of-concept, the inventors focused on Anaphase-Promoting Complex/Cyclosome (APC/C), Ub ligase targeting Securin (and other proteins) for proteasome degradation, and used rhodamine (Rd)-labeled-Ub extracts of HeLa S3 cells synchronized to Gl phase of the cell cycle (Gl extracts).

Preliminary off-chip experiments, illustrated in Figures 8B-8C and Figures 9A-9C, demonstrate validity of this approach. To which end, Securin-eGFP (Sec-eGFP) and its stable (non-degradable) mutant (A64Sec-eGFP), and p27 cell cycle protein were expressed and labeled in vitro (IVT) with ³⁵S-Met. IVT products were incubated in Gl extracts with energy- regeneration mix and Ub (0.4 mg/ml) supplemented with either APC/C-activating E2 enzyme UbcHIO (0.3 mg/ml) or the dominant-negative variant UbcHIO (UbcH10^DN) (0.3 mg/ml) or GST-tagged APC/C inhibitor Emil C terminus (0.48 mg/ml). Time-dependent degradation (27 °C) was analyzed by SDS-PAGE and autoradiography. Figure 8B shows that Sec-eGFP was degraded in Gl extracts, but the mutant A64Sec-eGFP and p27, whose turnover is regulated in the S-phase by the Ub ligase SCF^Skp2, remained stable. Importantly, Figure 8C shows that when Gl extracts were supplemented with various Ub or Rd-Ub concentrations, Sec-eGFP turnover was accelerated in a dose dependent manner (data were normalized to maximum signal at time t=0). Supplementary degradation assays illustrated in Figures 9A-9C supported the above results. Figure 9A shows that eGFP IVT product for itself (deposited on chip via anti-GFP antibodies) remained stable after incubation with Gl extracts supplemented with UbcHIO or UbcH10^DN as above. Figure 9B shows that the p27 IVT product was degraded by S-phase arrested HeLa S3 cells extracts but not asynchronous (unsync) HeLa S3 cells. Degradation plots of various assays are illustrated in Figure 9C. Taken together, these experiments support validity of the presently employed experimental approach for the detection of APC/C-mediated ubiquitination.

In the following on-chip analysis, IVT products of Sec-eGFP, A64Sec-eGFP, Myc/His double tagged-p27, and Kifcl-eGFP (a known APC/C target) were deposited on chip via anti-GFP or anti-His antibodies and incubated (10 min, RT) with Gl extracts supplemented with Rd-Ub (0.04 mg/ml), energy-regeneration mix, and either UbcHIO, UbcH10^DN or APC/C inhibitor Emil. Figure 8D shows average ratios of Rd-Ub/protein (n=20) as detected by GFP-tag or Cy2-labeled anti-Myc antibodies, representative raw data of Rd-Ub signals are shown below. Clear Rd signals were observed for Securin, but not for A64Sec-eGFP. Importantly, these signals were reduced in the presence of UbcH10^DN or Emil. The same reduction was observed for the other APC/C target Kifcl. As regards p27, while Rd signals were hardly detected in Gl extracts (left), they were clear with S-phase extracts (right), but remained unaffected by UbcH10^DN or Emil (right). A supplementary assay in Figure 10 shows that eGFP IVT product was undetected and remained unaffected by UbcHIO or UbcH10^DN. Figure 11 shows results for yet another APC/C target, i.e. Geminin-eGFP IVT product, deposited on chip and incubated with Gl extracts. In this case, the same reduction was observed in the presence of UbcH10°^N or Emil as for Sec-Egfp and Kifcl-eGFP. These results strongly support the notion that the observed Rd signals primarily resulted from APC/C-mediated ubiquitination, rather than non- specific ubiquitination or undesired protein- protein interactions with either Rd-Ub or any other ubiquitinated protein in the cell extracts. These experiments also emphasize advantageousness of IMPA in high throughput PTM analysis.

EXAMPLE 7

On-chip analysis of Ub-chain preference

IMPA was further tested for capability to distinguish Ub-chain preference. For proof-of- concept the inventors focused on the two major ubiquitinations responsible for protein degradation, Kl l -linked versus K48-linked ubiquitination. Securin degradation is known to be mediated by Kl l Ub-chains. Two strategies were developed for testing differential detection by IMPA, illustrated in Figures 12A-12IG and Figure 13.

The first involved use of Cy5-labeled Ub molecules, wherein Kl l or K48 was substituted with Arginine (R). Figure 12A shows a schematic on-chip Kl l -specific ubiquitination using K48R-Ub to form Kl l -Ub-chains. Figure 12B shows on-chip ubiquitination of Securin with Gl extracts supplemented with 0.04 mg/ml Cy5-labeled K48R-Ub or Kl lR-Ub. The histogram shows average Cy5-mutant-Ub/protein ratios («=20), representative raw data are on the right. Data clearly indicate that ubiquitination of Securin was more significant in the presence of Cy5-K48R-Ub. This experiment, however, could not exclude differential labeling efficiency of the two Ub mutants, which in principle could distort data analysis and interpretation.

