Glycan detection methods, compositions, and applications thereof

Abstract

Fusion proteins according to aspects of the present disclosure include: a glycan binding component linked to a detection moiety, the glycan binding component capable of specific binding to a glycosylation post-translational modification of a target protein. Fusion proteins according to aspects of the present disclosure are useful, for example, as detection tools for detection of glycosylation post-translational modification of proteins.

Description

GLYCAN DETECTION METHODS, COMPOSITIONS, AND APPLICATIONSTHEREOFGOVERNMENT FUNDING CLAUSE

[0001] This invention was made with government support under R35GM142637 awarded by the National Institute of General Medicine Sciences. The government has certain rights in the invention.REFERENCE TO RELATED APPLICATIONS

[0002] This application claims priority from U.S. Provisional Patent Application Serial No. 63 / 665,986, fded June 28, 2025, the entire content of which is incorporated herein by reference.SEQUENCE LISTING

[0003] The instant application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. Said XML format Sequence Listing, created on June 27, 2025, is named WAY16952-68476- O199.xml and is 53,747 bytes in size.BACKGROUND OF THE INVENTION

[0004] A fundamental mechanism that all eukaryotic cells use to adapt to their environment is dynamic protein modification with monosaccharide sugars. In humans, O- linked N-acetylglucosamine (O-GlcNAc) is rapidly added to and removed from diverse protein sites as a response to fluctuating nutrient levels, stressors, and signaling cues.

[0005] The O-GlcNAc (O-linked A-acetylglucosamine) modification on proteins is a nutrient- and condition-sensing post-translational modification essential for all mammalian cells to adapt to their microenvironment. Thousands of O-GlcNAc sites regulate cell biology7, including signaling and transcription, in both nutrient-driven and nutrient-independent roles. Protein O-GlcNAcylation is cycled by two proteins, O-GlcNAc transferase (OGT) and O- GlcNAcase (OGA) (Figure 1 A). The OGT gene can produce three isoforms, each of which is most active in a distinct cellular location: nucleocytoplasmic ncOGT is primarily found in thenucleus; mitochondrial mOGT is found in mitochondria; and short sOGT, which lacks a nuclear localization signal and is therefore mainly cytosolic. During insulin signaling, OGT is known to move to the plasma membrane, where it is then active on membrane proteins. Therefore, a crucial facet of O-GlcNAc regulation depends on the spatial location of target proteins in the cell and which isoform(s) of OGT is produced at a given time.

[0006] A second mechanism for O-GlcNAc regulation is time-based because O-GlcNAc modifications can be dynamically removed by OGA. In this vein, mammalian cells regulate the balance of OGT / OGA concerning overall O-GlcNAc levels, employing a variety of mechanisms including regulatory modifications, expression, as well as levels of OGT and OGA pre-mRNA transcripts. In particular, this mRNA regulation via alternative splicing enables cells to respond to O-GlcNAc perturbations within 30 min. During OGT / OGA rebalancing, O-GlcNAc events in this 30 min phase are increasingly recognized as critical for a wide range of cellular functions.

[0007] There is a continuing need for a compositions and methods specific for O- GlcNAc sugar modifications which allow detection of changes in space and time.SUMMARY OF THE INVENTION

[0008] Fusion proteins according to aspects of the present disclosure include: a glycan binding component linked to a detection moiety, the glycan binding component capable of specific binding to a glycosylation post-translational modification of a target protein. Fusion proteins according to aspects of the present disclosure are useful, for example, as detection tools for detection of glycosylation post-translational modification of proteins.

[0009] Fusion proteins according to aspects of the present disclosure include: a glycan binding component linked to a detection moiety, wherein the detection moiety is a reporter protein or peptide, the glycan binding component capable of specific binding to a glycosylation post-translational modification of a target protein.

[0010] Fusion proteins according to aspects of the present disclosure include: a glycan binding component linked to a detection moiety, the glycan binding component capable of specific binding to a glycosylation post-translational modification of a target protein, wherein the detection moiety is detectable by chemiluminescence, fluorescence, colorimetric reactions, antibody binding, inducible markers, and / or ligand binding assays when expressed.

[0011] Fusion proteins according to aspects of the present disclosure include: a glycan binding component linked to a detection moiety which is a reporter protein or peptide, theglycan binding component capable of specific binding to a glycosylation post-translational modification of a target protein, wherein the detection moiety is detectable by chemiluminescence, fluorescence, colorimetric reactions, antibody binding, inducible markers, and / or ligand binding assays when expressed.

[0012] Fusion proteins according to aspects of the present disclosure include: a glycan binding component linked to a detection moiety, wherein the detection moiety is a reporter protein or peptide, the glycan binding component capable of specific binding to a glycosylation post-translational modification of a target protein, wherein the reporter protein or peptide is selected from the group consisting of: green fluorescent protein (GFP); enhanced green fluorescent protein (eGFP); yellow fluorescent protein (YFP); enhanced yellow fluorescent protein (eYFP); hfYFP; mhYFP; LSSA12; LSSmGFP; cyan fluorescent protein (CFP); enhanced cyan fluorescent protein (eCFP); blue fluorescent protein (BFP); enhanced blue fluorescent protein (eBFP); MmGFP; dsRed; luciferase or a variant of any thereof.

[0013] Fusion proteins according to aspects of the present disclosure include: a glycan binding component linked to a detection moiety, wherein the detection moiety is a reporter protein or peptide, the glycan binding component capable of specific binding to a glycosylation post-translational modification of a target protein, wherein the reporter protein or peptide is selected from the group consisting of: Renilla luciferase variant Rluc8_S257G, Renilla luciferase variant Rluc8.6, beta-galactosidase (lacZ), and an epitope tag.

[0014] Fusion proteins according to aspects of the present disclosure include: a glycan binding component linked to a detection moiety, wherein the detection moiety7is a reporter protein or peptide, the glycan binding component capable of specific binding to a glycosylation post-translational modification of a target protein, wherein the reporter protein or peptide is an epitope tag is selected from the group consisting of: a FLAG tag, a human influenza tag, and a Myc tag.

[0015] Fusion proteins according to aspects of the present disclosure include: a glycan binding component linked to a detection moiety, the glycan binding component capable of specific binding to a glycosylation post-translational modification of a target protein, wherein the detection moiety is an antigen for antibody -based detection.

[0016] Fusion proteins according to aspects of the present disclosure include: a glycan binding component linked to a detection moiety, the glycan binding component capable of specific binding to a glycosylation post-translational modification of a target protein, whereinthe detection moiety is capable of emitting fluorescent and / or luminescent light when bound to a cognate detection moiety, the cognate detection moiety bound to an antibody which specifically recognizes the target protein.

[0017] Fusion proteins according to aspects of the present disclosure include: a glycan binding component linked to a detection moiety, the glycan binding component capable of specific binding to a glycosylation post-translational modification of a target protein, wherein the detection moiety / cognate detection moiety pair is split superfolder GFP (green fluorescent protein), N- and C-terminal sections; split Venus YFP (yellow fluorescent protein), N- and C- terminal sections; or split firefly luciferase, N- and C-terminal sections.

[0018] Fusion proteins according to aspects of the present disclosure include: a glycan binding component linked to a detection moiety, the glycan binding component capable of specific binding to a glycosylation post-translational modification of a target protein, wherein the glycan binding component is selected from the group consisting of: an enzyme, a lectin, a collectin, a ficolin, a C-reactive protein, and a carbohydrate-binding domain of any thereof.

[0019] Fusion proteins according to aspects of the present disclosure include: a glycan binding component linked to a detection moiety, the glycan binding component capable of specific binding to a glycosylation post-translational modification of a target protein, wherein the glycan binding component is selected from the group consisting of: an aptamer, an antibody, and an antigen-binding fragment of an antibody.

[0020] Fusion proteins according to aspects of the present disclosure include: a glycan binding component linked to a detection moiety, the glycan binding component capable of specific binding to a glycosylation post-translational modification of a target protein, wherein the glycan binding component is a mutant O-GlcNAcase enzyme derived from a member of the GH84 family of glycosylhydrolases and lacking enzymatic glycosylhydrolase activity.

[0021] Fusion proteins according to aspects of the present disclosure include: a glycan binding component linked to a detection moiety, the glycan binding component capable of specific binding to a glycosylation post-translational modification of a target protein, wherein the mutant O-GlcNAcase enzyme derived from a member of the GH84 family of glycosylhydrolases and lacking enzymatic glycosylhydrolase activity is a mutant human O- GlcNAcase (hOGA) enzyme, a glycan specific binding fragment thereof, or a variant of either thereof.

[0022] Fusion proteins according to aspects of the present disclosure include: a glycan binding component linked to a detection moiety, the glycan binding component capable ofspecific binding to a glycosylation post-translational modification of a target protein, wherein the mutant human O-GlcNAcase (hOGA) enzyme is a D174N mutant human O-GlcNAcase (hOGA) enzyme which includes the amino acid sequence:MVQKESQATLEERESELSSNPAASAGASLEPPAAPAPGEDNPAGAGGAAVAGAAGG ARRFLCGVVEGFYGRPWVMEQRKELFRRLQKWELNTYLYAPKDDYKHRMFWREM YSVEEAEQLMTLISAAREYEIEFIYAISPGLDITFSNPKEVSTLKRKLDQVSQFGCRSFA LLFNDIDHNMCAADKEVFSSFAHAQVSITNEIYQYLGEPETFLFCPTEYCGTFCYPNV SQSPYLRTVGEKLLPGIEVLWTGPKVVSKEIPVESIEEVSKIIKRAPVIWDNIHANDYD QKRLFLGPYKGRSTELIPRLKGVLTNPNCEFEANYVAIHTLATWYKSNMNGVRKDV VMTDSEDSTVSIQIKLENEGSDEDIETDVLYSPQMALKLALTEWLQEFGVPHQYSSR QVAHSGAKASVVDGTPLVAAPSLNATTVVTTVYQEPIMSQGAALSGEPTTLTKEEEK KQPDEEPMDMVVEKQEETDHKNDNQILSEIVEAKMAEELKPMDTDKESIAESKSPE MSMQEDCISDIAPMQTDEQTNKEQFVPGPNEKPLYTAEPVTLEDLQLLADLFYLPYE HGPKGAQMLREFQWLRANSSVVSVNCKGKDSEKIEEWRSRAAKFEEMCGLVMGM FTRLSNCANRTILYDMYSYVWDIKSIMSMVKSFVQWLGCRSHSSAQFLIGDQEPWAF RGGLAGEFQRLLPIDGANDLFFQPP (SEQ ID NO:4), or a variant of SEQ ID NO:4.

[0023] Fusion proteins according to aspects of the present disclosure include: a glycan binding component linked to a detection moiety, the glycan binding component capable of specific binding to a glycosylation post-translational modification of a target protein, wherein the glycan binding component is GafD lectin.

[0024] Fusion proteins according to aspects of the present disclosure include: a glycan binding component linked to a detection moiety7, the glycan binding component capable of specific binding to a glycosylation post-translational modification of a target protein, wherein the glycan binding component has a C-terminus and an N-terminus, the detection moiety is a protein having a C-terminus and an N-terminus, and the C-terminus of the glycan binding component is linked to the N-terminus of the detection moiety or the N-terminus of the glycan binding component is linked to the C-terminus of the detection moiety.

[0025] Fusion proteins according to aspects of the present disclosure include: a glycan binding component linked to a detection moiety, the glycan binding component capable of specific binding to a glycosylation post-translational modification of a target protein, wherein the glycan binding component is linked to the detection moiety by a linker disposed between the glycan binding component and the detection moiety.

[0026] Fusion proteins according to aspects of the present disclosure include: a glycan binding component linked to a detection moiety, the glycan binding component capable of specific binding to a glycosylation post-translational modification of a target protein, the fusion protein further including a localization signal peptide.

[0027] Fusion proteins according to aspects of the present disclosure include: a glycan binding component linked to a detection moiety, the glycan binding component capable of specific binding to a glycosylation post-translational modification of a target protein, the fusion protein further including a localization signal peptide capable of promoting localization of the fusion protein to a subcellular compartment selected from the group consisting of: nucleus, cytosol, mitochondria, endoplasmic reticulum, and plasma membrane.

[0028] Fusion proteins according to aspects of the present disclosure include: a glycan binding component linked to a detection moiety, the glycan binding component capable of specific binding to a glycosylation post-translational modification of a target protein, the fusion protein further including an exogenous detectable tag.

[0029] Methods of detecting glycosylated protein according to aspects of the present disclosure include: contacting a living cell with the fusion protein of the present disclosure under compatible biological conditions, whereby the fusion protein specifically binds to a glycosylation post-translational modification of a protein of the cell. According to aspects of the present disclosure, a stimulus is provided to the living cell, whereby the detection moiety emits fluorescent light and / or luminescent light in response to the stimulus; and a signal is detected from the detection moiety, thereby detecting a glycosylated protein bound to the fusion protein. According to aspects of the present disclosure contacting the living cell with the fusion protein comprises introducing an expression construct encoding the fusion protein into the cell, such that the fusion protein is expressed.

[0030] Expression constructs according to aspects of the present disclosure include a nucleic acid encoding fusion proteins according to aspects of the present disclosure which include: a glycan binding component linked to a detection moiety, the glycan binding component capable of specific binding to a glycosylation post-translational modification of a target protein. Cell of the present disclosure include expression constructs encoding fusion proteins according to aspects of the present disclosure which include: a glycan binding component linked to a detection moiety, the glycan binding component capable of specific binding to a glycosylation post-translational modification of a target protein.BRIEF DESCRIPTION OF THE DRAWINGS

[0031] Figure 1A diagrammatically illustrates that O-GlcNAc protein modifications change rapidly in cells via OGT and OGA activity in response to nutrients and signaling:

[0032] Figure I B diagrammatically illustrates GlycoLight tools according to aspects of the present disclosure for measuring global and protein-specific O-GlcNAc changes in cell lysates in plate-based assay format; square = O-GlcNAc abbreviation;

[0033] Figures 2A and 2B diagrammatically illustrate a method according to aspects of the present disclosure for expression of His-Gly coLight Protein in E. coli;

[0034] Figure 3 is an image of a Western blot showing results of preliminary expression condition screening for His6-hOGA*-Flag (“GlycoID2 ’) protein in E. coli; the desired size of 85 kDa was the major band detected using an anti-Flag antibody, note that ‘hOGA*"’ refers to D174N mutant human O-GlcNAcase of SEQ ID NO:4;

[0035] Figure 4 is an image of an immunoblot showing results of purification of His- hOGA*-Flag using cobalt affinity7chromatography; imaged using anti-Flag AF-488 antibody for green fluorescence detection;

[0036] Figure 5 diagrammatically illustrates a method according to aspects of the present disclosure including a Far Western blot experiment with purified His-hOGA*-Flag (His6- GlycoID-Flag) GlycoLight variant and detection with anti-Flag antibody conjugated to AlexaFluor488 (AF-488) fluorescent dye;

[0037] Figure 6 shows results of a Far western blot with His6-hOGA*-Flag protein (10 ug / mL) on U2OS cell lysates loaded with the indicated amount of total protein (10 ug or 25 ug). Left: anti-Flag-HRP secondary antibody was imaged by enhanced chemiluminescence detection. Right: anti-Flag-AF-488 secondary7antibody was imaged using green fluorescence after washing; also shown are diagrammatic representations of complexes formed;

[0038] Figure 7 is a set of four images of Far Western Blots with His-hOGA*-Flag (‘’GlycoID”); conditions used followed the above description, with amount of GlycoLight substrates changing over the experiment as indicated; and

[0039] Figure 8 is an image of protein blots in which the proteins were purified from lysed mouse C2C12 cells treated with or without OGT inhibitor 5SGlcNHex for 48 hours; protein extracts were separated on SDS-PAGE membranes, then probed by O-GlcNAc antibody as a positive control (left blot) or with GlycoLight fusion protein mNEON-GafD (right blot) to validating the activity7of GlycoLight fusion proteins of the present disclosure for detection of O-GlcNAc proteins.DETAILED DESCRIPTION

[0040] Scientific and technical terms used herein are intended to have the meanings commonly understood by those of ordinary skill in the art. Such terms are found defined and used in context in various standard references illustratively including J. Sambrook and D.W Russell, Molecular Cloning: A Laboratory7Manual, Cold Spring Harbor Laboratory' Press; 3rd Ed., 2001; F.M. Ausubel, Ed., Short Protocols in Molecular Biology, Current Protocols; 5th Ed., 2002; B. Alberts et al., Molecular Biology of the Cell, 4th Ed., Garland, 2002; CRISPR / Cas: A Laboratory Manual, Doudna and Mali (eds), Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2016; D.L. Nelson and M.M. Cox, Lehninger Principles of Biochemistry', 4th Ed., W.H. Freeman & Company, 2004; J.-H. Fuhrhop et al. (Eds.), Organic Synthesis, Concepts and Methods, 3rdEd., Wiley-VCH Verlag GmbH & Co. KGaA, 2003; Herdewijn, P. (Ed.), Oligonucleotide Synthesis: Methods and Applications, Methods in Molecular Biology, Humana Press, 2004; D. J. Taxman (ed.), siRNA Design, Methods and Protocols, Humana Press, 2012; Harlow, E. and Lane, D., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 1988; J. D. Pound (Ed.) Immunochemical Protocols, Methods in Molecular Biology, Humana Press, 2nd ed., 1998; Chu, E. and Devita, V.T., Eds., Physicians’ Cancer Chemotherapy Drug Manual, Jones & Bartlett Publishers, 2021; J.M. Kirkwood et al., Eds., Current Cancer Therapeutics, 4th Ed., Current Medicine Group, 2001; A Adejare (Ed.), Remington: The Science and Practice of Pharmacy, Elsevier, 23rd Ed., 2021; L.V. Allen, Jr. et al., Ansel's Pharmaceutical Dosage Forms and Drug Delivery Systems, 1 1th Ed., Wolters Kluwer, 2016; and L. Brunton et al., Goodman & Gilman’s The Pharmacological Basis of Therapeutics, McGraw-Hill Education, 13th Ed., 2018.

