US 20050186645 A1
The invention relates generally the structural analysis of glycomolecule-containing macromolecules, such as those that occur either attached to proteins (proteoglycans, glycoproteins), lipids, or as free saccharides.
1. A method for determining a glycomolecule fingerprint for a glycomolecule, the method comprising:
providing a glycomolecule, wherein said glycomolecule has been modified by desialylation or treatment with N-Glycosidase F (PNGaseF);
adding said glycomolecule to a substrate comprising a plurality of saccharide-binding agents;
detecting glycomolecule bound to saccharide-binding agents in the plurality; and
obtaining a fingerprint for the glycomolecule based on the binding of the glycomolecule to the saccharide-binding agents.
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39. A method for determining a glycomolecule fingerprint for a glycomolecule, the method comprising:
adding a glycomolecule to a substrate comprising a plurality of saccharide-binding agents;
detecting glycomolecule bound to saccharide-binding agents in the plurality; and
obtaining a fingerprint for the glycomolecule based on the binding of the glycomolecule to the saccharide-binding agents.
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56. A kit for analyzing a glycomolecule, the kit comprising
a glycomolecule modification agent selected from the group consisting of a desialidase and a PNGase F; and
a labeling agent for labeling a glycomolecule.
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an antibody specifically binds to a glycoprotein; and
a labeling agent for labeling a glycomolecule.
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This application claims the benefit of, and priority to, U.S. Ser. No. 60/531,578, filed Dec. 18, 2003. The contents of this application are incorporated herein by reference in their entirety.
The invention relates generally to the structural analysis of glycomolecules, which are molecules that contain carbohydrates and include carbohydrates attached to proteins (proteoglycans, glycoproteins), to lipids, or carbohydrates present as free polysaccharides.
Mammalian glycoprotein oligosaccharides are commonly built from a limited number of monosaccharides. Nevertheless, structural diversity is vast, mainly due to complex branching patterns. Glycosylation sites on glycoproteins commonly display microheterogeneity in that they can be fully or partially occupied by structurally diverse oligosaccharides. Consequently, a glycoprotein is not typically isolated as a single structural entity, but rather as a set of glycosylation variants known as glycoforms.
There is evidence that both the in vivo and in vitro properties of glycoproteins are affected by changes in occupancy and/or the precise oligosaccharide attached to a particular site. Distinct biological properties have been correlated with the presence of particular glycoforms.
A method for determining the composition and sequence of polysaccharides in a carbohydrate-containing molecule (“glycomolecule”) has been described (see, e.g., WO00/668688, WO01/84147, WO02/37106, and WO02/44714). In this method, termed UC-FINGERPRINT™ analysis (also known as GMID™ analysis), a carbohydrate-containing molecule is added to a substrate containing an array of saccharide-binding agents (typically antibodies or lectins). Saccharide-binding agents bound to the glycomolecule are identified, and the binding information is used to obtain composition and sequence information of the monosaccharide subunits in the polysaccharide.
The invention is based in part on the discovery of methods that facilitate the preparation of glycomolecules for subsequent analysis in UC-FINGERPRINT™ technology. The analysis provides glycan composition and sequence information, which is often referred to as a fingerprint of the glycomolecule.
Among the advantages of the method is simplified sample preparation and processing. The methods described herein eliminate the need for multiple pretreatment, treatment, purification, and buffer changing steps. In addition, the methods facilitate access to glycomolecules that are otherwise difficult to analyze. These glycans include, e.g., glycans in inter-subunit clefts or intra-subunit clefts of glycoproteins.
In one aspect, the invention provides a method for determining a glycomolecule fingerprint for a glycomolecule. In some embodiments, the glycomolecule is one whose native glycan structure has been modified. The method includes adding the glycomolecule to a substrate that includes a plurality of saccharide-binding agents. Glycomolecules bound to saccharide-binding agents on the substrate are detected. A fingerprint is obtained for the glycomolecule based on the binding of the glycomolecule to the saccharide-binding agents.
In some embodiments, the glycomolecule has been modified by desialylation. The extent of the desialylation can be modulated, so that in some embodiments, substantially all of the sialic acid residues have been removed from the glycomolecule. In other embodiments, less than all of the sialic acids have been removed from the glycomolecule.
A suitable method for desialylating the glycomolecule is by reacting the glycomolecule with a sialidase. The glycomolecule can optionally be reacted with the sialidase in the presence of a protease inhibitor.
The glycomolecule can alternatively, or in addition, be modified by treatment with PNGaseF. The extent of treatment with PNGaseF can be modulated, so that in some embodiments, substantially all of the bonds between the innermost GlcNAc and asparagine residues of high mannose, hybrid and complex oligosaccharides of the glycoprotein have been cleaved. In other embodiments, less than all of the N-Acetyl Glucosamine acid residues have been cleaved.
