WO2006114661A1 - High throughput glycan analysis for diagnosing and monitoring rheumatoid arthritis and other autoimmune diseases - Google Patents

High throughput glycan analysis for diagnosing and monitoring rheumatoid arthritis and other autoimmune diseases Download PDF

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WO2006114661A1
WO2006114661A1 PCT/IB2005/002885 IB2005002885W WO2006114661A1 WO 2006114661 A1 WO2006114661 A1 WO 2006114661A1 IB 2005002885 W IB2005002885 W IB 2005002885W WO 2006114661 A1 WO2006114661 A1 WO 2006114661A1
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glycans
autoimmune disease
glycoproteins
glycosylation
glycosylation profiles
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PCT/IB2005/002885
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French (fr)
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Raymond A. Dwek
Louise Royle
Pauline Rudd
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Dwek Raymond A
Louise Royle
Pauline Rudd
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Priority to US11/411,231 priority Critical patent/US20060269979A1/en
Publication of WO2006114661A1 publication Critical patent/WO2006114661A1/en

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    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G01N33/6842—Proteomic analysis of subsets of protein mixtures with reduced complexity, e.g. membrane proteins, phosphoproteins, organelle proteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6803—General methods of protein analysis not limited to specific proteins or families of proteins
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/68—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids
    • G01N33/6893—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving proteins, peptides or amino acids related to diseases not provided for elsewhere
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/10—Musculoskeletal or connective tissue disorders
    • G01N2800/101—Diffuse connective tissue disease, e.g. Sjögren, Wegener's granulomatosis
    • G01N2800/102—Arthritis; Rheumatoid arthritis, i.e. inflammation of peripheral joints
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N2800/00—Detection or diagnosis of diseases
    • G01N2800/24—Immunology or allergic disorders

Definitions

  • This invention is directed to diagnostic and monitoring methods for rheumatoid arthritis and other autoimmune diseases and, in particular, to diagnostic and monitoring methods for rheumatoid arthritis (RA) and other autoimmune diseases based on detailed glycosylation analysis of glycoprotein glycans.
  • RA rheumatoid arthritis
  • RA is generally considered a systemic inflammatory disease in which an immune response by the adaptive immune system translates into an attack on the diarthrodial joints (synovium, cartilage, and bone with attendant joint destruction) and less frequently on other anatomic sites.
  • lymphocytes in RA pathogenesis. Histologically, T- cells account for a portion of the mononuclear infiltrate in the synovial sublining, see Van Boxel, J. A., and S. A. Paget. Predominantly T-cell infiltrate in rheumatoid synovial membranes. New England Journal of Medicine 293:517, 1975.
  • the Sa system a novel antigen-antibody system specific for rheumatoid arthritis
  • J Rheumatol 21:1027, 1994 BiP (Blass, S., Novel 68 kDa autoantigen detected by rheumatoid arthritis specific antibodies. Ann Rheum Dis 54:355, 1995)
  • RA33 Hassfeld, W., G. Steiner, K. Hartmuth, G. Kolarz, O. Scherak, W. Graninger, N. Thumb, and J. S. Smolen. Demonstration of a new antinuclear antibody (anti-RA33) that is highly specific for rheumatoid arthritis.
  • T-lymphocytes B-cells are frequently found in the synovial mononuclear infiltrate in RA. With discrete differences, these lymphocytes can organize into aggregates similar to those found in lymph nodes and Peyer's patches (Rooney, M., A. et.al. The immunohistologic features of synovitis, disease activity and in vitro IgM rheumatoid factor synthesis by blood mononuclear cells in rheumatoid arthritis. Journal of Rheumatology 16:459, 1989). Taken together, these findings implicate autoimmunity involving T-lymphocytes, B-lymphocytes and IgG in the pathogenesis of RA.
  • immunoglobulin G of serum or other body fluid.
  • Methods for diagnosing and monitoring diseases based on mass-spectrometric measuring of glycosylation profiles of glycans released from purified glycoproteins are also disclosed in US patent application publication "Glycan Markers for Diagnosing and Monitoring Disease" No. 2004/0147033 to Shriver et. al. published on July 29, 2004.