[0041] The singular terms "a," "an," and "the" are not intended to be limiting and include plural referents unless explicitly stated otherwise or the context clearly indicates otherwise.

[0042] The terms “includes,” “comprises,” “including,” “comprising,” “has,” “having,” and grammatical variations thereof, when used in this specification, are not intended to be limiting, and specify the presence of stated features, elements, and / or components, but do not preclude the presence or addition of one or more other features, elements, components, and / or groups thereof.

[0043] The term “about” as used herein in reference to a number is used herein to include numbers which are greater, or less than, a stated or implied value by 1%, 5%, 10%, or 20%.

[0044] Particular combinations of features are recited in the claims and / or disclosed in the specification, and these combinations of features are not intended to limit the disclosure of various aspects. Combinations of such features not specifically recited in the claims and / or disclosed in the specification. Although each dependent claim listed below may directly depend on only one claim, the disclosure of various aspects includes each dependent claim in combination with every other claim in the claim set. As used herein, a phrase referring to "at least one of' a list of items refers to any combination of those items, including single members. As an example, "at least one of: a, b, or c" is intended to cover a alone; b alone; c alone, a and b, a, b, and c, b and c, a and c, as well as any combination with multiples of the same element, such as a and a; a, a, and a; a, a, and b; a, a, and c; a, b, and b; a, c, and c; and any other combination or ordering of a, b, and c).

[0045] The terms “first,” “second,” and the like are used herein to describe various features or elements, but these features or elements are not intended to be limited by these terms, but are only used to distinguish one feature or element from another feature or element. Thus, a first feature or element could be termed a second feature or element, and vice versa, without departing from the teachings of the present disclosure.

[0046] Fusion proteins are provided according to aspects of the present disclosure which include: a glycan binding component linked to a detection moiety, the glycan binding component capable of specific binding to a glycosylation post-translational modification of a target protein and the detection moiety capable of emitting fluorescent and / or luminescent light. Such fusion proteins are one aspect of assays and reagents called “Gly co-Light” herein.

[0047] Fusion proteins are provided according to aspects of the present disclosure which include: a glycan binding component linked to a detection moiety, the glycan binding component capable of specific binding to a glycosylation post-translational modification of a target protein and the detection moiety is an antigen for antibody-based detection. Such fusion proteins are one aspect of assays and reagents called “Gly co-Light” herein.

[0048] Fusion proteins are provided according to aspects of the present disclosure which include: a glycan binding component linked to a detection moiety, the glycan binding component capable of specific binding to a glycosylation post-translational modification of a target protein and the detection moiety capable of emitting fluorescent and / or luminescent light when bound to a cognate detection moiety, the cognate detection moiety bound to an antibody which specifically recognizes the target protein. Such fusion proteins are one aspectof assays and reagents called "Glyco-Light" herein in combination with the cognate detection moiety.

[0049] Figure IB shows an overview of compositions and methods according to aspects of the present disclosure.

[0050] The term “glycan binding component" as used herein refers to a binding agent characterized by specific binding to a specified glycan target. The phrase "specific binding" and grammatical equivalents as used herein in reference to binding of a binding agent to a specified glycan target refers to binding of the binding agent to the specified glycan target without substantial binding to other substances present in a cell which include a fusion protein according to aspects of the present disclosure. The term "binding" refers to a physical or chemical interaction between a binding agent and its target. Binding includes, but is not limited to, ionic bonding, non-ionic bonding, covalent bonding, hydrogen bonding, hydrophobic interaction, hydrophilic interaction, and Van der Waals interaction.

[0051] Specific binding refers to a binding agent that binds to a specified glycan target with greater affinity, greater avidity, and / or greater duration, than to other substances. According to aspects of the present disclosure, a binding agent specifically binds to its glycan target when it has an equilibrium dissociation constant, KD, for its target in the range of about IO"4to about IO’12, i.e. a KD of about 10'4, about 10’5, about 10’6, about IO"7, about IO'8, about 10’9, about 10'10, about 10'11, or about 10‘12. Binding affinity of a binding agent can be determined by Scatchard analysis such as described in P.J. Munson and D. Rodbard, Anal. Biochem., 107:220-239, 1980 or by other methods such as Biomolecular Interaction Analysis using plasmon resonance.

[0052] Binding agents specific for a specified glycan target may be obtained from commercial sources or generated for use in methods of the present disclosure according to well-known methodologies.

[0053] According to aspects of the present disclosure, the glycan binding component is a binding agent which is, or includes, an enzyme, a lectin, a collectin, a ficolin, a C-reactive protein, or a carbohydrate-binding domain of any thereof. According to aspects of the present disclosure, the glycan binding component is a binding agent which is or includes, an aptamer, antibody, or an antigen-binding fragment of an antibody.

[0054] The term "antibody1" is used herein in its broadest sense and includes antibodies, and antigen-binding fragments, characterized by specific binding to an antigen. An antibody included in methods according to aspects of the present disclosure may be a polyclonalantibody, a monoclonal antibody, a chimeric antibody, a humanized antibody, and / or an antigen-binding antibody fragment of any thereof. An antibody included in methods in particular aspects of the present disclosure includes a standard intact immunoglobulin having four polypeptide chains including two heavy chains (H) and two light chains (L) linked by disulfide bonds. An antibody included in methods in particular aspects of the present disclosure includes an antigen-binding antibody fragments illustratively include an Fab fragment, an Fab' fragment, an F(ab')2 fragment, an Fd fragment, an Fv fragment, an scFv fragment and a domain antibody (dAb), for example. In addition, the term antibody refers to antibodies of various classes including IgG, IgM, IgA, IgD and IgE, as well as subclasses, illustratively including for example human subclasses IgGl, IgG2, IgG3 and IgG4 and marine subclasses IgGl, IgG2, IgG2a. IgG2b, IgG3 and IgGM, for example.

[0055] In particular embodiments, an antibody which is characterized by specific binding to its target has a dissociation constant in the range of about 10'4to about 10'12, i.e. a K-D of about 10'4, about 10'5, about 10'6, about 10'7, about 10'8, about 10'9, about IO'10, about 10’11, or about 10‘12. Binding affinity of an antibody can be determined by Scatchard analysis such as described in P.J. Munson and D. Rodbard, Anal. Biochem, 107:220-239, 1980 or by other methods such as Biomolecular Interaction Analysis using plasmon resonance. Antibodies may be tested for specific binding to the target by methods illustratively including ELISA, Western blot, and immunocytochemistry.

[0056] Antibodies, antigen-binding fragments, and methods for their generation are known in the art, for instance, as described in Antibody Engineering, Kontemann, R. and Dubel, S. (Eds.), Springer, 2001; Harlow7, E. and Lane, D., Antibodies: A Laboratory’ Manual, Cold Spring Harbor Laboratory Press, 1988; Ausubel. F. et al., (Eds.), Short Protocols in Molecular Biology, Wiley, 2002; J. D. Pound (Ed.) Immunochemical Protocols, Methods in Molecular Biology7, Humana Press, 2nd ed., 1998; B. K. C. Lo (Ed ), Antibody Engineering: Methods and Protocols, Methods in Molecular Biology7, Humana Press, 2003; and Kohler, G. and Milstein, C., Nature, 256:495-497 (1975).

[0057] A binding agent according to aspects of the present disclosure may be an aptamer. The term "aptamer" refers to a nucleic acid or peptide that substantially specifically binds to a specified substance. In the case of a nucleic acid aptamer, the aptamer is characterized by binding interaction with a target other than Watson / Crick base pairing or triple helix binding with a second and / or third nucleic acid. Such binding interaction may include Van der Waals interaction, hydrophobic interaction, hydrogen bonding and / orelectrostatic interactions, for example. Techniques for identification and generation of aptamers is known in the art as described, for example, in F. M, Ausubel et al., Eds., Short Protocols in Molecular Biology, Current Protocols, Wiley, 2002; S. Klussman, Ed., The Aptamer Handbook: Functional Oligonucleotides and Their Applications, Wiley, 2006; and J. Sambrook and D. W. Russell, Molecular Cloning: A Laboratory' Manual, Cold Spring Harbor Laboratory Press, 3rd Ed., 2001.

[0058] According to aspects of the present disclosure, the glycan binding component capable of specific binding to a glycosylation post-translational modification of a target protein is a lectin.

[0059] According to aspects of the present disclosure, the glycan binding component capable of specific binding to a glycosylation post-translational modification of a target protein is a glycan binding enzyme, or a glycan specific binding fragment thereof.

[0060] According to aspects of the present disclosure, the glycan binding component is referred to as an O-GlcNAc binding protein.

[0061] According to aspects of the present disclosure, the glycan binding component capable of specific binding to a glycosylation post-translational modification of a target protein is a GafD lectin, or a glycan specific binding fragment thereof. GafD is E. coli N- acetyl-D-glucosamine-specific fimbrial lectin (adhesin) protein GafD is a protein which specifically binds to N-acetyl-D-glucosamine.

[0062] According to aspects of the present disclosure, an included GafD lectin includes the following sequence:

[0063] MTNFYKVCLAVFILVCCNISHAAVSFIGSTENDVGPSQGSYSSTHAMDNL PFVYNTGYNIGYQNANVWRISGGFCVGLDGKVDLPVVGSLDGQSIYGLTEEVGLLI WMGDTNYSRGTAMSGNSWENVFSGWCVGNYVSTQGLSVHVRPVILKRNSSAQYS VQKTSIGSIRMRPYNGSSAGSVQTTVNFSLNPFTLNDTVTSCRLLTPSAVNVSLAAISA GQLPSSGDEVVAGTTSLKLQCDAGVTVWATLTDATTPSNRSDILTLTGASTATGVGL RIYKNTDSTPLKFGPDSPVKGNENQWQLSTGTETSPSVRLYVKYVNTGEGINPGTVN GISTFTFSYQ (SEQ ID NO: 1) or a variant thereof.

[0064] GlcNAc-binding lectin GafD is described in detail in Saarela S et al., Infect. Immun 1996, 64, 2857-2860, PubMed: 8698525

[0065] GafD is selective for GlcNAc-linked molecules over other sugars, including > 10-fold binding selectivity over glucose-linked molecules, > 100-fold selectivity overGalNAc, and no detectable binding against mannose, fucose, galactose, or sialic acid sugars as detailed in Hsu K-L et al.. Mol. BioSyst 2008, 4, 654-662, PubMed: 18493664

[0066] A glycan specific binding fragment of GafD lectin is included according to aspects of the present disclosure.

[0067] According to aspects of the present disclosure, an included glycan specific binding fragment of GafD lectin includes the following sequence:

[0068] MAVSFIGSTENDVGPSQGSYSSTHAMDNLPFVYNTGYNIGYQNANVWRI SGGFCVGLDGKVDLPVVGSLDGQSIYGLTEEVGLLIWMGDTNYSRGTAMSGNSWE NVFSGWCVGNYVSTQGLSVHVRPVILKRNSSAQYSVQKTSIGSIRMRPYNGSS (SEQ ID NO:2) or a variant thereof.

[0069] According to aspects of the present disclosure, the glycan binding component capable of specific binding to a glycosylation post-translational modification of a target protein is a O-linked N-acetylglucos amine (O-GlcNAc) binding enzyme from the GH84 family of glycosylhydrolases, a glycan specific binding fragment thereof, or a variant of either thereof.

[0070] According to aspects of the present disclosure, the glycan binding component capable of specific binding to a glycosylation post-translational modification of a target protein is a O-GlcNAcase enzyme from the GH84 family of glycosylhydrolases, a glycan specific binding fragment thereof, or a variant of either thereof.

[0071] According to aspects of the present disclosure, the glycan binding component capable of specific binding to a glycosylation post-translational modification of a target protein is a human O-GlcNAcase (hOGA) enzyme, a glycan specific binding fragment thereof, or a variant of either thereof.

[0072] According to aspects of the present disclosure, the glycan binding component capable of specific binding to a glycosylation post-translational modification of a target protein is a human O-GlcNAcase (hOGA) enzyme which includes the amino acid sequence: MVQKESQATLEERESELSSNPAASAGASLEPPAAPAPGEDNPAGAGGAAVAGAAGG ARRFLCGVVEGFYGRPWVMEQRKELFRRLQKWELNTYLYAPKDDYKHRMFWREM YSVEEAEQLMTLISAAREYEIEFIYAISPGLDITFSNPKEVSTLKRKLDQVSQFGCRSFA LLFDDIDHNMCAADKEVFSSFAHAQVSITNEIYQYLGEPETFLFCPTEYCGTFCYPNV SQSPYLRTVGEKLLPGIEVLWTGPKVVSKEIPVESIEEVSKIIKRAPVIWDNIHANDYD QKRLFLGPYKGRSTELIPRLKGVLTNPNCEFEANYVAIHTLATWYKSNMNGVRKDV VMTDSEDSTVSIQIKLENEGSDEDIETDVLYSPQMALKLALTEWLQEFGVPHQYSSRQVAHSGAKASVVDGTPLVAAPSLNATTVVTTVYQEPIMSQGAALSGEPTTLTKEEEK KQPDEEPMDMVVEKQEETDHKNDNQ1LSE1VEAKMAEELKPMDTDKES1AESKSPE MSMQEDCISDIAPMQTDEQTNKEQFVPGPNEKPLYTAEPVTLEDLQLLADLFYLPYE HGPKGAQMLREFQWLRANSSVVSVNCKGKDSEKIEEWRSRAAKFEEMCGLVMGM FTRLSNCANRTILYDMYSYVWDIKSIMSMVKSFVQWLGCRSHSSAQFLIGDQEPWAF RGGLAGEFQRLLPIDGANDLFFQPPPLTPTSKVYTIRPYFPKDEASVYKICREMYDDG VGLPFQSQPDLIGDKLVGGLLSLSLDYCFVLEDEDGICGYALGTVDVTPFIKKCKISW IPFMQEKYTKPNGDKELSEAEKIMLSFHEEQEVLPETFLANFPSLIKMDIHKKVTDPS VAKSMMACLLSSLKANGSRGAFCEVRPDDKRILEFYSKLGCFEIAKMEGFPKDVVIL GRSL (SEQ ID NO: 39 full length) or a variant thereof.