In some embodiments, the method includes reacting the glycomolecule with a reducing agent and, preferably, an alkylating agent prior to obtaining the fingerprint. Examples of suitable reducing agents include mercaptoethanol, dithiothreitol, and mercaptethylamine. Examples of suitable alkylating agents iodoacetamide and iodoacetic acid.
In some embodiments, all steps of the method are performed in a single container.
In some embodiments, the glycomolecule is detected with a label associated with the glycomolecule. Examples of suitable labels include, e.g., a fluorescent label. The fluorescent label can be, e.g., fluorescein isothiocyanate (FITC), rhodamine, Texas Red, and Cy5.
The label can be added to the glycomolecule prior to, after, or while adding the glycomolecule to the substrate.
In some embodiments, the label is associated directly with the glycomolecule. In other embodiments, the label is associated with a second saccharide-binding agent that binds specifically to the glycomolecule. The second saccharide-binding agent can be, e.g., a lectin or an antibody. In some embodiments, the label is associated with an agent (such as an antibody) that binds specifically to a non-carbohydrate region of the glycomolecule.
In some embodiments, the method further includes purifying the glycomolecule prior to adding the glycomolecule to the substrate. The purification can be, e.g., by column chromatography ordialysis with a molecular cut off of a defined mass, e.g., 5000 kD The glycomolecule can be any saccharide-containing molecule. Examples include glycoprotein, polysaccharide, or glycolipid.
In some embodiments, the glycoprotein is obtained from a cell culture medium. In some embodiments, the glycoprotein is purified and/or concentrated prior to being used. In other embodiments the glycoprotein is obtained from the medium and used without purification or concentration.
In some embodiments, the method includes treating the glycomolecule with a detergent prior to obtaining the fingerprint. The detergent can be, e.g., a non-ionic detergent or anionic detergent. Examples of a suitable detergent include, e.g. sodium docecyl sulfate (SDS), Triton, and Tween80.
Examples of suitable glycoproteins include, eg., immunuglobuin molecules (including IgA, IgD, or IgG, or IgM isotypes) or fragments of immunoglobulin molecules. For example, the fragment can include an Fc region of an immunoglubulin.
In another aspect, the invention provides a kit that includes a glycomolecule modification agent selected that is a desialidase and/or a PNGase F, a labeling agent for labeling a glycomolecule, a container and, optionally instructions for using the kit to modify the glycomolecule The directions can be provided on a kit label or as a kit insert, which describe how to manipulate a glycomolecule using the methods described herein.
The kit may additionally contain a plurality of saccharide-binding agents, a reducing agent, a detergent and an alkylating agent. The labeling agent may bind directly to the glycomolecule. Alternatively, the labeling agent is present with, or capable of being associated with, a second saccharide-binding agent that binds to the glycomolecule. The second saccharide-binding agent can be, e.g., a lectin or an antibody.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. These include, but are not limited to, WO00/68688, WO01/84147, WO02/37106, and WO02/44714. In the case of conflict, the present Specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
Other features and advantages of the invention will be apparent from the following detailed description and claims.
The invention provides methods for determining the glycan composition and sequence of carbohydrate-containing molecules. In some embodiments, the methods include modifying a glycomolecule. These methods enhance the information obtained when the carbohydrate content of the carbohydrate-containing molecule is analyzed.
The methods for analyzing the glycomolecule are particularly suited for analyzing molecules in the UC-FINGERPRINT™ method, which is also referred to as a glycomolecule identification (GMID™) method. In this method, information about the carbohydrate content of a glycomolecule is obtained by adding a glycomolecule to a substrate to which is attached one or more saccharide-binding agents (also referred to herein as first saccharide-binding agents). The first saccharide-binding agents that have bound the glycomolecule are identified, and the resulting binding information is used to generate a fingerprint of the glycomolecule.
For example, one way to perform the method is with a set of 20-30 lectins printed on a membrane-coated glass slide in replicates of 4-8. A sample of intact glycoprotein is applied to the array, and its binding pattern is detected by either direct labeling of the glycoprotein using FITC, or by using an FITC-labeled probe that is directed at either the protein moiety—an antibody for example, or a carbohydrate moiety—a lectin. The resulting fingerprints are highly characteristic of the glycosylation pattern of the sample. The large number of lectins, each with its specific recognition pattern, ensures high sensitivity of the fingerprint to changes in the glycosylation pattern. Other fluorescent labels such as Cy3 Cy5 can also be used. In addition, labeling can be effected using biotin-avidin systems known in the art.