  • One embodiment of the invention is a method for diagnosing and monitoring an autoimmune disease comprising releasing glycans of glycoproteins from samples of a body fluid without purifying the glycoproteins, and without exposing the body fluid to hydrazinolysis; and quantitatively analyzing the glycans.
  • the method may further be used to improve therapy for an autoimmune disease by establishing optimal dosage for an existing therapeutic agent used to treat the autoimmune disease.
  • the glycosylation profile during treatment of an autoimmune disease patient is monitored to assess whether different dosages of a therapeutic agent change the glycosylation profile so that it moves closer to the glycosylation profile of a normal individual.
  • the method may further be used to screen for new therapeutic agents by generating a candidate agent to be assessed for therapeutic activity in the treatment of an autoimmune disease and determining whether the candidate agent changes the glycosylation profile in an autoimmune disease patient so that it moves closer to the glycosylation profile of a normal individual, hi this regard, combinatorial chemistry may be used to rapidly generate candidate agents for screening in the method of the present invention to determine therapeutic activity in the treatment of an autoimmune disease.
  • Another embodiment of the invention is a method of diagnosing and monitoring an autoimmune disease comprising measuring diseased glycosylation profiles and one or more control glycosylation profiles, wherein the diseased glycosylation profiles are glycosylation profiles of glycans of glycoproteins from autoimmune disease patients and the one or more control glycosylation profiles are glycosylation profiles of glycans of glycoproteins from patients without the autoimmune disease; and comparing peak ratios in the diseased glycosylation profiles and in the one or more control glycosylation profiles and selecting a ratio having a highest correlation with parameters of the autoimmune disease patients out of the peak ratios as a glycosylation marker of the autoimmune disease.
  • Yet another embodiment of the invention is a high throughput method for diagnosing and monitoring rheumatoid arthritis in a patient comprising releasing glycans of glycoproteins from a body fluid or a body tissue of the patient; and measuring a ratio between an amount of GO glycans and an amount of Gl glycans in the glycans.
  • FIGURE 1 shows sodium dodecyl sulphate polyacryl amide gel electrophoresis (SDS-PAGE) and normal phase high performance liquid chromatography (NP-HPLC) profiles of glycans released from purified immunoglobulin G (IgG) of samples GBRA13 and GBRAl.
  • SDS-PAGE sodium dodecyl sulphate polyacryl amide gel electrophoresis
  • NP-HPLC normal phase high performance liquid chromatography
  • FIGURE 2 shows NP-HPLC profiles of glycans released from purified IgG of sample GBRAl 5.
  • FIGURE 3 shows NP-HPLC profiles of control and sample GBRAl 5.
  • FIGURE 4 shows a correlation between GO/tripleGl versus GO as a percentage of total purified IgG glycans for purified IgG glycans.
  • FIGURE 5 shows a correlation between GO/tripleGl from serum versus purified IgG.
  • FIGURE 6 shows a correlation between GO/tripleGl for glycans released from whole serum and GO as a percentage of total glycans released from purified IgGs.
  • FIGURE 7 shows GO/tripleGl ratios in glycans released from whole serum using polyvinyldene fluoride (PVDF) membranes (serum PVDF) and in glycans released from purified IgG heavy chain gel bands (purified IgG heavy chain gel band).
  • PVDF polyvinyldene fluoride
  • the present invention is directed to diagnostic and monitoring methods for autoimmune diseases and, in particular, to diagnostic and monitoring methods for autoimmune diseases based on detailed glycosylation analysis of glycans of glycoproteins.
  • One embodiment of the invention is a method for diagnosing and monitoring an autoimmune disease comprising releasing glycans of glycoproteins from samples of body fluid without purifying the glycoproteins and without exposing the body fluid to hydrazinolysis; and quantitatively analyzing the glycans.
  • the method of the invention can be also used for prognosticating and predicting response to specific therapies in a patient of the autoimmune disease.