[0073] According to aspects of the present disclosure, the glycan binding component capable of specific binding to O-GlcNAc is a mutant O-GlcNAcase enzyme derived from a member of the GH84 family of glycosylhydrolases and lacking enzy matic glycosylhydrolase activity.

[0074] According to aspects of the present disclosure, the mutant O-GlcNAcase enzyme derived from a member of the GH84 family of glycosylhydrolases and lacking enzymatic glycosylhydrolase activity is a mutant human O-GlcNAcase (hOGA) enzyme which includes the amino acid sequence SEQ ID NO: 39 where any one, two, or all of D174, D175, D285 is substituted with any amino acid where at least one of D174, D175, and D285 is not D.

[0075] According to aspects of the present disclosure, the mutant O-GlcNAcase enzyme derived from a member of the GH84 family of glycosylhydrolases and lacking enzymatic glycosylhydrolase activity7is a D174N mutant human O-GlcNAcase (hOGA) enzyme which includes the amino acid sequence:MVQKESQATLEERESELSSNPAASAGASLEPPAAPAPGEDNPAGAGGAAVAGAAGG ARRFLCGVVEGFYGRPWVMEQRKELFRRLQKWELNTYLYAPKDDYKHRMFWREM YSVEEAEQLMTLISAAREYEIEFIYAISPGLDITFSNPKEVSTLKRKLDQVSQFGCRSFA LLFNDIDHNMCAADKEVFSSFAHAQVSITNEIYQYLGEPETFLFCPTEYCGTFCYPNV SQSPYLRTVGEKLLPGIEVLWTGPKVVSKEIPVESIEEVSKIIKRAPVIWDNIHANDYD QKRLFLGPYKGRSTELIPRLKGVLTNPNCEFEANYVAIHTLATWYKSNMNGVRKDV VMTDSEDSTVSIQIKLENEGSDEDIETDVLYSPQMALKLALTEWLQEFGVPHQYSSR QVAHSGAKASVVDGTPLVAAPSLNATTVVTTVYQEPIMSQGAALSGEPTTLTKEEEK KQPDEEPMDMVVEKQEETDHKNDNQILSEIVEAKMAEELKPMDTDKESIAESKSPE MSMQEDCISDIAPMQTDEQTNKEQFVPGPNEKPLYTAEPVTLEDLQLLADLFYLPYEHGPKGAQMLREFQWLRANSSVVSVNCKGKDSEKIEEWRSRAAKFEEMCGLVMGM FTRLSNCANRT1LYDMYSYVWD1KS1MSMVKSFVQWLGCRSHSSAQFLIGDQEPWAF RGGLAGEFQRLLPIDGANDLFFQPPPLTPTSKVYTIRPYFPKDEASVYKICREMYDDG VGLPFQSQPDLIGDKLVGGLLSLSLDYCFVLEDEDGICGYALGTVDVTPFIKKCKISW IPFMQEKYTKPNGDKELSEAEKIMLSFHEEQEVLPETFLANFPSLIKMDIHKKVTDPS VAKSMMACLLSSLKANGSRGAFCEVRPDDKRILEFYSKLGCFEIAKMEGFPKDVVIL GRSL (SEQ ID NO: 40 full length D174N) or a variant thereof.

[0076] According to aspects of the present disclosure, the mutant O-GlcNAcase enzy me derived from a member of the GH84 family of glycosylhydrolases and lacking enzymatic glycosylhydrolase activity is a D175N mutant human O-GlcNAcase (hOGA) enzyme which includes the amino acid sequence:MVQKESQATLEERESELSSNPAASAGASLEPPAAPAPGEDNPAGAGGAAVAGAAGG ARRFLCGVVEGFYGRPWVMEQRKELFRRLQKWELNTYLYAPKDDYKHRMFWREM YSVEEAEQLMTLISAAREYEIEFIYAISPGLDITFSNPKEVSTLKRKLDQVSQFGCRSFA LLFDNIDHNMCAADKEVFSSFAHAQVSITNEIYQYLGEPETFLFCPTEYCGTFCYPNV SQSPYLRTVGEKLLPGIEVLWTGPKVVSKEIPVESIEEVSKIIKRAPVIWDNIHANDYD QKRLFLGPYKGRSTELIPRLKGVLTNPNCEFEANYVAIHTLATWYKSNMNGVRKDV VMTDSEDSTVSIQIKLENEGSDEDIETDVLYSPQMALKLALTEWLQEFGVPHQYSSR QVAHSGAKASVVDGTPLVAAPSLNATTVVTTVYQEPIMSQGAALSGEPTTLTKEEEK KQPDEEPMDMVVEKQEETDHKNDNQILSEIVEAKMAEELKPMDTDKESIAESKSPE MSMQEDCISDIAPMQTDEQTNKEQFVPGPNEKPLYTAEPVTLEDLQLLADLFYLPYE HGPKGAQMLREFQWLRANSSVVSVNCKGKDSEKIEEWRSRAAKFEEMCGLVMGM FTRLSNCANRTILYDMYSYVWDIKSIMSMVKSFVQWLGCRSHSSAQFLIGDQEPWAF RGGLAGEFQRLLPIDGANDLFFQPPPLTPTSKVYTIRPYFPKDEASVYKICREMYDDG VGLPFQSQPDLIGDKLVGGLLSLSLDYCFVLEDEDGICGYALGTVDVTPFIKKCKISW IPFMQEKYTKPNGDKELSEAEKIMLSFHEEQEVLPETFLANFPSLIKMDIHKKVTDPS VAKSMMACLLSSLKANGSRGAFCEVRPDDKRILEFYSKLGCFEIAKMEGFPKDVVIL GRSL (SEQ ID NO: 41 full length D175N) or a variant thereof.

[0077] According to aspects of the present disclosure, the mutant O-GlcNAcase enzyme derived from a member of the GH84 family of glycosylhydrolases and lacking enzymatic glycosylhydrolase activity is a D285N mutant human O-GlcNAcase (hOGA) enzy me which includes the amino acid sequence:MVQKESQATLEERESELSSNPAASAGASLEPPAAPAPGEDNPAGAGGAAVAGAAGG ARRFLCGVVEGFYGRPWVMEQRKELFRRLQKWELNTYLYAPKDDYKHRMFWREM YSVEEAEQLMTLISAAREYEIEFIYAISPGLDITFSNPKEVSTLKRKLDQVSQFGCRSFA LLFDDIDHNMCAADKEVFSSFAHAQVSITNEIYQYLGEPETFLFCPTEYCGTFCYPNV SQSPYLRTVGEKLLPGIEVLWTGPKVVSKEIPVESIEEVSKIIKRAPVIWDNIHANNYD QKRLFLGPYKGRSTELIPRLKGVLTNPNCEFEANYVAIHTLATWYKSNMNGVRKDV VMTDSEDSTVSIQIKLENEGSDEDIETDVLYSPQMALKLALTEWLQEFGVPHQYSSR QVAHSGAKASVVDGTPLVAAPSLNATTVVTTVYQEPIMSQGAALSGEPTTLTKEEEK KQPDEEPMDMVVEKQEETDHKNDNQILSEIVEAKMAEELKPMDTDKESIAESKSPE MSMQEDCISDIAPMQTDEQTNKEQFVPGPNEKPLYTAEPVTLEDLQLLADLFYLPYE HGPKGAQMLREFQWLRANSSVVSVNCKGKDSEKIEEWRSRAAKFEEMCGLVMGM FTRLSNCANRTILYDMYSYVWDIKSIMSMVKSFVQWLGCRSHSSAQFLIGDQEPWAF RGGLAGEFQRLLPIDGANDLFFQPPPLTPTSKVYTIRPYFPKDEASVYKICREMYDDG VGLPFQSQPDLIGDKLVGGLLSLSLDYCFVLEDEDGICGYALGTVDVTPFIKKCKISW IPFMQEKYTKPNGDKELSEAEKIMLSFHEEQEVLPETFLANFPSLIKMDIHKKVTDPS VAKSMMACLLSSLKANGSRGAFCEVRPDDKRILEFYSKLGCFEIAKMEGFPKDVVIL GRSL (SEQ ID NO: 42 full length D285N) or a variant thereof.

[0078] According to aspects of the present disclosure, the glycan binding component capable of specific binding to a glycosylation post-translational modification of a target protein is a human O-GlcNAcase (hOGA) enzyme which includes the amino acid sequence: MVQKESQATLEERESELSSNPAASAGASLEPPAAPAPGEDNPAGAGGAAVAGAAGG ARRFLCGVVEGFYGRPWVMEQRKELFRRLQKWELNTYLYAPKDDYKHRMFWREM YSVEEAEQLMTLISAAREYEIEFIYAISPGLDITFSNPKEVSTLKRKLDQVSQFGCRSFA LLFDDIDHNMCAADKEVFSSFAHAQVSITNEIYQYLGEPETFLFCPTEYCGTFCYPNV SQSPYLRTVGEKLLPGIEVLWTGPKVVSKEIPVESIEEVSKIIKRAPVIWDNIHANDYD QKRLFLGPYKGRSTELIPRLKGVLTNPNCEFEANYVAIHTLATWYKSNMNGVRKDV VMTDSEDSTVSIQIKLENEGSDEDIETDVLYSPQMALKLALTEWLQEFGVPHQYSSR QVAHSGAKASVVDGTPLVAAPSLNATTVVTTVYQEPIMSQGAALSGEPTTLTKEEEK KQPDEEPMDMVVEKQEETDHKNDNQILSEIVEAKMAEELKPMDTDKESIAESKSPE MSMQEDCISDIAPMQTDEQTNKEQFVPGPNEKPLYTAEPVTLEDLQLLADLFYLPYE HGPKGAQMLREFQWLRANSSVVSVNCKGKDSEKIEEWRSRAAKFEEMCGLVMGM FTRLSNCANRTILYDMYSYVWDIKSIMSMVKSFVQWLGCRSHSSAQFLIGDQEPWAF RGGLAGEFQRLLPIDGANDLFFQPP (SEQ ID NO: 3) or a variant thereof.

[0079] According to aspects of the present disclosure, the glycan binding component capable of specific binding to O-GlcNAc is a mutant O-GlcNAcase enzyme derived from a member of the GH84 family of glycosylhydrolases and lacking enzymatic glycosylhydrolase activity.

[0080] According to aspects of the present disclosure, the mutant O-GlcNAcase enzyme derived from a member of the GH84 family of glycosylhydrolases and lacking enzymatic glycosylhydrolase activity is a mutant human O-GlcNAcase (hOGA) enzyme which includes the amino acid sequence SEQ ID NO: 3 where any one, two, or all of D174, D175, D285 is substituted with any amino acid where at least one of D174, D175, and D285 is not D.

[0081] According to aspects of the present disclosure, the mutant O-GlcNAcase enzyme derived from a member of the GH84 family of glycosylhydrolases and lacking enzymatic glycosylhydrolase activity is a D174N mutant human O-GlcNAcase (hOGA) enzyme which includes the amino acid sequence: MVQKESQATLEERESELSSNPAASAGASLEPPAAPAPGEDNPAGAGGAAVAGAAGG ARRFLCGVVEGFYGRPWVMEQRKELFRRLQKWELNTYLYAPKDDYKHRMFWREM YSVEEAEQLMTLISAAREYEIEFIYAISPGLDITFSNPKEVSTLKRKLDQVSQFGCRSFA LLFNDIDHNMCAADKEVFSSFAHAQVSITNEIYQYLGEPETFLFCPTEYCGTFCYPNV SQSPYLRTVGEKLLPGIEVLWTGPKVVSKEIPVESIEEVSKIIKRAPVIWDNIHANDYD QKRLFLGPYKGRSTELIPRLKGVLTNPNCEFEANYVAIHTLATWYKSNMNGVRKDV VMTDSEDSTVSIQIKLENEGSDEDIETDVLYSPQMALKLALTEWLQEFGVPHQYSSR QVAHSGAKASVVDGTPLVAAPSLNATTVVTTVYQEPIMSQGAALSGEPTTLTKEEEK KQPDEEPMDMVVEKQEETDHKNDNQILSEIVEAKMAEELKPMDTDKESIAESKSPE MSMQEDCISDIAPMQTDEQTNKEQFVPGPNEKPLYTAEPVTLEDLQLLADLFYLPYE HGPKGAQMLREFQWLRANSSVVSVNCKGKDSEKIEEWRSRAAKFEEMCGLVMGM FTRLSNCANRTILYDMYSYVWDIKSIMSMVKSFVQWLGCRSHSSAQFLIGDQEPWAF RGGLAGEFQRLLPIDGANDLFFQPP (SEQ ID NO: 4 D174N truncated) or a variant thereof.

[0082] According to aspects of the present disclosure, the mutant O-GlcNAcase enzyme derived from a member of the GH84 family of glycosylhydrolases and lacking enzymatic glycosylhydrolase activity is a D175N mutant human O-GlcNAcase (hOGA) enzyme which includes the amino acid sequence: MVQKESQATLEERESELSSNPAASAGASLEPPAAPAPGEDNPAGAGGAAVAGAAGG ARRFLCGVVEGFYGRPWVMEQRKELFRRLQKWELNTYLYAPKDDYKHRMFWREMYSVEEAEQLMTLISAAREYEIEFIYAISPGLDITFSNPKEVSTLKRKLDQVSQFGCRSFA LLFDN1DHNMCAADKEVFSSFAHAQVS1TNE1YQYLGEPETFLFCPTEYCGTFCYPNV SQSPYLRTVGEKLLPGIEVLWTGPKVVSKEIPVESIEEVSKIIKRAPVIWDNIHANDYD QKRLFLGPYKGRSTELIPRLKGVLTNPNCEFEANYVAIHTLATWYKSNMNGVRKDV VMTDSEDSTVSIQIKLENEGSDEDIETDVLYSPQMALKLALTEWLQEFGVPHQYSSR QVAHSGAKASVVDGTPLVAAPSLNATTVVTTVYQEPIMSQGAALSGEPTTLTKEEEK KQPDEEPMDMVVEKQEETDHKNDNQILSEIVEAKMAEELKPMDTDKESIAESKSPE MSMQEDCISDIAPMQTDEQTNKEQFVPGPNEKPLYTAEPVTLEDLQLLADLFYLPYE HGPKGAQMLREFQWLRANSSVVSVNCKGKDSEKIEEWRSRAAKFEEMCGLVMGM FTRLSNCANRTILYDMYSYVWDIKSIMSMVKSFVQWLGCRSHSSAQFLIGDQEPWAF RGGLAGEFQRLLPIDGANDLFFQPP (SEQ ID NO: 43 D175N truncated) or a variant thereof.

[0083] According to aspects of the present disclosure, the mutant O-GlcNAcase enzy me derived from a member of the GH84 family of glycosylhydrolases and lacking enzymatic glycosylhydrolase activity is a D285N mutant human O-GlcNAcase (hOGA) enzyme which includes the amino acid sequence:MVQKESQATLEERESELSSNPAASAGASLEPPAAPAPGEDNPAGAGGAAVAGAAGG ARRFLCGVVEGFYGRPWVMEQRKELFRRLQKWELNTYLYAPKDDYKHRMFWREM YSVEEAEQLMTLISAAREYEIEFIYAISPGLDITFSNPKEVSTLKRKLDQVSQFGCRSFA LLFDDIDHNMCAADKEVFSSFAHAQVSITNEIYQYLGEPETFLFCPTEYCGTFCYPNV SQSPYLRTVGEKLLPGIEVLWTGPKVVSKEIPVESIEEVSKIIKRAPVIWDNIHANNYD QKRLFLGPYKGRSTELIPRLKGVLTNPNCEFEANYVAIHTLATWYKSNMNGVRKDV VMTDSEDSTVSIQIKLENEGSDEDIETDVLYSPQMALKLALTEWLQEFGVPHQYSSR QVAHSGAKASVVDGTPLVAAPSLNATTVVTTVYQEPIMSQGAALSGEPTTLTKEEEK KQPDEEPMDMVVEKQEETDHKNDNQILSEIVEAKMAEELKPMDTDKESIAESKSPE MSMQEDCISDIAPMQTDEQTNKEQFVPGPNEKPLYTAEPVTLEDLQLLADLFYLPYE HGPKGAQMLREFQWLRANSSVVSVNCKGKDSEKIEEWRSRAAKFEEMCGLVMGM FTRLSNCANRTILYDMYSYVWDIKSIMSMVKSFVQWLGCRSHSSAQFLIGDQEPWAF RGGLAGEFQRLLPIDGANDLFFQPP (SEQ ID NO: 44 D285N truncated) or a variant thereof

[0084] A detection moiety is included in fusion proteins according to aspects of the present disclosure.