Modifications of Glycomolecules Prior to Fingerprinting Analysis
It has now been unexpectedly found that the modifications describe herein enhance the type and amount of information that can be obtained in UC-FINGERPRINT™ analysis. One modification is removing some or all of the sialic acid residues from glycomolecule, a process know as desialylation, prior to UC-FINGERPRINT™ analysis. Sialic acid residues are negatively charged residues that cap carbohydrate moieties attached to many sites on glycoproteins. Sialic acid residues can be removed using the enzyme sialidase, which is also known as neuraminidase. The extent of desialylation can be controlled by modulating the extent of digestion of the glycomolecule with the sialidase. In addition to sialidase, any other method that reduces the sialic acid content of a glycomolecule (including a glycoprotein) can be used.
Another modification that has been found to enhance the information revealed in a UC-FINGERPRINT™ analysis is digestion with PNGaseF, which is also known as N-glycosidase F. PNGase F cleaves N-linked glycoproteins between the innermost GlcNAc and asparagine residues of high mannose, hybrid and complex oligosaccharides. O-linked glycan residues (such as N-GlcNAc and O-Fuc) are not affected and are available for subsequent analysis. This modification is particularly suitable glycoproteins for which O-linked glycan composition is of interest. An example of such a protein is erythropoietin (EPO).
In addition to PNGaseF, a glycomolecule can be modified with other glycosidase-modifying anzymes known in the art, e.g., Endo F2, Endo F3, Endo H.
A further modification for preparing a glycomolecule for UC-FINGERPRINT™ analysis is to expose the glycomolecule to a reducing agent and, alkylating agent. Exposure to a reducing agent can disrupt intra- and inter-chain disulfide bonds and make available for analysis glycans that would not otherwise be detected. Suitable reducing agents include, e.g., β-mercaptoethanol, dithiothreitol, and mercaptethylamine. Alkylating agents include, e.g., iodoacetamide and iodoacetic acid.
A still further modification that has been found to enhance the information obtained by UC-FINGERPRINT™ analysis is to subject the glycomolecule to denaturing conditions. This modification is suitable for glycomolecules containing glycans that are hiden or obscured because of, e.g., protein aggregation. For example, a glycoprotein can be heated in the presence of a detergent prior to performing UC-FINGERPRINT™ analysis. The optimal conditions (including, e.g., selection of detergent, temperature, buffer composition and concentration, and pH) can be chosen based on the glycomolecule of interest, the first saccharide-binding agents that are immobilized on the substrate, and/or the labaeling scheme that is used to detect glycomolecules bound to the saccharide-binding agents on the substrate.
For example, to detect glycans associated with Fc subunits for IgG molecules, the glycoprotein can be treated in 0.01 to 075% SDS, more preferably 0.04-0.06% SDS, and most preferably about 0.05% SDS. The sample is in addition boiled for 5-15 minutes at 95-100° C., e.g., at 10 minutes at 100° C., or the equivalent.
The above-described modifications can be performed singly or in any desired combination. For example, a glycoprotein can be treated with salidase, and then subjected to reducing conditions prior to submtiting the modified glycoprotein to UC-FINGERPRINT™ analysis.
The methods described above facilitate identification of carbohydrate information for glycoproteins that contain glycans that are obscured by sialic acid residues, and/or obscured because the glycoproteins otherwise exist as present as multimers and/or are present in inter-subunit or intra-subunit clefts.
It has been further unexpectedly discovered that many of the manipulations described herein—modification, labeling, and reducing, for example—can be performed without the need for changing the buffer. This eliminates the need to subject a glycomolecule of interest to cumbersome, multi-step processing treatments. In addition, the method can be performed using less material, with less material loss and in a shorter time.
If desired, the modifications described above can be performed directly on glycomolecules (including glycoproteins) isolated directly from a culture medium.
Determining Fingerprints of Modified Glycomolecules
A “glycomolecule fingerprint” refers to the information provided by the amount of binding detected to one or more saccharide-binding agents for a glycomolecule of interest. The fingerprint can be expressed graphically by presenting as a histogram the relative binding intensities for multiple saccharide-binding agents. In some embodiments, the analysis of the glycomolecule includes determining a map of the glycomolecule. “Mapping”—means defining a sequential order of certain predefined patterns on the polysaccharide chain. The predefined patterns can correspond to of location(s) on the glycomolecule that bind to a saccharide-binding agent, and/or are substrates for a glycoside-cleaving enzyme.
A glycomolecule can include any molecule that includes a saccharide moiety. For example, a glycomolecule can includes carbohydrate-containing proteins (glycoproteins) or glycolipids, and free polysaccharides. Glycoproetins include, e.g., fetuin, α1 Acid GP, and tPA.