The second strategy involved use of an excess unlabeled mutant K48R-Ub or Kl lR-Ub to displace the endogenous Ub. Figure 12C shows an experiment using Gl extracts supplemented with 0.4 mg/ml Rb-Ub or Rb-Ub with an excess of unlabeled K48R-Ub or Kl lR-Ub. Protein degradation was assayed and quantified as above. Data show that an excess of Kl lR-Ub, but not K48R-Ub, delayed Securin degradation, suggesting replacement of the endogenous Ub wt with the mutant Ub. Figure 12D shows schematically that an excess of unlabeled K48R-Ub should displace the labeled Rd-Ub (wt) from Securin (dotted gray circle). Figure 12E shows on-chip ubiquitination of Sec-eGFP with 0.04 mg/ml Rd-Ub and 0.2 mg/ml unlabeled K48R-Ub or Kl lR-Ub. Representative raw data are shown on the right. These data shows that the employed approach was fully informative, as Rd signals in Securin chambers supplemented with K48R-Ub were less than 25% of signals obtained with Kl lR- Ub. These finding were further supported by similar results obtained for another Kl l-Ub target, Geminin. Figure 13 on-chip ubiqitination of Geminin-eGFP IVT product with Gl extracts (RT) supplemented with 0.04 mg/ml Rd-Ub and 0.2 mg/ml unlabeled K48R-Ub or Kl lR-Ub. The histogram represents the average ratios of Rd-Ub/protein (as detected by the GFP tag) (n=20).

The inventors tested performance of IMPA for detecting Ub-chain preference on the newly discovered APC/C target Kifcl, for which no Ub-chain preference information has been available so far. Figures 12F and 12G show on-chip ubiquitination of Kifcl using both of the above approaches, as in Figures 12A and 12D, representative raw data are shown on the right. By these data, ubiquitination of Kifcl is clearly preferential in favor of Kl lUb-chains over K48-chains. Figure 12G and 121 shows complementary degradation assays using on-chip Gl extracts (27 °C) supplemented with 0.4 mg/ml of Ub wt or mutant derivatives, as K11R, K48R, Kl l-only and K48-only (in the two latter, all K residues except Kl l or K48 are substituted with R). Time-dependent degradation of ³⁵S-Kifcl-eGFP was quantified in three independent experiments (Fig. 121), representative data raw are shown (Fig. 12H). Degradation assays were confirmatory of the above of findings of specificity to Kl l, Kifcl degradation was inhibited by an excess of Kl lR-Ub or K48-only-Ub (single K Ub mutants), but nearly unaffected by K11R- or Kl l -only- Ub. Altogether, these findings demonstrate capability of IMPA in the detection and refined analysis of complex PTMs, such as ubiquitination, in quasi-cellular and cellular environments.

Claims

1. A method for analyzing post translational modifications (PTMs) of proteins in a biological sample, the method comprising:

a. providing a plurality of tagged proteins or any peptides thereof;

b. measuring the signal intensity of a detectable tag for each protein of said plurality of tagged proteins;

c. contacting said sample or any functional extract thereof with each protein of said plurality of tagged proteins under conditions that allow individual PTM reactions;

d. measuring the signal intensity of a detectable label or moiety specific for PTM generated by each individual PTM reaction of step (c); and

e. normalizing for each protein the signal intensity measured in step (d) with the signal intensity measured in step (b), to obtain the PTM value for each protein of said plurality of tagged proteins; thereby analyzing PTMs of proteins in said sample.

2. The method according to claim 1 , wherein each of said plurality of tagged proteins is immobilized to a solid support in a predetermined position.

3. The method according to claim 1, wherein providing a plurality of tagged proteins according to step (a) comprises:

(i) providing a plurality of expression template molecules encoding said tagged proteins or any peptides thereof;

(ii) incubating said plurality of expression template molecules under conditions allowing in vitro translation of the expression template molecules into tagged proteins; and

(iii) immobilizing each of the tagged proteins obtained in step (ii) to a solid support in a predetermined position.

4. The method according to claim 3, wherein said expression template molecules are cDNA molecules, and wherein each of said cDNA molecules or tagged proteins is contained in an individual chamber.

5. The method according to any one of claims 1 to 4, wherein said proteins are at least one of membrane protein/s, nuclear protein/s and cytoplasmic protein/s.