[0085] According to aspects of the present disclosure, the detection moiety is a reporter protein or peptide encoded by a reporter gene. The term ‘'reporter gene” as used herein refers to gene that is easily detectable when expressed, for example by luminescence, fluorescence, colorimetric reactions, antibody binding, inducible markers, and / or ligand binding assays. Exemplary reporter proteins or peptides include, but are not limited to, green fluorescent protein (GFP); enhanced green fluorescent protein (eGFP); yellow fluorescent protein (YFP); enhanced yellow fluorescent protein (eYFP); hfYFP; mhYFP; LSSA12; LSSmGFP; cyan fluorescent protein (CFP); enhanced cyan fluorescent protein (eCFP); blue fluorescent protein (BFP); enhanced blue fluorescent protein (eBFP); MmGFP; dsRed; luciferase or a variant, such as but not limited to Renilla luciferase variants Rluc8_S257G and Rluc8.6, NanoLUC® luciferase derived from deep sea shrimp (Oplophorus gracilirostris) used with an imidazopyrazinone substrate furimazinem and luciferases from organisms such as Gaussia princeps, Metrida longa. Cypridinct noclilucc and Pyrocystis lunula, betagalactosidase (lacZ), beta-lactamase, beta-glucuronidase, horseradish peroxidase, alkaline phosphatase, chloramphenicol acetyl transferase; and an epitope tag such as a FLAG tag, a human influenza tag, or a Myc tag.

[0086] A detection moiety capable of emitting fluorescent and / or luminescent light is included in fusion proteins according to aspects of the present disclosure.

[0087] According to aspects of the present disclosure, a detection moiety is capable of emitting fluorescent and / or luminescent light when bound to a cognate detection moiety, the cognate detection moiety bound to an antibody which specifically recognizes the target protein. For example, a detection moiety / cognate detection moiety pair is split superfolder GFP (green fluorescent protein), N- and C-terminal sections; split Venus YFP (yellow fluorescent protein), N- and C-terminal sections; or split firefly luciferase, N- and C-terminal sections.

[0088] Detection of a detection moiety according to aspects of the present disclosure can be accomplished by providing an appropriate detection stimulus to a detection moiety resulting in a detectable signal.

[0089] According to aspects of the present disclosure, the detection moiety is a fluorescent protein, the detection stimulus is light having an excitation wavelength that causes the fluorophore of the fluorescent protein to become excited and emit fluorescent light, such that the detectable signal is fluorescent light.

[0090] According to aspects of the present disclosure, the detection moiety is a luminescent enzyme, such as a luciferase, the detection stimulus is a substrate for the luminescent enzy me (along with appropriate cofactors such as ATP, 02, and Mg2+) wherein the enzymatic action of the luciferase on the substrate results in emission of luminescent light, such that the detectable signal is luminescent light.

[0091] Detection of a detection moiety can be accomplished by visual observation and / or use of a detection device, such as, an electronic reader. An electronic reader used to detect results of a lateral flow assay is configured to detect the detection moiety used.

[0092] Any of a variety of detection moieties and their complementary detection modalities can be used when practicing the described methods, such as a fluorometer, a plate reader, a colorimeter, flow cytometer, a spectrophotometer, a luminometer, or a fluorescence detection microscope.

[0093] In a further example, where the detection moiety is fluorescent, a suitable excitation source and sensor of the resulting emitted signal may be used.

[0094] An electronic reader may generate data for analysis using one or more computer implemented methods. The electronic reader may include hardware and software components for computer implemented methods to analyze the resulting signal.

[0095] According to aspects of the present disclosure, methods of detecting glycosylated protein may be quantitative or semi-quantitative, for example, using a standard.

[0096] Standards are well-known in the art and one of skill in the art would readily recognize an appropriate standard and be able to determine an appropriate standard for a method of the present invention with no more than routine experimentation.

[0097] According to aspects of the present disclosure, the standard includes one or more known concentrations (e.g., serial dilutions) of a detectable moiety. According to aspects of the present disclosure, the concentrations of standard amounts of the detection moiety and the signals measured from the standard amounts are used to calculate a standard curve.

[0098] Amino acid sequences and nucleic acid sequences are shown or described herein. Methods and compositions of the present invention are not limited to particular amino acid sequences and nucleic acid sequences identified herein and variants of a reference amino acid or nucleic acid sequence are encompassed.

[0099] As used herein, the term "variant" defines either an isolated naturally occurring mutant of a protein or nucleic acid, or a recombinantly prepared mutant of a protein or nucleic acid, each of which contain one or more mutations compared to a correspondingreference sequence, such as a wild-type sequence. For example, such mutations in a protein sequence can be one or more amino acid substitutions, additions, and / or deletions. In a further example, For example, such mutations in a nucleic acid sequence can be one or more nucleotide substitutions, additions, and / or deletions. The term "variant" further refers to orthologues.

[0100] The term “wild-type” refers to a naturally occurring, or unmutated, protein or nucleic acid.

[0101] According to aspects of the present disclosure, a variant protein includes an amino acid sequence having at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or greater than 99%, identity with the reference amino acid sequence, and retains at least a substantial proportion (at least about 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or more) of the functional characteristics of the reference protein.

[0102] According to aspects of the present disclosure, a variant protein includes an amino acid sequence having at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% identity, or greater than 99%, identity with the reference amino acid sequence, and retains at least a substantial proportion (at least about 50%, 60%, 70%, 80%, 90%, 95%, 98%, 99% or more) of the functional characteristics of the reference protein.

[0103] To determine the percent identity of two amino acid sequences or of two nucleic acid sequences, the sequences are aligned for optimal comparison purposes (e.g., gaps can be introduced in the sequence of a first amino acid or nucleic acid sequence for optimal alignment with a second amino acid or nucleic acid sequence). The amino acid residues or nucleotides at corresponding amino acid positions or nucleotide positions are then compared. When a position in the first sequence is occupied by the same amino acid residue or nucleotide as the corresponding position in the second sequence, then the molecules are identical at that position. The percent identity betw een the two sequences is a function of the number of identical positions shared by the sequences (i.e., % identity=number of identical overlapping positions / total number of positions XI 00%). The two sequences compared are generally the same length or nearly the same length. Optionally, the two sequences are natural variants of a structural domain of a protein or two related proteins.

[0104] The determination of percent identity between two sequences can also be accomplished using a mathematical algorithm. A preferred, non-limiting example of a mathematical algorithm utilized for the comparison of two sequences is the algorithm ofKarlin and Altschul, 1990, PNAS 87:2264 2268, modified as in Karlin and Altschul, 1993, PNAS. 90:5873 5877. Such an algorithm is incorporated into the NBLAST and XBLAST programs of Altschul et al., 1990, J. Mol. Biol. 215:403. BLAST nucleotide searches are performed with the NBLAST nucleotide program parameters set, e.g., for score=100, wordlength=12 to obtain nucleotide sequences homologous to a nucleic acid molecules of the present invention. BLAST protein searches are performed with the XBLAST program parameters set, e.g., to score 50, wordlength=3 to obtain amino acid sequences homologous to a protein molecule of the present invention. To obtain gapped alignments for comparison purposes, Gapped BLAST are utilized as described in Altschul et al., 1997, Nucleic Acids Res. 25:3389 3402. Alternatively, PSI BLAST is used to perform an iterated search which detects distant relationships between molecules (Id.). When utilizing BLAST, Gapped BLAST, and PSI Blast programs, the default parameters of the respective programs (e.g., of XBLAST and NBLAST) are used (see, e.g., the NCBI website). Another preferred, nonlimiting example of a mathematical algorithm utilized for the comparison of sequences is the algorithm of Myers and Miller, 1988, CABIOS 4: 11 17. Such an algorithm is incorporated in the ALIGN program (version 2.0) which is part of the GCG sequence alignment software package. When utilizing the ALIGN program for comparing amino acid sequences, a PAM 120 weight residue table, a gap length penalty of 12, and a gap penalty of 4 is used.

[0105] The percent identity between two sequences is determined using techniques similar to those described above, with or without allowing gaps. In calculating percent identity, typically only exact matches are counted.

[0106] One of skill in the art will recognize that one or more nucleotide or amino acid mutations can be introduced without altering the functional properties of a given nucleic acid or protein, respectively.

[0107] Mutations can be introduced using standard molecular biology techniques, such as site-directed mutagenesis and PCR-mediated mutagenesis, to produce variants. For example, one or more amino acid substitutions, additions, or deletions can be made without altering the functional properties of a reference protein. When comparing a reference protein to a putative variant, amino acid similarity may be considered in addition to identity of amino acids at corresponding positions in an amino acid sequence. “Amino acid similarity” refers to amino acid identity and conservative amino acid substitutions in a putative variant compared to the corresponding amino acid positions in a reference protein.

[0108] Conservative amino acid substitutions can be made or may be present in reference proteins to produce or identify vanants.

[0109] Conservative amino acid substitutions are art recognized substitutions of one amino acid for another amino acid having similar characteristics. For example, each amino acid may be described as having one or more of the following characteristics: electropositive, electronegative, aliphatic, aromatic, polar / nonpolar, hydrophobic and hydrophilic. A conservative substitution is a substitution of one amino acid having a specified structural or functional characteristic for another amino acid having the same characteristic. Acidic amino acids include aspartate, glutamate; basic amino acids include histidine, lysine, arginine; aliphatic amino acids include isoleucine, leucine and valine; aromatic amino acids include phenylalanine, tyrosine and tryptophan; polar amino acids include aspartate, glutamate, histidine, lysine, asparagine, glutamine, arginine, serine, threonine and tyrosine; and hydrophobic amino acids include alanine, cysteine, phenylalanine, glycine, isoleucine, leucine, methionine, proline, valine and tryptophan; and conservative substitutions include substitution among amino acids within each group. Amino acids may also be described in terms of relative size; alanine, cysteine, aspartate, glycine, asparagine, proline, threonine, serine, valine are all typically considered to be small.

[0110] A variant can include sy nthetic amino acid analogs, amino acid derivatives and / or non-standard amino acids, illustratively including, without limitation, alphaaminobutyric acid, citrulline, canavanine, cyanoalanine, diaminobutyric acid, diaminopimelic acid, dihydroxy-phenylalanine, djenkolic acid, homoarginine, - 18 - 18 hydroxyproline, norleucine, non aline, 3 -phosphoserine, homoserine, 5- hydroxy try ptophan, 1 -methylhistidine, 3-methylhistidine, and ornithine.

[0111] It will be appreciated by those of ordinary skill in the art that, due to the degenerate nature of the genetic code, alternate nucleic acid sequences encode a specified protein such variant nucleic acid sequences may be used in compositions and methods described herein.

[0112] Protein variants are encoded by nucleic acids having a high degree of identity with a nucleic acid encoding a corresponding reference protein, such as a wild-type protein, or a corresponding portion thereof. The complement of a nucleic acid encoding a variant specifically hybridizes with a nucleic acid encoding a corresponding reference protein, such as a w ild-type protein, under high stringency conditions.

[0113] The term ‘“nucleic acid” refers to RNA or DNA molecules having more than one nucleotide in any form including single-stranded, double-stranded, oligonucleotide or polynucleotide. The term “‘nucleotide sequence” refers to the ordering of nucleotides in an oligonucleotide or polynucleotide in a single-stranded form of nucleic acid.

[0114] The term “complementary” refers to Watson-Crick base pairing between nucleotides and specifically refers to nucleotides hydrogen bonded to one another with thymine or uracil residues linked to adenine residues by two hydrogen bonds and cytosine and guanine residues linked by three hydrogen bonds. In general, a nucleic acid includes a nucleotide sequence described as having a “percent complementarity ” to a specified second nucleotide sequence. For example, a nucleotide sequence may have 80%, 90%, or 100% complementarity to a specified second nucleotide sequence, indicating that 8 of 10, 9 of 10 or 10 of 10 nucleotides of a sequence are complementary to the specified second nucleotide sequence. For instance, the nucleotide sequence 3’-TCGA-5’ is 100% complementary' to the nucleotide sequence 5’-AGCT-3’. Further, the nucleotide sequence 3'-TCGA- is 100% complementary’ to a region of the nucleotide sequence 5'-TTAGCTGG-3'.

[0115] The terms “hybridization” and “‘hybridizes” refer to pairing and binding of complementary’ nucleic acids. Hybridization occurs to varying extents between two nucleic acids depending on factors such as the degree of complementarity’ of the nucleic acids, the melting temperature, Tm, of the nucleic acids and the stringency of hybridization conditions, as is well known in the art. The term ‘“stringency of hybridization conditions” refers to conditions of temperature, ionic strength, and composition of a hybridization medium with respect to particular common additives such as formamide and Denhardt’s solution. Determination of particular hybridization conditions relating to a specified nucleic acid is routine and is well known in the art, for instance, as described in J. Sambrook and D.W. Russell, Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory’ Press; 3rd Ed., 2001; and F.M. Ausubel, Ed., Short Protocols in Molecular Biology, Current Protocols; 5th Ed., 2002. High stringency hybridization conditions are those which only allow hybridization of substantially complementary nucleic acids. Typically, nucleic acids having about 85-100% complementarity are considered highly complementary’ and hybridize under high stringency conditions. Intermediate stringency conditions are exemplified by conditions under which nucleic acids having intermediate complementarity’, about 50-84% complementarity, as well as those having a high degree of complementarity, hybridize. Incontrast, low stringency hybridization conditions are those in which nucleic acids having a low degree of complementarity hybridize.

[0116] The terms “specific hybridization” and “specifically hybridizes” refer to hybridization of a particular nucleic acid to a target nucleic acid without substantial hybridization to nucleic acids other than the target nucleic acid in a sample.

[0117] Stringency of hybridization and washing conditions depends on several factors, including the Tm of the probe and target and ionic strength of the hybridization and wash conditions, as is well-known to the skilled artisan. Hybridization and conditions to achieve a desired hybridization stringency are described, for example, in Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 2001; and Ausubel, F. et al., (Eds.), Short Protocols in Molecular Biology, Wiley, 2002.

[0118] An example of high stringency hybridization conditions is hybridization of nucleic acids over about 100 nucleotides in length in a solution containing 6X SSC, 5X Denhardt’s solution, 30% formamide, and 100 micrograms / ml denatured salmon sperm at 37°C overnight followed by washing in a solution of 0. IX SSC and 0. 1% SDS at 60°C for 15 minutes. SSC is 0.15M NaCl / 0.015M Na citrate. Denhardt’s solution is 0.02% bovine serum albumin / 0.02% FICOLL / 0.02% polyvinylpyrrolidone.

[0119] Nucleic acids encoding a protein, or a variant thereof, can be isolated or generated recombinantly or synthetically using well-known methodology.

[0120] A linker is disposed between, and linked to each of, two components of a fusion protein according to aspects of the present disclosure, thereby linking the two components through the linker.

[0121] According to aspects of the present disclosure, an included linker is, or includes about 1 to 100 amino acids, such as about 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, 35, 40, 45, 50, 55, 60, 65, 70, 75, 80, 85,90, 95, or 100 amino acids. According to aspects of the present disclosure, is a peptide including about 1 to 100 amino acids, such as about 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, 35, 40, 45, 50, 55, 60, 65, 70, 75,80, 85, 90, 95, or 100 amino acids.

[0122] According to aspects of the present disclosure, the linker can be , or include, a bond, an atom, a multi-atom group, or a chain of atoms. Non-limiting examples of a linker which is an atom are oxygen, and sulfur. A non-limiting example of a linker which is a multiatom group is C(O).