The modified glycomolecules are added to a substrate that includes one or more first saccharide-binding agents. The first saccharide-binding agent may be immobilized to a substrate using any art-recognized method. For example, immobilization may utilize functional groups of the protein, such as amino, carboxy, hydroxyl, or thiol groups. For instance, a glass support may be functionalized with an epode group by reaction with silane. The epode group reacts with amino groups such as the free ε-amino groups of lysine residues. Another mechanism consists in covering a surface with electrophilic materials such as gold. As such materials form stable conjugates with thiol groups, a protein may be linked to such materials directly by free thiol groups of cysteine residues. Alternatively, thiol groups may be introduced into the protein by conventional chemistry, or by reaction with a molecule that contains one or more thiol groups and a group reacting with free amino groups, such as the N-hydroxyl succinimidyl ester of cysteine. Also thiol-cleavable cross-linkers, such as dithiobis(succinimidyl propionate) may be reacted with amino groups of a protein. A reduction with sulfhydryl agent will then expose free thiol groups of the cross-linker.
For some applications, it is preferable to design a substrate that contains a plurality of saccharide-binding agents known to bind, or suspected of binding, to a particular glycomolecule of interest. For example, heparin, heparin sulfate, or fragments (such as those produced by heparanase digestion), as well as variant forms of these polysaccharides can be screened for their ability to bind to one or more proteins such as, e.g., αFGF, βFGF, PDGF, VEGF, VEGF-R, HGF, EGF, TGF-beta, MCP-1, -2 and -3, IL-1, -2, -3, -6, -7. -8, -10, and -12, annexin IV, V, and VI, MIP-1 alpha, MIP-1 beta, ecotaxin, thrombospondin, PF-4, IP-10, interferon alpha, interferon gamma, selectin L and selectin P, antithrombin, plasminogen activator, vitronectin, CD44, SOD, lipoprotein lipase, ApoE, fibronectin, and laminin. These putative agents can be attached to a surface (i.e., can be first saccharide binding agents).
The substrate can be conveniently provided on a membrane disposed on a supporting surface. For example, the saccharide-binding agents can be provided on a nitrocellulose filter on a glass slide. Alternatively, the substrate can be a microsphere, or bead. In various embodiments, one or more distinct saccharide-binding agents are provided on a single microsphere.
A suitable saccharide-binding agent is any agent that binds specifically to a carbohydrate-portion of a glycomolecule. Suitable saccharide-binding agents include, e.g., lectins, antibodies that recognize carbohydrate-containing epitopes, and carbohydrate-modifying enzymes, such as glycosidases.
Lectins are proteins isolated from plants that bind saccharides. For the purpose of this application, the term “lectin” also encompasses saccharide-binding proteins from animal species (e. g.“mammalian lectins”). Examples of lectins include lectins isolated from the following plants: Conavalia ensiformis, Anguilla anguilla, Tritium vulgaris, Datura stramonium, Galnthus nivalis, Maackia amurensis, Arachis hypogaea, Sambucus nigra, Erythtina cristagalli, Sambucis nigra, Erythrina cristagalli, Lens culinaris, Glycine max, Phaseolus vulgaris Allomyrina dichotoma, Dolichos biflorus, Lotus tetragonolobus, Ulex europaeus, and Ricinus commurcis. Other biologically active compounds such as cytokines, chemokines and growth factors also bind glycomolecules, and hence, for the purposes of the present invention are considered to be lectins.
Examples of glycosidases include a-Galactosidase, (3-Galactosidase, N-acetylhexosaminidase, α:-mannosidase, β-mannosidase, and α-Fucosidase.
Detecting Bound Glycomolecules
Glycomolecules that have bound to a saccharide-binding agent on a substrate can be detected using any method that will result in detection of the bound glycomolecule. For example, the glycomolecule can be directly labeled before, during, or after it is added to the substrate. Examples of direct labeling include, e.g., FITC labeling.
Alternatively, the bound glycomolecule can be detected with a label associated with an agent that specifically recognizes the bound glycomolecule. The agent can recognize a carbohydrate-containing region of the molecule. When the agent has this specificity it is referred to as a second saccharide-binding agent. The second saccharide-binding agent can be an antibody or a lectin, including the antibodies and lectins described above.
In some embodiments, the agent recognizes a non-carbohydrate portion of the glycomolecule. An example of such an agent is an antibody that recognizes a peptide epitope in a glycoprotein.
If desired, bound glycomolecules can be detected using a series of agents. For example, desialo darbepoetin alfa (ARASNEP™) bound to glycans can be detected using anti-human EPO monoclonal mouse antibody followed by an anti-mouse IgG-FITC-labeled antibody.