  • the autoimmune disease can be, for example, rheumatoid arthritis, osteoarthritis, juvenile chronic arthritis, systematic lupus erythematosus, Sjogren's syndrome, ankylosing spondylitis, psoriatic arthritis, multiple sclerosis, inflammatory bowel disease, graft-vs-host disease or scleroderma.
  • the methodology of the invention can be also applied to other diseases associated with glycosylation changes, for example, to congenital disorders of glycosylation and cancers.
  • Glycans can be released from a sample of a body fluid or a body tissue, such as a sample of whole serum, blood plasma, urine, seminal fluid, seminal plasma, feces or saliva.
  • the released glycans can be N-glycans or O-glycans.
  • releasing a glycan pool of glycoproteins from a sample of a body fluid or a body tissue can be carried out without purifying the glycoproteins.
  • the released glycans are glycans of all or substantially all of the glycoproteins present in the sample of a body fluid or a body tissue rather than of one or more purified and isolated glycoproteins.
  • substantially all of the glycoproteins can mean all the glycoproteins that are recovered, yet in some embodiments substantially all of the glycoproteins can mean all the glycoproteins except those that are specifically removed.
  • Releasing glycans can be carried out without exposing a sample of a body fluid or a body tissue to hydrazinolysis. In some embodiments, releasing glycans can be carried out from a very small sample of a body fluid. In some embodiments, samples of a body fluid can be less than 100 microliters, yet preferably less than 50 microliters, yet more preferably less than 20 microliters, yet more preferably less than 10 microliters, yet most preferably less than 5 microliters.
  • releasing glycans can comprise releasing glycans from total glycoproteins of a body fluid or a body tissue in solution.
  • releasing glycans can comprise immobilizing total glycoproteins of a body fluid or a body tissue, for example, on protein binding membrane or in a gel.
  • IProtein binding membrane can be any protein binding membrane, for example, polyvinyldene fluoride (PVDF) membrane, nylon membrane or Polytetrafiuoroethylene (PTFE) membrane.
  • releasing glycans can further comprise releasing glycans from the total glycoproteins immobilized on the protein binding membrane or in the gel.
  • releasing glycans from the immobilized glycoproteins can be carried out using enzymatic release with, for example, peptide N glycosidase F.
  • releasing glycans can comprise separating the gel into a plurality of bands and selecting one or more bands from the plurality of bands from which the glycans are subsequently released (in gel band method).
  • releasing glycans from the gel can be carried out from the total gel, i.e. without separating gel into the bands.
  • releasing glycans is carried out by chemical release methods, such as / ⁇ -elimination or ammonia-based / ⁇ -elimination, which can be used for releasing JV-linked or O-linked glycans from glycoproteins in solution or from glycoproteins immobilized on protein binding membrane.
  • chemical release methods such as / ⁇ -elimination or ammonia-based / ⁇ -elimination, which can be used for releasing JV-linked or O-linked glycans from glycoproteins in solution or from glycoproteins immobilized on protein binding membrane.
  • In-gel-band This method can be used for N-glycan release from single glycopeptides in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE) gel bands and is based on the method described in Kuster, B., Wheeler, S. F., Hunter, A. P., Dwek, R. A. and Harvey, D. J. (1997) "Sequencing of N-linked oligosaccharides directly from protein gels: in-gel deglycosylation followed by matrix-assisted laser desorption/ionization mass spectrometry and normal-phase high- performance liquid chromatography.” Anal-Biochem 250: 82-101, incorporated hereby by reference in its entirety.
  • Samples can be reduced and alkylated by adding 4 ⁇ l of 5X sample buffer (5X sample buffer: 0.04g Bromophenol blue, 0.625ml 0.5M Tris (6g for 100ml) adjusted to pH 6.6 with HCl, ImI 10%SDS, 0.5ml glycerol, in 2.875ml water), 2 ⁇ l of 0.5M dithiothreitol (DTT) and water to make up to 20 ⁇ l in total, incubated at 7O 0 C for lOmin, then alkylated by addition of 2 ⁇ l of 10OmM iodoacetamide and incubated for 30 min in the dark at room temperature.