[0123] According to aspects of the present disclosure, an included linker is, or includes, a chain of atoms such as, branched or linear chain of 2- 20, or more, atoms. According to aspects of the present disclosure, a linker is, or includes, a linear chain of 3- 20, or more, atoms. According to aspects of the present disclosure, a linker is, or includes, a linear chain of 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 atoms. According to aspects of the present disclosure, a linker is, or includes, a linear chain of 6, 7, 8, 9, 10, 11, or 12 atoms. According to aspects of the present disclosure, a linker is, or includes, a linear chain of 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 atoms. According to aspects of the present disclosure, a linker is, or includes, a linear chain of 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, or 20 atoms. According to aspects of the present disclosure, a linker is, or includes, a linear chain of 10, 11, 12, 13, 14, or 15 atoms.

[0124] According to aspects of the present disclosure, an included linker is, or includes, a chain of atoms such as, but not limited to, substituted or unsubstituted C1-C20 alkyl, substituted or unsubstituted C2-C20 alkenyl, substituted or unsubstituted C2-C20 alkynyl, substituted or unsubstituted C6-C12 aryl, substituted or unsubstituted C3-C12 cycloalkyl, substituted or unsubstituted C5-C12 heteroaryl, or substituted or unsubstituted C5-C12 heterocyclyl. According to aspects of the present disclosure, the chain includes at least one hydroxyl functional group, and preferably at least one terminal hy droxyl functional group. The term "terminal hydroxyl functional group" as used herein refers to a hydroxyl group on the final atom of a chain of atoms in a linker, i.e. the atom of the chain of atoms which is furthest from the magnetic particle, or within no more than 2, 3, or 4 atoms from the final atom of the chain of atoms in a linker.

[0125] A linker can be a poly(Glycine)-Serine peptide linker characterized by a pentapeptide amino acid sequence GGGGS (GlyGlyGlyGlySer) (SEQ ID NO:20), repeated “n” times, “G4S(n)” where n is 1 , 2, 3, 4, 5, 6 or more, e.g. GGGGS (SEQ ID NO:20), GGGGSGGGGS (SEQ ID NO:32), GGGGS GGGGS GGGGS (SEQ ID NO:33), GGGGSGGGGSGGGGSGGGGS (SEQ ID NO:34),GGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO:35),GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 18). The sequence G4S(n)may itself constitute the entire linker or it may be part of a larger linker.

[0126] According to aspects of the present disclosure, a linker is disposed between, and linked to each of, a glycan binding component and a detection moiety, thereby linking the glycan binding component and the detection moiety through the linker.

[0127] According to aspects of the present disclosure, the glycan binding component is linked to the detection moiety by a linker disposed between the glycan binding component and the detection moiety.

[0128] According to aspects of the present disclosure, the fusion protein includes a localization signal peptide. According to aspects of the present disclosure, the localization signal peptide is capable of promoting localization of the fusion protein to a subcellular compartment selected from the group consisting of nucleus, cytosol, mitochondria, endoplasmic reticulum, and plasma membrane. One or more localization signal peptides are included in a fusion protein according to aspects of the present disclosure. Optionally, a linker is disposed between a localization signal peptide and the glycan binding component and / or linker is disposed between the localization signal peptide and the detection moiety. When two or more localization signal peptides are included in a fusion protein according to aspects of the present disclosure, a linker is optionally disposed between the two or more localization signal peptides.

[0129] According to aspects of the present disclosure, the localization signal peptide is a mitochondrial localization signal. An exemplary mitochondrial localization signal is MLGFVGRVAAAPASGALRRLTPSASLPPAQLLLRAAPTAVHPVRDYA (SEQ ID NO: 5), or a variant thereof.

[0130] According to aspects of the present disclosure, the localization signal peptide is a plasma membrane localization signal. An exemplary plasma membrane localization signal is MGCINSKRK, (SEQ ID NO: 6), or a variant thereof.

[0131] According to aspects of the present disclosure, the localization signal peptide is a nuclear localization signal. An exemplary nuclear localization signal (NLS) is PKKKRKV (SEQ ID NO: 7), or a variant thereof.

[0132] According to aspects of the present disclosure, the localization signal peptide is a cytoplasm localization signal. An exemplary cytoplasm localization signal (NES) is LPPLERTL, (SEQ ID NO: 8), or a variant thereof.

[0133] According to aspects of the present disclosure, the fusion protein includes an exogenous detectable tag, such as a V5 tag or HA tag, or a variant of either thereof. An exemplary V5 tag is GKPIPNPLLGLDST, (SEQ ID NO: 9), or a variant thereof. An exemplary7HA tag is YPYDVPDYA (SEQ ID NO: 10), or a variant thereof.

[0134] An expression construct is provided according to aspects of the present disclosure which includes a nucleic acid encoding a fusion protein including a glycan bindingcomponent linked to a detection moiety, the glycan binding component capable of specific binding to a glycosylation post-translational modification of a target protein.

[0135] The term '‘expression construct” is used herein to refer to a double-stranded recombinant DNA molecule containing a nucleic acid desired to be expressed and containing appropriate regulatory elements necessary' or desirable for the transcription of the operably linked nucleic acid sequence in vitro or in vivo. The term “recombinant” is used to indicate a nucleic acid construct in yvhich two or more nucleic acids are linked and w hich are not found linked in nature. The term “expressed” refers to transcription of a nucleic acid to produce a corresponding mRNA and / or translation of the mRNA to produce the corresponding protein. Expression constructs can be generated recombinantly or synthetically or by DNA synthesis using well-known methodology.

[0136] An expression construct is introduced into a cell using well-known methodology, such as, but not limited to, by introduction of a vector containing the expression construct into the cell. A “vector” is a nucleic acid that transfers an inserted nucleic acid into and / or between host cells becoming self-replicating. The term includes vectors that function primarily for insertion of a nucleic acid into a cell, replication of vectors that function primarily for the replication of nucleic acid, and expression vectors that function for transcription and / or translation of a nucleic acid. Also included are vectors that provide more than one of the above functions.

[0137] Vectors include plasmids, viruses, BACs, YACs, and the like. Particular viral vectors illustratively include those derived from adenovirus, adeno-associated virus and lentivirus.

[0138] The term “regulatory element” as used herein refers to a nucleotide sequence which controls some aspect of the expression of an operably linked nucleic acid. Exemplary regulatory elements illustratively include an enhancer, an internal ribosome entry site (IRES), an intron; an origin of replication, a polyadenylation signal (pA), a promoter, a transcription termination sequence, and an upstream regulatory domain, which contribute to the replication, transcription, post-transcriptional processing of a nucleic acid. Those of ordinary skill in the art are capable of selecting and using these and other regulatory elements in an expression construct with no more than routine experimentation.

[0139] The term “promoter” as used herein refers to a DNA sequence operably linked to a nucleic acid to be transcribed such as a nucleic acid encoding a desired molecule. Apromoter is generally positioned upstream of a nucleic acid sequence to be transcribed and provides a site for specific binding by RNA polymerase and other transcription factors.

[0140] In addition to a promoter, one or more enhancer sequences may be included such as, but not limited to, cytomegalovirus (CMV) early enhancer element and an SV40 enhancer element. Additional included sequences are an intron sequence such as the beta globin intron or a generic intron, a transcription termination sequence, and an mRNA polyadenylation (pA) sequence such as, but not limited to SV40-pA, beta-globin-pA, the human growth hormone (hGH) pA and SCF-pA. The term '‘poly A” or “p(A)” or “pA” refers to nucleic acid sequences that signal for transcription termination and mRNA polyadeny lation. The polyA sequence is characterized by the hexanucleotide motif AAUAAA. Commonly used polyadenylation signals are the SV40 pA, the human growth hormone (hGH) pA, the beta-actin pA, and betaglobin pA. The sequences can range in length from 32 to 450 bp. Multiple pA signals may be used.

[0141] The term “operably linked’" as used herein refers to a nucleic acid in functional relationship with a second nucleic acid. The term “operably linked” encompasses functional connection of two or more nucleic acids, such as an oligonucleotide or polynucleotide to be transcribed and a regulatory element such as a promoter or an enhancer element, which allows transcription of the nucleic acid to be transcribed.

[0142] An expression construct provided according to aspects of the present disclosure includes a nucleic acid encoding a fusion protein including a glycan binding component linked to a detection moiety, the glycan binding component capable of specific binding to a glycosylation post-translational modification of a target protein a detection moiety is or includes a nucleic acid sequence encoding:

[0143] MVQKESQATLEERESELSSNPAASAGASLEPPAAPAPGEDNPAGAGGAA VAGAAGGARRFLCGVVEGFYGRPWVMEQRKELFRRLQKWELNTYLYAPKDDYKH RMFWREMYSVEEAEQLMTLISAAREYEIEFIYAISPGLDITFSNPKEVSTLKRKLDQVS QFGCRSFALLFNDIDHNMCAADKEVFSSFAHAQVSITNEIYQYLGEPETFLFCPTEYC GTFCYPNVSQSPYLRTVGEKLLPGIEVLWTGPKVVSKEIPVESIEEVSKIIKRAPVIWD NIHANDYDQKRLFLGPYKGRSTELIPRLKGVLTNPNCEFEANYVAIHTLATWYKSNM NGVRKDVVMTDSEDSTVSIQIKLENEGSDEDIETDVLYSPQMALKLALTEWLQEFGV PHQYSSRQVAHSGAKASVVDGTPLVAAPSLNATTVVTTVYQEPIMSQGAALSGEPTT LTKEEEKKQPDEEPMDMVVEKQEETDHKNDNQILSEIVEAKMAEELKPMDTDKESI AESKSPEMSMQEDCISDIAPMQTDEQTNKEQFVPGPNEKPLYTAEPVTLEDLQLLADLFYLPYEHGPKGAQMLREFQWLRANSSVVSVNCKGKDSEKIEEWRSRAAKFEEMC GLVMGMFTRLSNCANRT1LYDMYSYVWDIKSIMSMVKSFVQWLGCRSHSSAQFLIG DQEPWAFRGGLAGEFQRLLPIDGANDLFFQPP (SEQ ID NO:4), or a variant thereof, and a detection moiety, wherein the two component proteins are linked directly or through a linker and wherein the expression construct optionally further encodes a localization signal peptide and / or exogenous tag in operable linkage with one or both component proteins.

[0144] An expression construct provided according to aspects of the present disclosure includes a nucleic acid encoding a fusion protein including a glycan binding component linked to a detection moiety, the glycan binding component capable of specific binding to a glycosylation post-translational modification of a target protein and the detection moiety is or includes a nucleic acid sequence encoding:MVQKESQATLEERESELSSNPAASAGASLEPPAAPAPGEDNPAGAGGAAVAGAAGG ARRFLCGVVEGFYGRPWVMEQRKELFRRLQKWELNTYLYAPKDDYKHRMFWREM YSVEEAEQLMTLISAAREYEIEFIYAISPGLDITFSNPKEVSTLKRKLDQVSQFGCRSFA LLFNDIDHNMCAADKEVFSSFAHAQVSITNEIYQYLGEPETFLFCPTEYCGTFCYPNV SQSPYLRTVGEKLLPGIEVLWTGPKVVSKEIPVESIEEVSKIIKRAPVIWDNIHANDYD QKRLFLGPYKGRSTELIPRLKGVLTNPNCEFEANYVAIHTLATWYKSNMNGVRKDV VMTDSEDSTVSIQIKLENEGSDEDIETDVLYSPQMALKLALTEWLQEFGVPHQYSSR QVAHSGAKASVVDGTPLVAAPSLNATTVVTTVYQEPIMSQGAALSGEPTTLTKEEEK KQPDEEPMDMVVEKQEETDHKNDNQILSEIVEAKMAEELKPMDTDKESIAESKSPE MSMQEDCISDIAPMQTDEQTNKEQFVPGPNEKPLYTAEPVTLEDLQLLADLFYLPYE HGPKGAQMLREFQWLRANSSVVSVNCKGKDSEKIEEWRSRAAKFEEMCGLVMGM FTRLSNCANRTILYDMYSYVWDIKSIMSMVKSFVQWLGCRSHSSAQFLIGDQEPWAF RGGLAGEFQRLLPIDGANDLFFQPP (SEQ ID NO:4), or a variant thereof, and a detection moiety, wherein the two component proteins are linked directly or through a linker, and wherein the expression construct optionally further encodes a localization signal peptide and / or exogenous tag in operable linkage with one or both component proteins.

[0145] A nucleic acid encoding a NLS is:GACCCCAAGAAGAAGAGGAAGGTGGACCCCAAGAAGAAGAGGAAGGTGGACCC CAAGAAGAAGAGGAAGGTG (SEQ ID NO: 14).

[0146] A nucleic acid encoding an NES is:CTGCCTCCCCTGGAGCGCCTGACCCTGGAC (SEQ ID NO: 15).

[0147] Nucleic acids encoding a linker are: GCGGCCGCCACCATGTACCCGTATGATGTTCCGGATTACGCTGGCTATCCCTACG ACGTGCCCGACTATGCCGGGTACCCCTATGACGTCCCAGACTACGCAGCTAGC (SEQ ID NO: 16), CTGCAG, and AAGCTTGCGGCCGCCACCATGGGCAAGCCCATCCCCAACCCCCTGCTGGGCCTG GACAGCACCGCTAGC (SEQ ID NO: 17).

[0148] A tag can be included such as a 6X tag:GGGGSGGGGSGGGGSGGGGSGGGGSGGGGS (SEQ ID NO: 18).

[0149] A flag tag can be included, such as DYKDDDDK (SEQ ID NO: 19). A 3X flag tag can be included, such as DYKDHDGDYKDHDIDYKDDDDK (SEQ ID NO: 36).

[0150] Nucleic acids encoding a protein, or a variant thereof, can be isolated or generated recombinantly or synthetically using well-known methodology.

[0151] According to aspects of the present disclosure, an expression construct encoding the fusion protein is introduced into host cells to express the fusion protein. The fusion protein can then be isolated from the host cells. The isolated fusion protein can be used in assays for detection of glycosylation post-translational modification of a proteins.

[0152] The expression construct may be transfected into host cells using well-known methods, such as electroporation, calcium-phosphate precipitation transfection, and lipofection. The host cells are screened for presence and / or integration by DNA analysis, such as PCR, Southern blot or sequencing. Host cells with the expression construct can be screened for functional expression, for example ELISA or Western blot analysis. Host cells can be mammalian cells, bacterial cells, yeast, insect cells, or any other cell suitable for expression of exogenous protein from an expression construct.

[0153] Cell-free protein expression systems may be used to express fusion proteins according to aspects of the present disclosure.

[0154] The expressed fusion protein can be isolated from cells by standard purification methods. The term "purification" in the context of purifying the expressed fusion protein refers to separation of the expressed fusion protein from at least one other component present in the system in which the expressed fusion protein was produced. For example, expressed fusion proteins are separated from cells in which they are expressed, generating purified expressed fusion protein.

[0155] According to aspects, the purified expressed fusion proteins make up at least about 0.01 - 100% of the mass, by weight, such as about 0.01%, 0.1%, 1%, 5%, 10%, 25%,50%, 75%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than about 99% of the mass, by weight, of material in a sample of purified expressed fusion protein. Such purification is achieved by techniques illustratively including salt, pH, hydrophobic or affinity precipitation, electrophoretic methods such as gel electrophoresis and 2-D gel electrophoresis; chromatography methods such as HPLC, ion exchange chromatography, affinity chromatography, size exclusion chromatography, thin layer and paper chromatography.

[0156] Cells are provided according to aspects of the present disclosure which include the expression construct which includes a nucleic acid encoding a fusion protein including a glycan binding component linked to a detection moiety, the glycan binding component capable of specific binding to a glycosylation post-translational modification of a target protein.

[0157] Methods of detecting a glycosylated protein according to aspects of the present disclosure include: providing a fusion protein including a glycan binding component linked to a detection moiety, the glycan binding component capable of specific binding to a glycosylation post-translational modification of a protein; contacting a cell lysate of a sample, or purified fraction thereof, containing one or more proteins which are putatively glycosylated by post-translational modification with the fusion protein under binding conditions, whereby the fusion protein specifically binds to one or more glycosylated proteins of the cell; and detecting a signal from the detection moiety, thereby detecting glycosylated proteins bound to the glycan binding component.