The label can be any label that is detected, or is capable of being detected. Examples of suitable labels include, e.g., chromogenic label, a radiolabel, a fluorescent label, and a biotinylated label. Thus, the label can be, e.g., colored lectins, fluorescent lectins, biotin-labeled lectins, fluorescent labels, fluorescent antibodies, biotin-labeled antibodies, and enzyme-labeled antibodies. In preferred embodiments, the label is a chromogenic label. The term “chromogenic binding agent” includes all agents that bind to saccharides and which have a distinct color or otherwise detectable marker, such that following binding to a saccharide, the saccharide acquires the color or other marker. In addition to chemical structures having intrinsic, readily-observable colors in the visible range, other markers used include fluorescent groups, biotin tags, enzymes (that may be used in a reaction that results in the formation of a colored product), magnetic and isotopic markers, and so on. The foregoing list of detectable markers is for illustrative purposes only, and is in no way intended to be limiting or exhaustive. In a similar vein, the term “color” as used herein (e.g. in the context of step (e) of the above described method) also includes any detectable marker.
The label may be attached to the agent using methods known in the art. Labels include any detectable group attached to the glycomolecule, or detection agent that does not interfere with its function. Labels may be enzymes, such as peroxidase and phosphatase. In principle, also enzymes such as glucose oxidase and β-galactosidase could be used. It must then be taken into account that the saccharide may be modified if it contains the monosaccharide units that react with such enzymes. Further labels that may be used include fluorescent labels, such as Fluorescein, Texas Red, Lucifer Yellow, Rhodamine, Nile-red, tetramethyl-rhodamine-5-isothiocyanate, 1,6-diphenyl-1,3,5-hexatriene, cis-Parinaric acid, Phycoerythrin, Allophycocyanin, 4′,6-diamidino-2-phenylindole (DAPI), Hoechst 33258, 2-aminobenzamide, and the like. Further labels include electron dense metals, such as gold, ligands, haptens, such as biotin, radioactive labels.
The agent can additionally be detected using enzymatic labels. The detection of enzymatic labels is well known in the art. Examples include, e.g., ELISA and other techniques where enzymatic detection is routinely used. The enzymes are available commercially, e.g., from companies such as Pierce.
In some embodiments, the label is detected using fluorescent labels. Fluorescent labels require an excitation at a certain wavelength and detection at a different wavelength. The methods for fluorescent detection are well known in the art and have been published in many articles and textbooks. A selection of publications on this topic can be found at p. O-124 to O-126 in the 1994 catalog of Pierce. Fluorescent labels are commercially available from Companies such as SIGMA, or the above-noted Pierce catalog.
The agent may itself contain a carbohydrate moiety and/or protein. Coupling labels to proteins and sugars are techniques well known in the art. For instance, commercial kits for labeling saccharides with fluorescent or radioactive labels are available from Oxford Glycosystems, Abingdon, UK, and ProZyme, San Leandro, Calif. USA). Reagents and instructions for their use for labeling proteins are available from the above-noted Pierce catalog.
Coupling is usually carried out by using functional groups, such as hydroxyl, aldehyde, keto, amino, sulfhydryl, carboxylic acid, or the like groups. A number of labels, such as fluorescent labels, are commercially available that react with these groups. In addition, bifunctional cross-linkers that react with the label on one side and with the protein or saccharide on the other may be employed. The use of cross-linkers may be advantageous in order to avoid loss of function of the protein or saccharide.
While the labeling has been described with respect to modified glycomolecules, the invention also encompasses these labeling methods when used to perform UC-FINGERPRINT™ analysis of unmodified glycomolecules.
Obtaining a Fingerprint
The intensity of label associated with bound glycomolecules can be detected using methods known in the art. Some detection methods are described in WO 93/22678. Particularly suitable for the method of the present invention is the CCD detector method. This method may be used in combination with labels that absorb light at certain frequencies, and so block the path of a test light source to the VLSI surface, so that the CCD sensors detect a diminished light quantity in the area where the labeled agent has bound. The method may also be used with fluorescent labels, making use of the fact that such labels absorb light at the excitation frequency. Alternatively, the CCD sensors may be used to detect the emission of the fluorescent label, after excitation. Separation of the emission signal from the excitation light may be achieved either by using sensors with different sensitivities for the different wavelengths, or by temporal resolution, or a combination of both.
The fingerprint is preferably determined by correcting for the glucose concentration of the media from which the glycomolecule is taken.
The acquired binding information can be used directly, e.g., following visual inspection of the binding pattern. Alternatively, the binding information can be stored, e.g., as a photograph or digitized image. If desired, the binding information can be stored in a database. Interpretation of binding information is also discussed in, e.g., WO00/68688, WO01/84147, WO02/37106, and WO02/44714, the contents of which are incorporated by reference herein in their entirety.