6. The method according to claim 5, wherein said proteins are membrane proteins.

7. The method according to claim 1 , wherein said tagged proteins comprise at least two different tagging molecules.

8. The method according to claim 1, wherein conditions that allow PTM reactions to occur include supplementing said sample or any functional extract thereof with at least one of reagent/s, enzyme/s, substrate/s, co-factor/s and inhibitor/s, prior to or during said PTM reaction.

9. The method according to claim 1, wherein said PTM reactions comprise at least one of phosphorylation, ubiquitination, sumoylation, neddylation, fatylation, and ISGylation .

10. The method according to claim 9, wherein said PTM reaction is ubiquitination.

11. The method according to any one of claims 1, 8 and 10, wherein said sample is supplemented with at least one of ubiquitin ligase/s, mutated ubiquitin ligase/s, ubiquitin ligase inhibitor/s, wild type ubiquitin, mutated ubiquitin, wild type ubiquitin comprising a detectable label or moiety and mutated ubiquitin comprising a detectable label or moiety.

12. The method according to claims 11, wherein said sample is supplemented with ubiquitin that comprises a detectable label or moiety and wherein the intensity of said detectable label is measured in step (d) of claim 1.

13. The method according to claims 11 , wherein said method is for determining Ub-chain preference of a protein.

14. The method according to claim 13, wherein said sample is supplemented with at least one of wild type ubiquitin, mutated ubiquitin, wild type ubiquitin comprising a detectable label or moiety and mutated ubiquitin comprising a detectable label or moiety.

15. The method according to claim 9, wherein said PTM reaction is phosphorylation.

16. The method according to any one of claims 1, 8 and 15, wherein said sample is supplemented with at least one of kinase/s, mutated kinase/s, kinase inhibitor/s, phosphatase inhibitor/s, kinase substrate/s, and ATP.

17. The method according to claim 15, wherein said method is for detecting at least one of phosphorylation, autophosphorylation and dephosphorylation of a protein in a sample.

18. The method according to any one of claims 1 and 3, for screening and identification of protein targets for specific PTM enzyme/s, the method comprising:

a. providing a plurality of candidate tagged target proteins or any peptides thereof;

b. measuring the signal intensity of a detectable tag for each protein of said plurality of candidate target tagged proteins;

c. contacting a sample or any functional extract thereof supplemented with said PTM enzyme/s with each protein of said candidate tagged proteins under conditions that allow PTM reactions;

d. measuring the signal intensity of a detectable label or moiety specific for PTM generated by each individual PTM reaction of step (c);

e. normalizing for each protein the signal intensity measured in step (d) with the signal intensity measured in step (b), to obtain the PTM value for each protein of said plurality of candidate target tagged proteins; and

f. determining if the PTM value for each candidate protein obtained in step (e) is any one of positive or negative with respect to a PTM value of at least one of positive control protein/s and negative control protein/s.

19. The method according to claim 1, for screening and identification of inhibitors of specific PTM enzyme/s.

20. The method according to any one of claims 1 and 3, for determining PTM state of at least one protein in a subject, the method comprising:

a. providing a plurality of tagged proteins or any peptides thereof;

b. measuring the signal intensity of a detectable tag for each protein of said plurality of tagged proteins; c. contacting a biological sample of said subject or any functional extract of said sample with each protein of said plurality of tagged proteins under conditions that allow individual PTM reactions;

e. normalizing for each protein the signal intensity measured in step (d) with the signal intensity measured in step (b), to obtain the PTM value for each protein of said plurality of tagged proteins.

21. The method according to claim 20, for diagnosing a pathologic condition associated with alteration of PTM of at least one protein, the method further comprising: (f) determining if the PTM value for each candidate protein obtained in step (e) is any one of positive or negative with respect to a predetermined standard PTM value of said protein in at least one of a population of subjects suffering from said condition and in a population of subjects that do not suffer from said condition or to a PTM value of said protein in at least one control sample.

22. The method according to any one of claims 1 to 21, wherein said method is implemented using a microfluidic device comprising at least one chip comprising plurality of single assay units, wherein each of said units comprise at least one DNA chamber and at least one protein chamber, said chambers are isolated from one another by micromechanical valve/s.

23. A diagnostic method for detecting a pathological condition associated with altered PTM state of at least one protein in a subject, the method comprising:

a. providing a plurality of tagged proteins or any peptides thereof;

c. contacting a biological sample of said subject or any functional extract of said sample with each protein of said plurality of tagged proteins under conditions that allow individual PTM reactions;

d. measuring the signal intensity of a detectable label or moiety specific for PTM generated by each individual PTM reaction of step (c); e. normalizing for each protein the signal intensity measured in step (d) with the signal intensity measured in step (b), to obtain the PTM value for each protein of said plurality of tagged proteins; and

f. determining if the PTM value for each protein obtained in step (e) is any one of positive or negative with respect to a predetermined standard PTM value of said protein at least one of a population of subjects suffering from said condition and in a population of subjects that do not suffer from said condition or to a PTM value of said protein in at least one control sample.