[0158] The term ‘"under binding conditions” refers to conditions which are compatible with specific binding of the glycan binding component and a glycosylation post-translational modification of a protein. Physiological conditions is a general term signifying compatible binding conditions and such conditions are well-known.

[0159] According to aspects of the present disclosure, detecting the glycosylated proteins includes purifying the glycosylated proteins from a sample and detecting the purified glycosylated proteins.

[0160] A sample putatively containing post-translationally glycosylated proteins can be any type of sample, particularly biological samples, including biological samples obtained or derived from a subject, such as a human or non-human animal. According to aspects of the present disclosure, the subject is a human, non-human primate, or other mammal, such as rodents, dogs, cats, horses, cattle, sheep, rabbits, pigs, and goats. A subject can be a nonmammal such as a reptile, amphibian, fish or bird.

[0161] The sample can be any sample putatively containing post-translationally glycosylated proteins such as a bodily fluid or tissue sample or fraction of any thereof, such as, but not limited to, blood, plasma, serum, cerebrospinal fluid, saliva, urine, semen, milk, nasal fluid, vaginal fluid, bronchial fluid, mucus, pus, wound exudate, tumor tissue or exudate, blood cells, brain cells, meninges, astrocytes, spinal cord cells, nerve cells organ tissue such as, but not limited to, kidney, liver, pancreas, ovary, testicle, uterus, prostate, gall bladder, spleen, lung, heart, skin, muscle, tendon, bone, parathyroid, and thyroid.

[0162] The sample can be obtained directly from a subject according to aspects of the present disclosure, such as by blood draw or biopsy for example. The sample may be cultured ex vivo prior to assay if desired.

[0163] The term "purifying" in the context of purifying the glycosylated proteins for detection refers to separation of the glycosylated proteins from at least one other component present in the system in which the glycosylated proteins were produced. For example, glycosylated proteins are separated from cells in which they are produced, generating purified glycosylated proteins.

[0164] According to aspects, the purified glycosylated proteins make up at least about 0.01 - 100% of the mass, by weight, such as about 0.01%, 0.1%, 1%, 5%, 10%, 25%, 50%, 75%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or greater than about 99% of the mass, byweight, of material in a sample of purified glycosylated proteins. Such purification is achieved by techniques illustratively including salt, pH, hydrophobic or affinity precipitation, electrophoretic methods such as gel electrophoresis and 2-D gel electrophoresis; chromatography methods such as HPLC, ion exchange chromatography, affinity chromatography, size exclusion chromatography, thin layer and paper chromatography.

[0165] According to aspects of the present disclosure, detecting the purified glycosylated proteins includes mass spectrometry.

[0166] According to aspects of the present disclosure, mass spectrometry is used in a method for detecting the purified glycosylated proteins. A variety of configurations of mass spectrometers can be used in a method of the present disclosure. In general, a mass spectrometer has the following major components: a sample inlet, an ion source, a mass analyzer, a detector, a vacuum system, and instrument-control system, and a data system. Common mass analyzers include a quadrupole mass filter, ion trap mass analyzer and time- of-flight mass analyzer.

[0167] The ion formation process is a starting point for mass spectrum analysis and several ionization methods are available. For example, electrospray ionization (ESI) can be used. Generally described, in ESI a solution containing the material to be analyzed is passed through a fine needle at high potential which creates a strong electrical field resulting in a fine spray of highly charged droplets that is directed into the mass spectrometer. Other ionization procedures include, for example, fast-atom bombardment (FAB) which uses a high-energy beam of neutral atoms to strike a solid sample causing desorption and ionization. Matrix-assisted laser desorption ionization (MALDI) is a method in which a laser pulse is used to strike a sample that has been crystallized in an UV-absorbing compound matrix. Other ionization procedures known in the art include, for example, plasma and glow discharge, plasma desorption ionization, resonance ionization, and secondary ionization.

[0168] Electrospray ionization (ESI) has several properties that are useful for methods of assessing an analyte of the present disclosure. For example, the efficiency of ESI can be very high which provides the basis for highly sensitive measurements. Furthermore, ESI produces charged molecules from solution, which is convenient for analyzing analytes and standards that are in solution. In contrast, ionization procedures such as MALDI require crystallization of the material to be analyzed prior to ionization.

[0169] Since ESI can produce charged molecules directly from solution, it is compatible with samples from liquid chromatography systems. In liquid chromatography with tandem mass spectrometry (LC-MS-MS), the inlet can be a capillary-column liquid chromatography source. For example, a mass spectrometer can have an inlet for a liquid chromatography system, such as an HPLC, so that fractions flow from the chromatography column into the mass spectrometer. This in-line arrangement of a liquid chromatography system and mass spectrometer is sometimes referred to as LC-MS. An LC-MS system can be used, for example, to separate analytes and standards from complex mixtures before mass spectrometry analysis. In addition, chromatography can be used to remove salts or other buffer components from the sample before mass spectrometry analysis. For example, desalting of a sample using a reversed-phase HPLC column, in-line or off-line, can be used to increase the efficiency of the ionization process and thus improve sensitivity of detection by mass spectrometry.

[0170] A variety of mass analyzers are available that can be paired with different ion sources. Different mass analyzers have different advantages as known to one skilled in the art and as described herein. The mass spectrometer and methods chosen for detection depends on the particular assay, for example, a more sensitive mass analyzer can be used when a smallamount of ions are generated for detection. Several types of mass analyzers and mass spectrometry methods are described below.

[0171] Quadrupole mass spectrometry utilizes a quadrupole mass filter or analyzer. This type of mass analyzer is composed of four rods arranged as two sets of two electrically connected rods. A combination of rf and de voltages are applied to each pair of rods which produces fields that cause an oscillating movement of the ions as they move from the beginning of the mass filter to the end. The result of these fields is the production of a high- pass mass filter in one pair of rods and a low-pass filter in the other pair of rods. Overlap between the high-pass and low-pass filter leaves a defined m / z that can pass both filters and traverse the length of the quadrupole. This m / z is selected and remains stable in the quadrupole mass filter while all other m / z have unstable trajectories and do not remain in the mass filter. A mass spectrum results by ramping the applied fields such that an increasing m / z is selected to pass through the mass filter and reach the detector. In addition, quadrupoles can also be set up to contain and transmit ions of all m / z by applying a rf-only field. This allows quadrupoles to function as a lens or focusing system in regions of the mass spectrometer where ion transmission is needed without mass filtering. This will be of use in tandem mass spectrometry as described further below.

[0172] A quadrupole mass analyzer, as well as the other mass analyzers described herein, can be programmed to analyze a defined m / z or mass range. Since the mass range of analytes and standards will be known prior to an assay, a mass spectrometer can be programmed to transmit ions of the projected correct mass range while excluding ions of a higher or lower mass range. The ability to select a mass range can decrease the background noise in the assay and thus increase the signal-to-noise ratio as well as increasing the specificity of the assay. Therefore, the mass spectrometer can accomplish an inherent separation step as well as detection and identification of analytes and standards.

[0173] Ion trap mass spectrometry utilizes an ion trap mass analyzer. In these mass analyzers, fields are applied so that ions of all m / z are initially trapped and oscillate in the mass analyzer. Ions enter the ion trap from the ion source through a focusing device such as an octapole lens system. Ion trapping takes place in the trapping region before excitation and ejection through an electrode to the detector. Mass analysis is accomplished by sequentially applying voltages that increase the amplitude of the oscillations in a way that ejects ions of increasing m / z out of the trap and into the detector. In contrast to quadrupole mass spectrometry, all ions are retained in the fields of the mass analyzer except those with theselected m / z. One advantage to ion traps is that they have very high sensitivity, as long as one is careful to limit the number of ions being tapped at one time. Control of the number of ions can be accomplished by varying the time over which ions are injected into the trap. The mass resolution of ion traps is similar to that of quadrupole mass fdters, although ion traps do have low m / z limitations.

[0174] Time-of-flight mass spectrometry utilizes a time-of-flight mass analyzer. For this method of m / z analysis, an ion is first given a fixed amount of kinetic energy by acceleration in an electric field (generated by high voltage). Following acceleration, the ion enters a field- free or "drift" region where it travels at a velocity that is inversely proportional to its m / z. Therefore, ions with low m / z travel more rapidly than ions with high m / z. The time required for ions to travel the length of the field-free region is measured and used to calculate the m / z of the ion. One consideration in this type of mass analysis is that the set of ions being studied be introduced into the analyzer at the same time. For example, this type of mass analysis is well suited to ionization techniques like MALDI which produces ions in short well-defined pulses. Another consideration is to control velocity spread produced by ions that have variations in their amounts of kinetic energy. The use of longer flight tubes, ion reflectors, or higher accelerating voltages can help minimize the effects of velocity spread. Time-of-flight mass analyzers have a high level of sensitivity and a wider m / z range than quadrupole or ion trap mass analyzers. Also data can be acquired quickly with this type of mass analyzer because no scanning of the mass analyzer is necessary.

[0175] Tandem mass spectrometry can utilize combinations of the mass analyzers described above.

[0176] Tandem mass spectrometers can use a first mass analyzer to separate ions according to their m / z in order to isolate an ion of interest for further analysis. The isolated ion of interest is then broken into fragment ions, called collisionally activated dissociation or collisionally induced dissociation, and the fragment ions are analyzed by the second mass analyzer. These types of tandem mass spectrometer systems are called tandem in space systems because the two mass analyzers are separated in space, usually by a collision cell. Tandem mass spectrometer systems also include tandem in time systems where one mass analyzer is used, however the mass analyzer is used sequentially to isolate an ion, induce fragmentation, and then perform mass analysis.

[0177] Mass spectrometers in the tandem in space category have more than one mass analyzer. For example, a tandem quadrupole mass spectrometer system can have a firstquadrupole mass filter, followed by a collision cell, followed by a second quadrupole mass filter and then the detector. Another arrangement is to use a quadrupole mass filter for the first mass analyzer and a time-of-flight mass analyzer for the second mass analyzer with a collision cell separating the two mass analyzers.

[0178] Other tandem systems are known in the art including reflection-time-of-flight, tandem sector and sector-quadrupole mass spectrometry.

[0179] Mass spectrometers in the tandem in time category have one mass analyzer that performs different functions at different times. For example, an ion trap mass spectrometer can be used to trap ions of all m / z. A series of rf scan functions are applied which ejects ions of all m / z from the trap except the m / z of ions of interest. After the m / z of interest has been isolated, an rf pulse is applied to produce collisions with gas molecules in the trap to induce fragmentation of the ions. Then the m / z values of the fragmented ions are measured by the mass analyzer. Ion cyclotron resonance instruments, also known as Fourier transform mass spectrometers, are an example of tandem-in-time systems.

[0180] Several types of tandem mass spectrometry experiments can be performed by controlling the ions that are selected in each stage of the experiment. The different types of experiments utilize different modes of operation, sometimes called "scans," of the mass analyzers. In a first example, called a mass spectrum scan, the first mass analyzer and the collision cell transmit all ions for mass analysis into the second mass analyzer. In a second example, called a product ion scan, the ions of interest are mass-selected in the first mass analyzer and then fragmented in the collision cell. The ions formed are then mass analyzed by scanning the second mass analyzer. In a third example, called a precursor ion scan, the first mass analyzer is scanned to sequentially transmit the mass analyzed ions into the collision cell for fragmentation. The second mass analyzer mass-selects the product ion of interest for transmission to the detector. Therefore, the detector signal is the result of all precursor ions that can be fragmented into a common product ion. Other experimental formats include neutral loss scans where a constant mass difference is accounted for in the mass scans. The use of these different tandem mass spectrometry scan procedures can be advantageous when large sets of analytes are measured in a single experiment.

[0181] In view of the above, those skilled in the art recognize that different mass spectrometry methods, for example, quadrupole mass spectrometry, ion trap mass spectrometry, time-of-flight mass spectrometry and tandem mass spectrometry, can use various combinations of ion sources and mass analyzers which allows for flexibility indesigning customized detection protocols. In addition, mass spectrometers can be programmed to transmit all ions from the ion source into the mass spectrometer either sequentially or at the same time. Furthermore, a mass spectrometer can be programmed to select ions of a particular mass for transmission into the mass spectrometer while blocking other ions. The ability to precisely control the movement of ions in a mass spectrometer allows for greater options in detection protocols which can be advantageous when a large number of analytes are being analyzed.

[0182] Different mass spectrometers have different levels of resolution, that is, the ability' to resolve peaks between ions closely related in mass. The resolution is defined as R=m / delta m, where m is the ion mass and delta m is the difference in mass between two peaks in a mass spectrum. For example, a mass spectrometer with a resolution of 1000 can resolve an ion with a m / z of 100.0 from an ion with a m / z of 100.1. Those skilled in the art will therefore select a mass spectrometer having a resolution appropriate for the analyte(s) to be detected.

[0183] Mass spectrometers can resolve ions with small mass differences and measure the mass of ions with a high degree of accuracy. Therefore, analytes of similar masses can be used together in the same experiment since the mass spectrometer can differentiate the mass of even closely related molecules. The high degree of resolution and mass accuracy achieved using mass spectrometry methods allows the use of large sets of analytes because they can be distinguished from each other.

[0184] Mass spectrometry devices and general methods of their use are w ell known in the art as exemplified in McMaster, M., LC / MS A Practical User’s Guide, 2005, John Wiley & Sons, USA; and Hoffmann and Stroobant, Mass Spectrometry Principles and Applications, 2007, John Wiley & Sons, England.

[0185] According to aspects of the present disclosure, detecting the purified glycosylated proteins includes chromatography. According to aspects of the present disclosure, detecting the purified glycosylated proteins includes gel electrophoresis. According to aspects of the present disclosure, detecting the purified glycosylated proteins includes gel electrophoresis and transfer of the electrophoresed purified glycosylated proteins to a membrane.

[0186] A membrane or other solid support containing proteins to be assayed for post- translational glycoslylation is contacted with a fusion protein according to aspects of the present disclosure under binding conditions to allow for specific binding of the fusion protein with the proteins. Following removal of and unbound fusion protein, any bound fusionprotein is detected by providing a detection stimulus and detecting a signal emitted from the bound fusion protein.

[0187] One or more controls or standards can be used to detect glycosylated proteins and / or compare one or more glycosylated proteins obtained under different conditions, e.g. before and after treatment of cells with a test substance.

[0188] A test substance may be a natural or synthetic chemical compound, nucleic acid, peptide, protein, saccharide, oligosaccharide, polysaccharide, lipid, or combination of any two or more thereof. Extracts of plants which contain several characterized or uncharacterized components may be a test substance. According to aspects, the test substance is an antisense molecule, an aptamer, siRNA, shRNA, miRNA, a DNAzyme, or a ribozyme.

[0189] Embodiments of inventive compositions and methods are illustrated in the following examples. These examples are provided for illustrative purposes and are not considered limitations on the scope of inventive compositions and methods.

[0190] Examples

[0191] GlycoLight tools for quantitative O-GlcNAc assays

[0192] Cells use protein post-translational modifications (PTMs) to rapidly control protein functions as dynamic adaptations to their environments. A fundamental need for cells is to regulate metabolism under changing nutrient conditions. One nutrient-sensing PTM is the dynamic glycosylation of proteins with O-linked N-acetylglucosamine (O-GlcNAc) on serine and threonine residues. Just two proteins cycle O-GlcNAc PTMs onto and off of proteins, the O-GlcNAc transferase (OGT) and the O-GlcNAcase (OGA) (Figure 1A). Despite this simplicity7, OGT and OGA both act on 1000s of intracellular substrates, making this system difficult to study on specific proteins. Depending on the context of an O- GlcNAcylated protein, the effect of these O-GlcNAc sugars can regulate activity, interactions, stability7, and subcellular location. Therefore, O-GlcNAc PTMs perform crucial regulatory events that connect cellular nutrient levels, stressors, and signaling activities with functional implications on many cellular proteins.