The invention additionally provides kits for modifying glycomolecules and then subjecting them to UC-FINGERPRINT™ analysis. The contents of a kit can include one or more of a modification agent(s), a labeling reagent for detecting a glycomolecule that is bound to a saccharide-binding agent, and, if desired, a substrate that contains or is capable of attaching to one or more saccharide-binding agents. The substrate can be, e.g., a microsphere.
Each kit preferably includes saccharide-binding agent or agents. The reagent is preferably supplied in a solid form or liquid buffer that is suitable for inventory storage, and later for exchange or addition into the reaction medium when the test is performed. Suitable packaging is provided. The kit may optionally provide additional components that are useful in the procedure. These optional components include buffers, capture reagents, developing reagents, labels, reacting surfaces, means for detection, control samples, instructions, and interpretive information.
The kit may optionally include a detectable second saccharide-binding agent and, if desired, reagents of detecting the second binding agent. The plurality of first saccharide-binding agents is preferably attached at predetermined location on the substrate and a detectable second saccharide-binding agent. In other embodiments, the kit is provided with a substrate and first saccharide-binding agents that can be attached to the substrate, as well as second saccharide-binding agents.
If desired, a slide holder as shown in
The invention will be further illustrated in the following non-limiting examples.
The glycomolecule fingerprint of human milk lactoferrin (hmLF) before and after disialyzation was examined. hmLF is a glycoprotein with a relatively simple glycosylation structure (Spik et al. Eur J Biochem. 1982;121(2):413-9). The hmLF structure includes two glycosylation sites that are occupied by any of 5 major glycans, resulting in 25 possible glycoforms. All of these glycans are of the complex bi-antennary type, containing a core fucose and differing in their levels of sialylation and the variable presence of antennary fucose (
The fingerprints (
10 mg/ml of hmLactoferrin was desialylated using 50 mU/ml Neuraminidase from Arthrobacter ureafaciens (Roche cat #269611). Galactose was removed using 20 mU/ml beta 1,4-Galactosidase from Streptococcus pneumoniae (Calbiochem, cat #345806). N-acetylglucosamine was removed with 20 U/ml of beta 1-2,3,4,6-N-Acetylglucosaminidase from Streptococcus pneumoniae (Calbiochem cat #110116). All cleavage reactions were performed in the present of 50 mM phosphate buffer at pH 6 containing protease inhibitors (PI Cocktail Set I -Calbiochem cat #539131) for 19 hours at 37° C.
The fingerprint of the native sample is virtually identical to that observed in the experiments of
The galactose-recognizing lectins demonstrate a more complex behavior. The signals from these lectins increase differentially, demonstrating the differential sensitivity of these lectins to the presence of sialic acid: lectin 11 is able to bind the non-sialylated antenna of a mono-sialylated glycan and thus the small increase in signal from this lectin following desialylation indicates a low level of di-sialylated structures in the native sample. In contrast, the affinity of lectin 15 towards mono-sialylated glycans is significantly decreased in comparison to fully desialylated glycans, and thus the large increase in the signal of this lectin indicates that the native protein contains a low level of neutral glycans. In addition, the large difference in the signals observed on these lectins in response to the desialylated sample demonstrates that their affinity towards galactose differs significantly.
The signal from lectin 1 increases following the removal of the terminal-galactose, demonstrating increased accessibility of the lectin to the tri-mannosyl core. The signals from the galactose recognizing lectins disappear, and a signal from a lectin that recognizes the newly exposed terminal N-acetyl-glucoseamine (GlcNAc) is evident. The signals from the fucose recognizing lectins remain unchanged. Following the removal of the GlcNAc only the signals from lectin 1, recognizing the tri-mannosyl core, and from the fucose recognizing lectins are observed
These results clearly demonstrate the sensitivity of the bound lectins to changes in the glycan structures. The fingerprints of
Deconvolution of the fingerprints is optimally performed by acquring a detailed understanding of lectin glycan recognition. This is complicated by the broadness of lectin specificities towards glycans, and by the fact that the affinities, both within and between the groups, differ markedly and are unknown. Measurement of these affinities are hampered by the inability to obtain a single-glycan-type glycoprotein for each glycan type. Mathematically, this translates into uncertainties in the conditional probabilities of observing a signal for a particular lectin, when the presence of a particular glycan is known. This limits the use of probabilistic-based algorithms.
An alternative approach using a rule-based expert system (Castillo et al., “Expert Systems and Probabilistic Network Models”—(Monographs in Computer Science) Springer-Verlag, New-York 1997) for fingerprint deconvolution was chosen. The rule base consists of lectin-glycan recognition rules that were extracted from the literature and further refined by manual curation of fingerprints that were run on a large set of well-characterized glycoproteins. Examples of these rules include:
The rules are written in a natural language form and are thus easily edited and optimized. In the example above, LEC1-LEC5 represent particular lectins; the first rule reads “if a signal is observed on LEC1 then there is either a tri-antennary or a tetra-antennary glycan present in the sample”. As emphasized above, knowledge of the relative probabilities of each of these epitopes is not available, and thus a straightforward inference from the set of rules relevant for any particular fingerprint is not possible.