  • 5X sample buffer 0.04g Bromophenol blue, 0.625ml 0.5M Tris (6g for 100ml) adjusted to pH 6.6 with HCl, ImI 10%SDS, 0.5ml glycerol, in 2.875ml water
  • DTT dithiothreitol
  • Samples can be then separated on SDS-PAGE gels after which the proteins are stained with Coomassie brilliant blue, the band of interest is excised and destained. Subsequently, the gel band can be cut into lmm pieces and frozen for 2 hours or more (this can help break down the gel matrix). This gel band can be then washed alternatively with ImI of acetonitrile then ImI of digestion buffer (2OmM NaHCO 3 pH 7), which can be repeated twice before the gel plug can be then dried. PNGase F buffer solution (30 ⁇ l of 100 U/ml) is added (this is enough for 10- 15mm 3 gel), more enzyme solution is added if larger gel bands can be used. The PNGaseF and gel pieces can be incubated overnight at 37 0 C.
  • the supernatant can be recovered along with 3 x 200 ⁇ l water washes (with sonication with gel pieces for 30 mins each) followed by an acetonitrile wash (to squeeze out the gel), another water wash and a final acetonitrile wash.
  • Samples can be desalted using, for example, 50 ⁇ l of activated AG-SO(H + ), filtered through a 0.45 ⁇ m LH Millipore filter and dried down for fluorescent labeling.
  • an in-gel-block release from protein mixtures can be used. Briefly, the whole protein mixture (e.g. serum or plasma) can be reduced and alkylated as in the In-gel oligosaccharide release described above, then set into 15% SDS-gel mixture but without bromophenol blue. A total volume of gel of 185 ⁇ l can be used (initially set into a 48 well plate, then removed for cutting up) with 300 ⁇ l of 100 U/ml of PNGaseF. The washing procedures can be similar to those used for in-gel-band release.
  • the whole protein mixture e.g. serum or plasma
  • a total volume of gel of 185 ⁇ l can be used (initially set into a 48 well plate, then removed for cutting up) with 300 ⁇ l of 100 U/ml of PNGaseF.
  • the washing procedures can be similar to those used for in-gel-band release.
  • This procedure can be more suitable for automated glycan release than in-solution PNGaseF release, and can be the preferred method for high throughput glycan analysis.
  • This system can be easily further modified to work with smaller amounts of gel set into a 96 well plate. Enzymatic release ofN-glycansfrom PVDF membranes
  • the glycoproteins in reduced and denatured serum samples can be attached to a hydrophobic PVDF membrane in a 96 well plate by simple filtration.
  • the samples can be then washed to remove contaminates, incubated with PNGaseF to release the glycans based on the methods described in Papac, D. L, et. al. Glycobiology 8: 445- 54, 1998, and in Callewaert, N., et. al. Electrophoresis 25: 3128-31, 2004, both incorporated hereby by reference in their entirety.
  • the iV-glycans can be then washed from the bound protein, collected and dried down ready for fluorescent labeling.
  • N- glycans can be released in situ from the glycoproteins by incubation with PNGaseF and by chemical means. Chemical release of N- and 0-glycans
  • Ammonia-based ⁇ -elimination can be used to release both N- and O-glycans by a modification of the classical ⁇ -elimination (Huang, Y. et. al. Analytical Chemistry 73: 6063-6069, 2001) which can be applied to glycoproteins in solution or on PVDF membranes. Ammonia-based ⁇ -elimination can be done from PVDF membranes. This strategy, can be optimized for high throughput, and can provide a powerful approach for releasing both N- and O-glycans in their correct molar proportions and in an open ring form suitable for post-release labeling.