[0193] A primary challenge in studying O-GlcNAcylation dynamics is that the regulation of OGT and OGA activity and substrate selectivity is not fully known. Inhibition or knockdown of either protein leads to a cell-wide, global changes in O-GlcNAc levels because of the wide substrate scope of OGT and OGA. To date, substrate-selective inhibitors are not known. Adding to this difficulty, there are very few protein- or site-specific O- GlcNAc antibodies that have been generated. Chemical biology tools have enabled a protein-specific detection of fluorescent proteins using metabolic O-GlcNAc labeling with azide sugars, intracellular click chemistry to add a second, fluorescent resonance energy transfer (FRET)-compatible fluorophore for imaging studies. However, this approach is tedious to perform and requires the use of modified proteins. This lack of direct assays for global and protein-specific O-GlcNAc quantitation adds to the difficulty in elucidating O-GlcNAc-based mechanisms of nutrient and signaling regulation in cells.

[0194] Herein is disclosed a simple, cellular lysate-based homogenous assay platform for detecting changes in either global O-GlcNAc levels or protein-specific O-GlcNAcylation directly from cells. Because O-GlcNAc is part of the response to numerous signaling, nutrient sensing, and cell stress responses, compositions and methods according to aspects of the present are useful in analysis of these and other metabolic processes.

[0195] MethodsConstruct design. Plasmid constructs encoding the following protein sequences were synthesized by Twist Biosciences. GlycoLight plasmids encoded 3 segments: 1) a glycan binding domain, specifically a modified human O-GlcNAcase 1-706 with D174N mutation, the E. coli GafD residues 23- 178,23or the mutant C. perfringens OGA D298N (cpOGA*).322) a short flexible linker of GGGGS (SEQ ID NO: 20) repeats as spacing units, and 3) a detection domain, with either a luminescent protein like NanoLuc,61a fluorescent protein like mNEONgreen,62or a FLAG epitope tag for detection with antibodies. Each construct was tagged with an N-terminal polyhistidine sequence for affinity purification and was cloned into the pET28a bacterial expression vector.MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGS (SEQ ID NO: 37) included in several exemplary GlycoLight Sequences shown herein wherein MGSS (SEQ ID NO:38) includes the start site and a short linker; HHHHHH (SEQ ID NO: 11) is a His tag (abbreviated "His” and “His6” herein in the context of constructs) used for purification; SSGLVPRGSH (SEQ ID NO: 12) is a thrombin protease cleavage site; MASMTGGQQMGR (SEQ ID NO: 13) is a T7 tag sequence; and the final GS comes from a BamHI cloning site.

[0196] GlycoLight Sequences:

[0197] His-hOGA*-linker-FlagMGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSMVQKESQATLEERESELSSNP AASAGASLEPPAAPAPGEDNPAGAGGAAVAGAAGGARRFLCGVVEGFYGRPWVME QRKELFRRLQKWELNTYLYAPKDDYKHRMFWREMYSVEEAEQLMTLISAAREYEIE FIYAISPGLDITFSNPKEVSTLKRKLDQVSQFGCRSFALLFNDIDHNMCAADKEVFSSFAHAQVSITNEIYQYLGEPETFLFCPTEYCGTFCYPNVSQSPYLRTVGEKLLPGIEVLW TGPKVVSKEIPVES1EEVSKI1KRAPV1WDNIHANDYDQKRLFLGPYKGRSTELIPRLK GVLTNPNCEFEANYVAIHTLATWYKSNMNGVRKDVVMTDSEDSTVSIQIKLENEGS DEDIETDVLYSPQMALKLALTEWLQEFGVPHQYSSRQVAHSGAKASVVDGTPLVAA PSLNATTVVTTVYQEPIMSQGAALSGEPTTLTKEEEKKQPDEEPMDMVVEKQEETD HKNDNQILSEIVEAKMAEELKPMDTDKESIAESKSPEMSMQEDCISDIAPMQTDEQT NKEQFVPGPNEKPLYTAEPVTLEDLQLLADLFYLPYEHGPKGAQMLREFQWLRANS SVVSVNCKGKDSEKIEEWRSRAAKFEEMCGLVMGMFTRLSNCANRTILYDMYSYV WDIKSIMSMVKSFVQWLGCRSHSSAQFLIGDQEPWAFRGGLAGEFQRLLPIDGANDL FFQPPGGGGSGGGGSGGGGSDYKDDDDK (SEQ ID N0:21)

[0198] His-hOGA*-linker-NanoLucMGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSMVQKESQATLEERESELSSNP AASAGASLEPPAAPAPGEDNPAGAGGAAVAGAAGGARRFLCGVVEGFYGRPWVME QRKELFRRLQKWELNTYLYAPKDDYKHRMFWREMYSVEEAEQLMTLISAAREYEIE FIYAISPGLDITFSNPKEVSTLKRKLDQVSQFGCRSFALLFNDIDHNMCAADKEVFSSF AHAQVSITNEIYQYLGEPETFLFCPTEYCGTFCYPNVSQSPYLRTVGEKLLPGIEVLW TGPKVVSKEIPVESIEEVSKIIKRAPVIWDNIHANDYDQKRLFLGPYKGRSTELIPRLK GVLTNPNCEFEANYVAIHTLATWYKSNMNGVRKDVVMTDSEDSTVSIQIKLENEGS DEDIETDVLYSPQMALKLALTEWLQEFGVPHQYSSRQVAHSGAKASVVDGTPLVAA PSLNATTVVTTVYQEPIMSQGAALSGEPTTLTKEEEKKQPDEEPMDMVVEKQEETD HKNDNQILSEIVEAKMAEELKPMDTDKESIAESKSPEMSMQEDCISDIAPMQTDEQT NKEQFVPGPNEKPLYTAEPVTLEDLQLLADLFYLPYEHGPKGAQMLREFQWLRANS SVVSVNCKGKDSEKIEEWRSRAAKFEEMCGLVMGMFTRLSNCANRTILYDMYSYV WDIKSIMSMVKSFVQWLGCRSHSSAQFLIGDQEPWAFRGGLAGEFQRLLPIDGANDL FFQPPGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSFDVFTLEDFVGDWRQTAGYN LDQVLEQGGVSSLFQNLGVSVTPIQRIVLSGENGLKIDIHVIIPYEGLSGDQMGQIEKIF KVVYPVDDHHFKVILHYGTLVIDGVTPNMIDYFGRPYEGIAVFDGKKITVTGTLWNG NKIIDERLINPDGSLLFRVTINGVTGWRLCERILATG (SEQ ID NO:22)

[0199] His-hOGA*-linker-mNEONMGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSMVQKESQATLEERESELSSNP AASAGASLEPPAAPAPGEDNPAGAGGAAVAGAAGGARRFLCGVVEGFYGRPWVME QRKELFRRLQKWELNTYLYAPKDDYKHRMFWREMYSVEEAEQLMTLISAAREYEIE FIYAISPGLDITFSNPKEVSTLKRKLDQVSQFGCRSFALLFNDIDHNMCAADKEVFSSFAHAQVSITNEIYQYLGEPETFLFCPTEYCGTFCYPNVSQSPYLRTVGEKLLPGIEVLWTGPKVVSKEIPVES1EEVSKI1KRAPV1WDNIHANDYDQKRLFLGPYKGRSTELIPRLKGVLTNPNCEFEANYVAIHTLATWYKSNMNGVRKDVVMTDSEDSTVSIQIKLENEGSDEDIETDVLYSPQMALKLALTEWLQEFGVPHQYSSRQVAHSGAKASVVDGTPLVAAPSLNATTVVTTVYQEPIMSQGAALSGEPTTLTKEEEKKQPDEEPMDMVVEKQEETDHKNDNQILSEIVEAKMAEELKPMDTDKESIAESKSPEMSMQEDCISDIAPMQTDEQTNKEQFVPGPNEKPLYTAEPVTLEDLQLLADLFYLPYEHGPKGAQMLREFQWLRANSSVVSVNCKGKDSEKIEEWRSRAAKFEEMCGLVMGMFTRLSNCANRTILYDMYSYVWDIKSIMSMVKSFVQWLGCRSHSSAQFLIGDQEPWAFRGGLAGEFQRLLPIDGANDLFFQPPGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEFVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYEELNLKSTKGDLQFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGASLTVNYRYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIISTFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQKAFTDVMGMDELYKTG (SEQID NO:23)

[0200] His-GafDshort-linker-FlagMGSSHHHFIHHSSGLVPRGSHMASMTGGQQMGRGSMAVSFIGSTENDVGPSQGSYSSTHAMDNLPFVYNTGYNIGYQNANVWRISGGFCVGLDGKVDLPVVGSLDGQSIYGLTEEVGLLIWMGDTNYSRGTAMSGNSWENVFSGWCVGNYVSTQGLSVHVRPVILKRNSSAQYSVQKTSIGSIRMRPYNGSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSEFDYKDHDGDYKDHDIDYKDDDDKTG (SEQ ID NO:24)

[0201] His-Flag-linker-CpQGA*MGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSMEFMDYKDHDGDYKDHDIDYKDDDDKTGGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSVGPKTGEENQVLVPNLNPTPENLEVVGDGFKITSS1NLVGEEEADENAVNALREFLTANN1EINSENDPNSTTLIIGEVDDDIPELDEALNGTTAENLKEEGYALVSNDGKIAIEGKDGDGTFYGVQTFKQLVKESNIPEVNITDYPTVSARGIVEGFYGTPWTHKDRLDQIKFYGENKLNTYIYAPKDDPYHREKWREPYPENEMQRMQELIDASAENKVDFVFGISPGIDIRFDGEAGEEDFNHLIAKAESLYDMGVRSFAIYWDNIQDKSAAKHAQVLNRFNEEFVKAKGDVKPLITVPTEYDTGAMVSNGQPRTTTRIFAETVDPSIEVMWTGPGVVTNEIPLSDAQLISGIYNRNMAVWWNYPVTDYFKGKLALGPMHGLDKGLNQYVDFFTVNPMEHAELSKISIHTAADYSWNMDNYDYDKAWNRAIDMLYGDLAEDMKVFANHSTRMDNKTWAKSGREDAPELRAKMDEL WNKLS SKED AS ALIEELYGEF ARMEEACNNLKANLPEVALEEC SRQLDELITLAQGDKASLDMIVAQLNEDTEAYESAKEIAQNKLNTALSSFAVISEKVAQSFIQEALS (SEQ ID NO:25)

[0202] Split variants

[0203] Split superfolder GFP (green fluorescent protein), N- and C-terminal sections:

[0204] Linker-sfGFPnGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSMSKGEELFTGVVPILVELDGDVNGHKFSVRGEGEGDATNGKLTLKFICTTGKLPVPWPTLVTTLTYGVQCFSRYPDHMKRHDFFKSAMPEGYVQERTISFKDDGTYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNFNSHNVYITADKQKNGIKANFKIRHNVEDGSVQLADHYQQNTPIGDGPVLLPDNHYLSTQSVLSKDPNEK (SEQ ID NO:26)

[0205] Linker-sfGFPcGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSRDHMVL LEFVTAAGI (SEQ IDNO:27)

[0206] Split Venus YFP (yellow fluorescent protein), N- and C-terminal sections:Linker- YFPnGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSMVSKGEELFTGVVPILVELDGDVNGHKFSVSGEGEGDATYGKLTLKLICTTGKLPVPWPTLVTTLGYGLQCFARYPDHMKQHDFFKSAMPEGYVQERTIFFKDDGNYKTRAEVKFEGDTLVNRIELKGIDFKEDGNILGHKLEYNYNSHNVYITA (SEQ ID NO:28)Linker- YFPcGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSDKQKNGIKANFKIRHNIEDGGVQLADHYQQNTPIGDGPVLLPDNHYLSYQSALSKDPNEKRDHMVLLEFVTAAGITLGMDELYK (SEQ ID NO: 29)

[0207] Split firefly luciferase, N- and C-terminal sections:Linker-FLuc-NGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSMEDAKNIKKGPAPFYPLEDGTAGEQLHKAMKRYALVPGTIAFTDAHIEVNITYAEYFEMSVRLAEAMKRYGLNTNHRIVVCSENSLQFFMPVLGALFIGVAVAPANDIYNERELLNSMNISQPTVVFVSKKGLQKILNVQKKLPIIQKIIIMDSKTDYQGFQSMYTFVTSHLPPGFNEYDFVPESFDRDKTIALIMNSSGSTGSPKGVALPHRTACVRFSHARDPIFGNQIIPDTAILSVVPFHHGFGMFTTLGYLICGFRVVLMYRFEEELFLRSLQDYKIQSALLVPTLFSFFAKSTLIDKYDLSNLHEIASGGAPLSKEVGEAVAKRFHLPGIRQGYGLTETTSAILITPEGDDKPGAVGKVVPFFEAKVVDLDTGKTLGVNQRGELCVRGPMIMSGYVNDPEATNALIDKDG (SEQ ID NO:30)Linker-FLuc-CGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSWLHSGDIAYWDEDEHFFIVDRLKSLI KYKGCQVAPAELESILLQHPNIFDAGVAGLPGDDAGELPAAVVVLEHGKTMTEKEIV DYVASQVTTAKKLRGGVVFVDEVPKGLTGKLDARKIREILIKAKKGGKSKL (SEQ ID N0:31)

[0208] GafD-linker-mNEONMGSSHHHHHHSSGLVPRGSHMASMTGGQQMGRGSMAVSFIGSTENDVGPSQGSYS STHAMDNLPFVYNTGYNIGYQNANVWRISGGFCVGLDGKVDLPVVGSLDGQSIYGL TEEVGLLIWMGDTNYSRGTAMSGNSWENVFSGWCVGNYVSTQGLSVHVRPVILKR NSSAQYSVOKTSIGSIRMRPYNGSSGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSE FVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYEELNLKSTKGDL QFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGASLTVNY RYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIISTFKWSYTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQK AFTDVMGMDELYKTG (SEQ ID NO:45)

[0209] mNEON-linker-GafDMVSKGEEDNMASLPATHELHIFGSINGVDFDMVGQGTGNPNDGYEELNLKSTKGDL QFSPWILVPHIGYGFHQYLPYPDGMSPFQAAMVDGSGYQVHRTMQFEDGASLTVNY RYTYEGSHIKGEAQVKGTGFPADGPVMTNSLTAADWCRSKKTYPNDKTIISTFKWS YTTGNGKRYRSTARTTYTFAKPMAANYLKNQPMYVFRKTELKHSKTELNFKEWQK AFTDVMGMDELYKTGGGGGSGGGGSGGGGSGGGGSGGGGSGGGGSMAVSFIGSTE NDVGPS QGS YS STH AMDNLPFVYNTGYNIGYQNANVWRIS GGFC VGLDGKVDLPV VGSLDGQSIYGLTEEVGLLIWMGDTNYSRGTAMSGNSWENVFSGWCVGNYVSTQGLSVHVRPVILKRNSSAQYSVQKTSIGSIRMRPYNGSS (SEQ ID NO:46)

[0210] The linker length and the orientation (N- and C-terminal fusions) can be varied.

[0211] Expression of GlycoLight constructs, using His-hOGA*-Flag as an example

[0212] Plasmids were transformed into E. coli (BL21) and selected on Luria broth agar plate with kanamycin (Figure 2a). A colony was selected from the overnight growth of the bacteria with the plasmid and used for protein expression. As a preliminary step to select which expression condition is best, the expression of protein was induced at room temperature and at 16 °C. Immunoblot results show’ improved expression of protein when induced with IPTG but no significant difference when protein was expressed at roomtemperature or at 16 °C (Figure 2a). The desired protein was expressed at room temperature in subsequent experiments (Figure 3).