The algorithmic solution adopted is an inference engine based on the Dempster-Shafer theory of evidence (Shafer, Probability judgment in artificial intelligence. In L. N. Kanal and J. F. Lemmer, editors, Uncertainty in Artificial Intelligence. North-Holland, New-York, 1986; Lefevre et al. A Generic Framework for Resolving the Conflict in the Combination of Belief Structures. FUSION 2000—3rd International Conference on Information Fusion. July 2000, Paris, France; Ronald R. Yager (Editor), Janusz Kacprzyk (Editor), Mario Fedrizzi (Editor). Advances in the Dempster-Shafer Theory of Evidence. Wiley & Sons 1994). This framework is powerful in situations where many pieces of evidence (observations) must be weighted in order to determine a single most probable model, and there is uncertainty in the system. Here, the lectin signals are the pieces of evidence, having uncertainties that stem from the broad specificities of the lectins as well as the multiplicity of glycans, glycoforms, and lectin recognition epitopes. The inputs to the inference engine are the lectin binding signals (the fingerprint) and the set of interpretation rules. The inference engine translates the rules into “evidence” based on signal intensities. The three rules shown in the example above are translated into:
Since each lectin can provide evidence for more than one glycan, the signals are iteratively processed until the inference engine converges to the following glycan profile:
Thus, the output is a set of glycan descriptors and quantitative estimates of the relative abundances of each descriptor in the analyzed sample. A careful choice of lectins allows for sufficient data for fingerprint deconvolution. For example, an analysis of an array of 25 lectins produces 25 signals. The required output is commonly a set of 5-10 glycan descriptors (major glycan structures and various additional epitopes). Mathematically, this indicates a problem whose number of equations is considerably larger than the number of variables. Thus, as long as the lectin binding patterns are sufficiently unique, we can expect the fingerprint to yield a solution.
Table 1 tabulates the deconvolution of fingerprints of 5 well-characterized glycoproteins: human milk lactoferrin (hmLF), bovine fetuin, a Bowes melanoma cell line derived tissue plasminogen activator (tPA), porcine thyroglobulin, and human al1-acid glycoprotein. The interpretation is based on fingerprints obtained by direct labeling of the samples. Deconvolution of the fingerprints is by the Dempster-Shafer rule-based inference algorithm. The numbers in brackets indicate percentages of each epitope as reported in the literature and verified by mass-spectrometry analysis (data not shown). NA indicates not applicable. * indicates that due to lack of multiple standards with varying levels of O-linked fucosylation, the quantitation of this epitope was not parameterized, and thus the output only detects its existence. ** is in accordance with convention, N-linked and O-linked glycans distributions were treated independently; the overall ratio of N/O-linked glycans is directly estimated from the relative intensities of the signals obtained from N-linked glycan and O-linked glycan specific lectins. * * * indicates an estimate of average level of sialylated glycans. A comparison of observed values to values compiled from the literature and verified by mass-spectroscopy (data not shown) is also reported.
Interpretation of the hmLF fingerprints results in the correct glycan profile (leftmost column of Table 1). Interpretation of the fingerprints demonstrates the same overall glycan structures following removal of sialic acid, galactose and GlcNAc, and detects the removal of sialic acid and appearance of terminal GlcNAc. Bovine fetuin contains 3 N-linked glycosylation sites, invariably occupied, and 3 partially occupied O-linked glycosylation sites (Green et al. J Biol Chem. 1988;263(34):18253-68; Edge and Spiro, J Biol Chem. 1987;262(33):16135-41; Spiro and Bhoyroo, J Biol Chem. 1974;249(18):5704-17; Yet et al. J Biol Chem. 1988;263(1): 111-7). Approximately 80% of the glycans are N-linked complex glycans, and the remaining 20% are O-linked. Interpretation of fetuin fingerprints results in the correct identification of the structures of both the N- and O-linked glycans, and their relative abundance (N- and O-linked glycans each calculated separately in accordance with convention, and their relative abundance is estimated from the relative intensities of lectins that preferentially recognize either N- or O-linked glycans).