  • Samples of glycoprotein, mixtures of glycoproteins, whole serum or other body fluids are reduced and alkylated by adding 4 ⁇ l of 5X sample buffer (5X sample buffer: 0.625ml 0.5M Tris (6g for 100ml) adjusted to pH 6.6 with HCl, ImI 10%SDS, 0.5ml glycerol, in 2.875ml water), 2 ⁇ l of 0.5M dithiothreitol (DTT) and water to make up to 20 ⁇ l in total, incubated at 7O 0 C for lOmin, then alkylated by addition of 2 ⁇ l of 10OmM iodoacetamide and incubated for 30 min in the dark at room temperature.
  • 5X sample buffer 0.625ml 0.5M Tris (6g for 100ml) adjusted to pH 6.6 with HCl, ImI 10%SDS, 0.5ml glycerol, in 2.875ml water
  • DTT dithiothreitol
  • Protein binding PVDF membranes (Durapore 13mm x 0.45 ⁇ m HVHP, Millipore) in Swinnex filter holders (Millipore) are pre-washed with 2 x 2.5 ml water using an all-polypropylene 2.5 ml syringe (Sigma), followed by a syringe full of air to remove most of the liquid from the membrane.
  • the reduced and alkylated sample is then applied directly to the membrane and left to bind for 5 min before washing by pushing through 2 x 2.5 ml water slowly with a syringe, followed by a syringe full of air to remove most of the liquid from the membrane.
  • the filter with the bound glycoprotein samples is then carefully removed from the filter holder and placed in a 1.5 ml screw capped polypropylene tube with a molded PTFE cap.
  • 1 ml of ammonium carbonate saturated 29.2% aqueous ammonium hydroxide, plus lOOmg ammonium carbonate is added to the tube. This is incubated for 40 hours at 6O 0 C, then cooled in the fridge. The liquid is then transferred to a clean tube and evaporated to dryness.
  • the released glycans are re-dissolved in water and re-dried until most of the salts are removed.
  • 100 ⁇ l of 0.5M boric acid is added to the glycans and incubated at 37 0 C for 30 min. The glycans are then dried under vacuum, ImI methanol added, re-dried, a further 1 ml methanol added and re-dried to remove the boric acid.
  • Labeling of glycans Upon releasing the glycans can be labeled with, for example, a fluorescent label or a radioactive label.
  • the fluorescent label can be, for example, 2- aminopyridine (2-AP), 2-aminobenzamide (2-AB), 2-aminoanthranilic acid (2-AA), 2-aminoacridone (AMAC) or 8-aminonaphthalene-l,3,6-trisulfonic acid (ANTS). Labeling of glycans with fluorescent labels is described, for example, by Bigge, J. C, et. al.
  • Fluorescent labels can label all glycans efficiently and non-selectively and can enable detection and quantification of glycans in the sub picomole range.
  • the choice of fluorescent label depends on the separation technique used. For example, a charged label is specifically required for capillary electrophoresis.
  • 2 AB label can be preferred for chromatographic, enzymatic and mass spectroscopic processes and analyses, while 2- AA label can be preferred for electrophoretic analyses.
  • Unlabelled glycans can be also detected by, for example, mass spectrometry, however, fluorescent labelling may aid glycan ionisation, see e.g. Harvey, D. J. (1999). "Matrix-assisted laser desorption/ionization mass spectrometry of carbohydrates.” Mass Spectrom Rev 18: 349-450.; Harvey, D. J. (2000). Electrospray mass spectrometry and fragmentation of N-linked carbohydrates derivatized at the reducing terminus. J Am Soc Mass Spectrom 11 : 900-915.
  • Quantitatively analyzing the glycans can be carried out, for example, by chromatography, mass spectrometry, electrophoresis or a combination thereof.
  • the chromatographic technique can be high performance anion exchange chromatography (HPAEC), weak ion exchange chromatography (WAX), gel permeation chromatography (GPC), high performance liquid chromatography (HPLC), normal phase high performance liquid chromatography (NP-HPLC), reverse phase HPLC (RP-HPLC), or porous graphite carbon HPLC (PGC-HPLC).
  • the mass spectrometry technique can be, for example, matrix assisted laser desorption/ ionization time of flight mass spectrometry (MALDI-TOF-MS), electrospray ionization time of flight mass spectrometry (ESI-TOF-MS), or liquid chromatography mass spectrometry (LC-MS).