24. The method according to claim 23, wherein providing a plurality of tagged proteins according to step (a) comprises:

(i) providing a plurality of expression template molecules encoding tagged proteins or any peptides thereof;

25. The method according to any one of claims 23 to 24, wherein said proteins are at least one of membrane protein/s, nuclear protein/s and cytoplasmic protein/s.

26. The method according to claim 23, wherein conditions that allow PTM reactions to occur include supplementing said sample or any functional extract thereof with at least one of reagent/s, enzyme/s, substrate/s, co-factor/s and inhibitor/s, prior to or during said PTM reaction.

27. The method according to claim 23, wherein said PTM reactions comprise at least one of phosphorylation, ubiquitination, sumoylation, neddylation, fatylation and ISGylation.

28. The method according to claim 27, wherein said PTM reaction is ubiquitination.

29. The method according to any one of claims 23, 26 and 28, wherein said sample is supplemented with at least one of ubiquitin ligase/s, mutated ubiquitin ligase/s, ubiquitin ligase inhibitor/s, wild type ubiquitin, mutated ubiquitin, wild type ubiquitin comprising a detectable label or moiety and mutated ubiquitin comprising a detectable label or moiety.

30. The method according to claims 29, wherein said sample is supplemented with ubiquitin that comprises a detectable label or moiety and wherein the intensity of said detectable label is measured in step (d) of claim 24.

31. The method according to claims 29, wherein said method determines Ub-chain preference of a protein in a subject.

32. The method according to claim 31 , wherein said sample is supplemented with at least one of wild type ubiquitin, mutated ubiquitin, wild type ubiquitin comprising a detectable label or moiety and mutated ubiquitin comprising a detectable label or moiety.

33. The method according to claim 27, wherein said PTM reaction is phosphorylation.

34. The method according to any one of claims 23, 26 and 33, wherein said sample is supplemented with at least one of kinase/s, mutated kinase/s, kinase inhibitor/s, phosphatase inhibitor/s, kinase substrate/s, and ATP.

35. The method according to claim 33, wherein said method is for detecting at least one of phosphorylation, autophosphorylation and dephosphorylation of a protein in a sample.

36. The method according to any one of claims 23 to 35, wherein said method is implemented using a microfluidic device comprising at least one chip comprising plurality of single assay units, wherein each of said units comprise at least one DNA chamber and at least one protein chamber, said chambers are isolated from one another by micromechanical valve/s.

37. A kit for use in analyzing PTMs of proteins in a biological sample, the kit comprising:

(a) at least one of a plurality of expression template molecules encoding tagged proteins or any peptides thereof; and a plurality of tagged proteins or any peptides thereof; and

(b) at least one of reagent/s, enzyme/s, substrate/s, co-factor/s and inhibitor/s required for a PTM reaction.

38. The kit according to claim 37, wherein said expression template molecules are cDNA molecules and wherein each of said plurality of tagged proteins and/or plurality of cDNA molecules are immobilized to a solid support in a predetermined position.

39. The kit according to claim 38, wherein each of said cDNA molecules or tagged proteins is contained in an individual chamber.

40. The kit according to claim 37, wherein said tagged proteins comprise at least two different tagging molecules.

41. The kit according to any one of claims 37 to 40, wherein said proteins are at least one of membrane protein/s, nuclear protein/s and cytoplasmic protein/s.

42. The method according to claim 41, wherein said proteins are membrane proteins.

43. The kit according to claim 37, wherein said PTM reactions comprise at least one of phosphorylation, ubiquitination, sumoylation, neddylation, fatylation and ISGylation.

44. The kit according to claim 43, wherein said PTM reaction is ubiquitination.

45. The kit according to claim 44 comprising at least one of ubiquitin ligase/s, mutated ubiquitin ligase/s, ubiquitin ligase inhibitor/s, wild type ubiquitin, mutated ubiquitin, wild type ubiquitin comprising a detectable label or moiety and mutated ubiquitin comprising a detectable label or moiety.

46. The kit according to claim 43, wherein said PTM reaction is phosphorylation.

47. The kit according to claim 46 comprising with at least one of kinase/s, mutated kinase/s, kinase inhibitor/s, phosphatase inhibitor/s, kinase substrate/s, and ATP.

48. The kit according to any one of claims 37 to 47, wherein said kit further comprises a microfluidic device comprising at least one chip comprising plurality of single assay units, wherein each of said units comprise at least one DNA chamber and at least one protein chamber, said chambers are isolated from one another by micromechanical valve/s.

49. The kit according to any one of claims 37 to 48, for use in a method for analyzing PTMs of proteins in a biological sample as define in any one of claims 1 to 22.