[0213] Purification of GlycoLight constructs

[0214] Following expression in the optimized conditions, bacterial cells were lysed with bacterial protein extraction reagent (ThermoFisher #78248) to isolate the expressed proteins. The crude protein was subsequently purified using a Cobalt HisPur column (ThermoFisher #89968). Cobalt ions have a very strong affinity for His6 tagged proteins, which can be subsequently eluted using a buffer containing competing chelating agents like imidazole. Wash and elution were done twice, while a buffer containing a higher concentration of imidazole was used as the elution solvent. SDS-PAGE experiment was conducted on these purified proteins, and the result showed distinct bands that correspond to the molecular size of the desired protein (Figure 4). The interaction of the purified protein with a fluorescent antibody, DYKDDDDK (SEQ ID NO: 19) Tag Monoclonal Antibody (L5)-Alexa Fluor™ 488 (AF488 was tested). A standard western blot protocol was followed and the membrane was incubated with AF488 at 4°C overnight with rocking. The membrane was imaged with chemiluminescence, and the final signal gave the expression of the interaction between the purified protein and the antibody (Fig.6). Fluorescent bands were observed that correspond to the molecular size of His6-GlycoID2-Flag. The purified protein was subsequently dialyzed using Dialysis Cassettes (ThermoFisher Scientific Slide-A-Lyzer) to remove excess imidazole before downstream applications. The protein content was quantified using a BCA assay.

[0215] Detection of O-GlcNAc proteins in lysates with GlycoLight, using His-hOGA*- Flag as an example

[0216] A far western blot experiment was performed using purified His-hOGA*-Flag protein and anti-Flag — AlexaFluor 488 conjugated antibody as a detection probe (Figure 5). Human U2OS cells were cultured in standard conditions in 6-well plates, then harvested with 100 pL RIPA buffer (150 mM NaCl, 0.5 mM tris, 1% NP40, 0.1% SDS) and were lysed via passage through a needle (at least 10 passes). Cellular protein lysates were quantified, and 10 and 25 micrograms of total protein were loaded into wells of an SDS-PAGE gel. The lysate proteins were electrophoresed, then transferred to a PVDF western blot membrane. Membranes were blocked with 5% bovine serum albumin (BSA). A “far western blot protocol'’ was followed as shown in Figure 5. Briefly, 10 ug / mL of GlycoLight in Trisbuffered sahne / Tween 20 (TBST) was applied to the membrane and incubated for 24 hours at4 °C. After rinsing 3 times with TBST for 5 minutes / rinse, an anti-Flag secondary antibody was applied for 1 hour at room temp at the recommended dilution. Either anti-Flag-AF488 (green fluorescence) or anti-Flag-horseradish peroxidase (HRP, luminescence) was used for detection. Unbound secondary antibody was washed 3 times in TBST, then the membrane was detected for fluorescence or luminescence, accordingly (Figure 6).

[0217] His-hOGA*-Flag protein was optimized in far western blot detection modes

[0218] The protein lysates were held constant, but the amount of His-hOGA*-Flag loading was varied from 10-100 pg / mL (Figure 7). Little change was observed from 10 pg / mL to 100 pg / mL loading, suggesting that the O-GlcNAc binding is complete at 10 pg / mL loading.

[0219] Controls with low vs. high O-GlcNAc levels (overnight treatment of cells with nothing (negative control), 50 pM OSMI-4, or 10 pM Thiamet-G. Then loading 25 pg per condition and binding with 10 pg / mL GlycoLight fusion protein.

[0220] Expression and purification of GlycoLight-mNEON and GlycoLight-NLuc constructs and showing expression / purification by western blot -> green fluorescent detection or addition of the NanoLuc substrate, Furimazine.

[0221] GlycoLight fusion protein detection of O-GlcNAc proteins in cell extracts with high specificity

[0222] Mouse C2C12 cells were treated for 48 hours with or without the O-GlcNAc Transferase (OGT) inhibitor 5SGlcNHex at the specified concentrations shown in Figure 8. Mouse C2C 12 cells incubated with or without O-GlcNAc transferase inhibitors, were expected to give a specific negative signal for GlycoLight detection.

[0223] Protein extraction: The mouse C2C12 cells were lysed with cell lysis (RIP A) buffer and protein extracts were separated by size on a SDS-PAGE gel. Proteins were transferred to nitrocellulose membrane for western blotting. The membranes were then blocked with a blocking reagent (bovine serum albumin), washed, and 6.5 ug / rnL GlycoLight fusion protein mNEON-linker-GafD in TBST buffer was applied for overnight staining. Fluorescent signal on the membrane was detected using a fluorescent electronic reader.

[0224] Figure 8 shows results of assays in which the protein extracts from the treated mouse C2C12 cells were separated on SDS-PAGE membranes, then probed by O-GlcNAc antibody as positive control (left blot) or with GlycoLight fusion protein mNEON-linker- GafD (SEQ ID N0:xx, “mNEON-GafD’' in Figure 8) (right blot), validating the activity of GlycoLight fusion proteins for detection of O-GlcNAc proteins.

[0225] Split versions of these assays are performed with split yellow fluorescent protein,63split green fluorescent protein,64and split luciferase65protein pairs, such as shown herein as SEQ ID NO:26 + SEQ ID NO:27; SEQ ID NO:28 + SEQ ID NO:29; and SEQ ID NO:30 + SEQ ID NO:31.

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[0227] Item List

[0228] Item 1. A fusion protein comprising: a glycan binding component linked to a detection moiety, the glycan binding component capable of specific binding to a glycosylation post-translational modification of a target protein.

[0229] Item 2. The fusion protein of item 1, wherein the detection moiety is a reporter protein or peptide.

[0230] Item 3. The fusion protein of item 2, wherein the detection moiety is detectable by chemiluminescence, fluorescence, colorimetric reactions, antibody binding, inducible markers, and / or ligand binding assays when expressed.

[0231] Item 4. The fusion protein of item 2 or item 3, wherein the detection moiety is selected from the group consisting of: green fluorescent protein (GFP); enhanced greenfluorescent protein (eGFP); yellow fluorescent protein (YFP); enhanced yellow fluorescent protein (eYFP); hfYFP; mhYFP; LSSA12; LSSmGFP; cyan fluorescent protein (CFP); enhanced cyan fluorescent protein (eCFP); blue fluorescent protein (BFP); enhanced blue fluorescent protein (eBFP); MmGFP; dsRed; luciferase or a variant of any thereof.

[0232] Item 5. The fusion protein of item 4, wherein the detection moiety is selected from the group consisting of: Renilla luciferase variant Rluc8_S257G, Renilla luciferase variant Rluc8.6, beta-galactosidase (lacZ), and an epitope tag.

[0233] Item 6. The fusion protein of item 5, wherein the epitope tag is selected from the group consisting of: a FLAG tag, a human influenza tag, and a Myc tag.

[0234] Item 7. The fusion protein of item 1, wherein the detection moiety is an antigen for antibody-based detection.

[0235] Item 8. The fusion protein of item 1, wherein the detection moiety is capable of emitting fluorescent and / or luminescent light when bound to a cognate detection moiety, the cognate detection moiety bound to an antibody which specifically recognizes the target protein.

[0236] Item 9. The fusion protein of item 8, wherein the detection moiety / cognate detection moiety pair is split superfolder GFP (green fluorescent protein), N- and C-terminal sections; split Venus YFP (yellow fluorescent protein), N- and C-terminal sections; or split firefly luciferase, N- and C-terminal sections.

[0237] Item 10. The fusion protein of any of items 1 to 9, wherein the glycan binding component is selected from the group consisting of: an enzy me, a lectin, a collectin, a ficolin, a C-reactive protein, and a carbohydrate-binding domain of any thereof.

[0238] Item 11. The fusion protein of any of items 1 to 10, wherein the glycan binding component is selected from the group consisting of: an aptamer, an antibody, and an antigenbinding fragment of an antibody.

[0239] Item 12. The fusion protein of any of items 1 to 11, wherein the glycan binding component is a mutant O-GlcNAcase enzyme derived from a member of the GH84 family of glycosylhydrolases and lacking enzymatic glycosylhydrolase activity.

[0240] Item 13. The fusion protein of any of items 1 to 12, wherein the mutant O- GlcNAcase enzyme derived from a member of the GH84 family of glycosylhydrolases and lacking enzymatic glycosylhydrolase activity is a mutant human O-GlcNAcase (hOGA) enzyme, a glycan specific binding fragment thereof, or a variant of either thereof.

[0241] Item 14. The fusion protein of any of items 1 to 13, wherein the mutant human O- GlcNAcase (hOGA) enzyme includes the amino acid sequence selected from the group consisting of: SEQ ID NO:4, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO:44, and a variant of any thereof.

[0242] Item 15. The fusion protein of any of items 1 to 10, wherein the glycan binding component is GafD lectin.

[0243] Item 16. The fusion protein of any of items 1 to 1 1, wherein the glycan binding component is GafD lectin of SEQ ID NO: 1, SEQ ID NO:2, or a variant of either thereof.

[0244] Item 17. The fusion protein of any one of items 1 to 16, wherein the glycan binding component has a C-terminus and an N-terminus, the detection moiety is a protein having a C-terminus and an N-terminus, and the C-terminus of the glycan binding component is linked to the N-terminus of the detection moiety or the N-terminus of the glycan binding component is linked to the C-terminus of the detection moiety.

[0245] Item 18. The fusion protein of any one of items 1 to 17, wherein the glycan binding component is linked to the detection moiety by a linker disposed between the glycan binding component and the detection moiety.

[0246] Item 19. The fusion protein of any one of items 1 to 18, further comprising a localization signal peptide.

[0247] Item 20. The fusion protein of any one of items 1 to 19, further comprising a localization signal peptide capable of promoting localization of the fusion protein to a subcellular compartment selected from the group consisting of: nucleus, cytosol, mitochondria, endoplasmic reticulum, and plasma membrane.

[0248] Item 21. The fusion protein of any one of items 1 to 20, further comprising an exogenous detectable tag.

[0249] Item 22. A method of detecting glycosylated protein, comprising: contacting a cell lysate or purified fraction thereof containing one or more proteins which are putatively glycosylated by post-translational modification with the fusion protein according to any one of items 1 to 21 under binding conditions, whereby the fusion protein specifically binds to a glycosylation post-translational modification of the one or more proteins of the cell, producing a complex of the fusion protein specifically bound to the glycosylation post- translational modification of the one or more proteins; providing a detection stimulus to produce a signal from the detection moiety of the fusion protein; and detecting the signal of the detection moiety, thereby detecting a glycosylated protein bound to the fusion protein.

[0250] Item 23. The method of item 1, wherein the detection moiety is a fluorescent protein and the signal is fluorescent light.

[0251] Item 24. The method of item 1, wherein the detection moiety is a luciferase and the signal is luminescent light.

[0252] Item 25. An expression construct comprising a nucleic acid encoding the fusion protein according to any one of items 1 to 21.

[0253] Item 26. A host cell comprising the expression construct of item 25.

[0254] Item 27. A composition substantially as shown or described herein.

[0255] Item 28. A method of detecting proteins proximal to a target protein substantially as shown or described herein.

[0256] Item 29. A method of detecting glycosylated proteins substantially as shown or described herein.

[0257] Any patents or publications mentioned in this specification are incorporated herein by reference to the same extent as if each individual publication is specifically and individually indicated to be incorporated by reference.

[0258] The compositions and methods described herein are presently representative of preferred embodiments, exemplary, and not intended as limitations on the scope of the invention. Changes therein and other uses will occur to those skilled in the art. Such changes and other uses can be made without departing from the scope of the invention as set forth in the claims.

Claims

CLAIMS1. A fusion protein comprising: a glycan binding component linked to a detection moiety, the glycan binding component capable of specific binding to a glycosylation post- translational modification of a target protein.

2. The fusion protein of claim 1, wherein the detection moiety is a reporter protein or peptide.

3. The fusion protein of claim 2, wherein the detection moiety is detectable by chemiluminescence, fluorescence, colorimetric reactions, antibody binding, inducible markers, and / or ligand binding assays when expressed.

4. The fusion protein of claim 2 or claim 3, wherein the detection moiety is a fluorescent protein.

5. The fusion protein of claim 4, wherein the detection moiety is a luciferase.

6. The fusion protein of claim 5, wherein the detection moiety is an epitope tag.

7. The fusion protein of claim 1, wherein the detection moiety is an antigen for antibody-based detection.

8. The fusion protein of claim 1, wherein the detection moiety is capable of emitting fluorescent and / or luminescent light when bound to a cognate detection moiety, the cognate detection moiety bound to an antibody which specifically recognizes the target protein.

9. The fusion protein of claim 8, wherein the detection moiety / cognate detection moiety pair is split superfolder GFP (green fluorescent protein), N- and C-terminal sections; split Venus YFP (yellow fluorescent protein), N- and C-terminal sections; or split firefly luciferase, N- and C-terminal sections.

10. The fusion protein of any of claims 1 to 9, wherein the glycan binding component is selected from the group consisting of: an enzyme, a lectin, a collectin, a ficolin, a C-reactive protein, and a carbohydrate-binding domain of any thereof.

11. The fusion protein of any of claims 1 to 10, wherein the glycan binding component is selected from the group consisting of: an aptamer, an antibody, and an antigenbinding fragment of an antibody.

12. The fusion protein of any of claims 1 to 11, wherein the glycan binding component is a mutant O-GlcNAcase enzyme derived from a member of the GH84 family of glycosylhydrolases and lacking enzymatic glycosylhydrolase activity.

13. The fusion protein of any of claims 1 to 12, wherein the mutant O-GlcNAcase enzyme derived from a member of the GH84 family of glycosylhydrolases and lacking enzymatic glycosylhydrolase activity is a mutant human O-GlcNAcase (hOGA) enzyme, a glycan specific binding fragment thereof, or a variant of either thereof.

14. The fusion protein of any of claims 1 to 13, wherein the mutant human O- GlcNAcase (hOGA) enzyme includes the amino acid sequence selected from the group consisting of:SEQ ID NO:4, SEQ ID NO:40, SEQ ID NO:41, SEQ ID NO:42, SEQ ID NO:43, SEQ ID NO: 44, and a variant of any thereof.

15. The fusion protein of any of claims 1 to 10, wherein the glycan binding component is GafD lectin.

16. The fusion protein of any of claims 1 to 11, wherein the glycan binding component is GafD lectin of SEQ ID NO: 1, SEQ ID NO:2, or a variant of either thereof.

17. The fusion protein of any one of claims 1 to 16, wherein the glycan binding component has a C-terminus and an N-terminus, the detection moiety is a protein having a C-terminus and an N-terminus, and the C-terminus of the glycan binding component is linked to the N-terminus of the detection moiety or the N-terminus of the glycan binding component is linked to the C-terminus of the detection moiety.

18. The fusion protein of any one of claims 1 to 17, wherein the glycan binding component is linked to the detection moiety by a linker disposed between the glycan binding component and the detection moiety.

19. The fusion protein of any one of claims 1 to 18, further comprising a localization signal peptide.

20. The fusion protein of any one of claims 1 to 19, further comprising a localization signal peptide capable of promoting localization of the fusion protein to a subcellular compartment selected from the group consisting of nucleus, cytosol, mitochondria, endoplasmic reticulum, and plasma membrane.

21. The fusion protein of any one of claims 1 to 20, further comprising an exogenous detectable tag.

22. A method of detecting glycosylated protein, comprising: contacting a cell lysate or purified fraction thereof containing one or more proteins which are putatively glycosylated by post-translational modification with the fusion protein according to any one of claims 1 to 21 under binding conditions, whereby the fusion protein specifically binds to a glycosylation post-translational modification of the one or more proteins of the cell, producing a complex of the fusion protein specifically bound to the glycosylation post-translational modification of the one or more proteins; providing a detection stimulus to produce a signal from the detection moiety of the fusion protein; and detecting the signal of the detection moiety, thereby detecting a glycosylated protein bound to the fusion protein.

23. The method of claim 1, wherein the detection moiety is a fluorescent protein and the signal is fluorescent light.

24. The method of claim 1 , wherein the detection moiety is a luciferase and the signal is luminescent light.

25. An expression construct comprising a nucleic acid encoding the fusion protein according to any one of claims 1 to 21.

26. A host cell comprising the expression construct of claim 25.

27. A composition substantially as shown or described herein.

28. A method of detecting proteins proximal to a target protein substantially as shown or described herein.

29. A method of detecting glycosylated proteins substantially as shown or described herein.

Images