Tissue plasminogen activator (tPA) (Pohl et al. Biochemistry. 1984;23(16):3701-7; Jaques et al. Biochem J. 1996;316 (Pt 2):427-37; Chan et al. Glycobiology. 1991 Mar;1(2):173-85) contains 3 N-linked glycosylation sites, 2 of which are fully occupied, one invariably by a high-mannose glycan and the other by a complex glycan. The third site shows partial occupancy by an additional complex glycan. In the sample analyzed (derived from a Bowes melanoma cell-line) the level of occupancy of the third site is low, as deduced from the abundance of the high-mannose glycans. In addition, an O-linked fucose is present. This demonstrates that the profiling of the tPA glycans is accurate, and includes the detection of the O-linked fucose, which cannot be readily detected using standard mass-spectrometry methods. Porcine thyroglobulin (Ronin et al. J Biol Chem. 1986;261(16):7287-93; de Waard et al. J Biol Chem. 1991;266(7):4237-43; Spiro and Bhoyroo, J Biol Chem. 1984;259(15):9858-66) contains, in addition to the major structures detailed in Table 1, low levels of Galα(1-3)Gal, an epitope produced by all mammalians excluding higher primates and man, and which is highly antigenic in humans. The detection of this epitope and its quantification are important to manufacturers of biological therapeutics. The UC-FINGERPRINT™ technology correctly detects the level of this epitope. α1-acid glycoprotein (Sei et al. J Chromatogr A. 2002;958(1-2):273-81) contains bi-, tri- and tetra-antennary glycans. The ability of the fingerprint to resolve the antennarity of these is evident from the accurate estimations—all within 10% of those obtained by chromatographic and mass spectroscopy techniques.
Fingerprint patterns obtained using direct labeling of the samples were compared to fingerprint patterns obtained using a labeled lectin to detect glycomolecules bound to a substrate.
The fingerprint in
These results demonstrate the power of using lectins as probes, and reveal the existence of a single high-mannose site in tPA. The fact that no signals are observed from lectins that recognize the high-mannose type glycans indicates that the glycoforms bound to these lectins (evident in panels a and b) do not have an additional high-mannose type lectin available for interaction with the labeled probe.
FIGS. 3A-C depicts fingerprints of de-sialylated bovine fetuin, obtained by using three probes that recognize terminal galactose (
The fingerprints were obtained using a glucose/mannose recognizing lectin probe. The fingerprints were comparable to the fingerprint obtained with a purified sample of the protein.
The ability of small quantities of a glycoprotein to be labeled using UC-FINGERPRINT™ technology was examined.
The results are shown in
These results demonstrate that UC-FINGERPRINT™ technology can be performed with glycan structures on intact glycoproteins with minimal sample pretreatment. Thus, the method can provide a high-throughput solution for accurate analysis of protein glycosylation. The analysis can additionally be preformed on crude samples in growth media, obviating the need for time-consuming purification and degradation steps. Less than 200 μl of sample volume with protein concentrations of <0.3 μM are sufficient to produce a quantitative analysis. This renders the technology applicable to all stages of development of protein therapeutics: clone selection and optimization, process development, growth condition monitoring, manufacturing and Quality Control. Additionally, the methods can be used without purification steps, which can introduce bias into the resulting glycoform population (Bond et al. Journal of Immunological Methods, 1993;166: 27-33).
0.667 μg/μl of Fc-Chimeric protein was desialized for about 16.5 hours at 37° C. in 50 mM NaAc pH 4.99, and protease inhibitor and 100 Units sialidase. The desialylated protein was then labeled at 25° C. for 2 hours with agitation in the absence of light in a volume of 500 μl at a concentration of about 0.667 μg/μl. The reaction included 80 μl 0.2M 2M K2HPO4pH 9.18 and 21.5 μg/μl Flourescein (2 mg/ml in DMSO).
The desialylated, labeled FC-chimeric protein was made 0.2M Tris-Cl pH 8.0 and 1 mM DDT and incubated for 10 minutes at 80° C. Iodoacetic acid was then added to a final concentration of 22 mM.
Free sialic acid, fluorescein, DDT, and iodoacetic acid were removed by DG-10 chromatography. Labeling of the protein was confirmed by measuring absorbance at 280 nm and 495 nm was measured for various collected fractions.
The UC-FINGERPRINT™ profile of PNGase treated native or denatured erythropoietin (EPO) was determined. Denatured EPO was prepared using SDS and β-mercaptoethanol and heating to 100° C. for 10 minutes. After cooling, Triton 1% was added along with PNGaseF (0.5 U/μl).
UC-FINGERPRINT™ profiles on multiple lectins were prepared for native and denatured EPO, and for PNGaseF-treated native and denatured EPO. The fingerprints obtained for each were distinct, demonstrating that denaturing the protein and subjecting the protein to PNGase F treatment reveals glycan information for EPO that is not detected when native, denatured EPO is used.
The descriptions given are intended to exemplify, but not limit, the scope of the invention. Additional embodiments are within the claims.