  • the electrophoretic technique can be, for example, gel electrophoresis or capillary electrophoresis. The use of these separation techniques for analyzing glycans is described, for example, in the following publications:
  • Quantitatively analyzing the glycans can be determining particular glycan structures present in the released glycans.
  • the particular glycan structure can be detected down to subpicomolar levels.
  • the results of quantitatively analyzing the glycans can be presented as glycosylation profiles.
  • the glycosylation profiles can comprise a plurality of peaks corresponding to the glycan structures present in the released glycans.
  • glycans of glycoproteins can be released from two groups of samples: 1) samples from a body fluid of patients diagnosed with the autoimmune disease and 2) control samples.
  • Control samples can be samples of a body fluid of a single healthy patient or from a pool of healthy patients. Healthy in context of the previous sentence means not diagnosed with the autoimmune disease.
  • Quantitatively analyzing the glycans in this embodiment comprises quantitatively analyzing glycans released from the control samples and quantitatively analyzing glycans released from the diseased samples.
  • Quantitatively analyzing the glycans can further comprise comparing the glycan profiles of the diseased glycans and the control glycans to determine a glycosylation marker of the autoimmune disease.
  • comparing the glycan profiles of the diseased glycans and the control glycans to determine a glycosylation marker of the autoimmune disease can comprise comparing peak ratios in the diseased glycan profiles and in the controlled glycan profiles.
  • One or more of the peak ratios with the highest correlation with parameters of the diseased patents can be selected as the glycosylation marker of the autoimmune disease.
  • the parameters of the autoimmune disease patients can be, for example, diagnosis age, sex, disease stage, disease activity, disease severity, disease prognosis, remission, response to therapy or medication.
  • the glycosylation marker can be applied for diagnosing, monitoring, prognosticating the autoimmune disease or predicting a response to a specific therapy or medication in one or more new patients in a high throughput fashion.
  • Applying the glycosylation marker to diagnosing and monitoring the autoimmune disease in new patients can be carried out, for example, by releasing glycans of glycoproteins from a body fluid of a new patient, quantitatively analyzing the glycans from the new patient and determining a relative level of the glycosylation marker in the glycosylation profile of the glycans from the new patient.
  • One embodiment of the present invention is a method of diagnosing and monitoring an autoimmune disease comprising measuring diseased glycosylation profiles and one or more control glycosylation profiles, wherein the diseased glycosylation profiles are glycosylation profiles of glycans of glycoproteins from autoimmune disease patients and the one or more control glycosylation profiles are glycosylation profiles of glycans of glycoproteins from patients without the autoimmune disease; and comparing peak ratios in the diseased glycosylation profiles and in the one or more control glycosylation profiles and selecting a ratio having a highest correlation with parameters of the autoimmune disease patients out of the peak ratios as a glycosylation marker of the autoimmune disease.
  • Measuring the glycosylation profiles in this embodiment can be carried out by any of the described above methods. Most preferably, quantification of glycans is carried out by HPLC or by HPLC in combination with mass spectrometry.
  • both glycans from autoimmune disease patients and control glycans can be released without purifying the glycoproteins. Both glycans from autoimmune disease patients and control glycans can be also released from purified glycoproteins.
  • purified glycoproteins can be serum immunoglobulin G (IgG), serum immunoglobulin A (IgA), IgM, complement components, or inflammatory markers.
  • Glycans from autoimmune disease patients and control glycans can be also released from a body fluid without purifying the glycoproteins. Glycans can be released by the described above techniques.
  • the method of this embodiment can further comprise applying the glycosylation marker to diagnosing the autoimmune disease, monitoring the autoimmune disease, prognosticating the autoimmune disease, or. predicting a response to a therapy in one or more new patients.
  • Yet another embodiment of the present invention is a high throughput method for diagnosing and monitoring rheumatoid arthritis in a patient comprising releasing glycans of glycoproteins from a body fluid or a body tissue of the patient; and measuring galactosylation of the released glycans.
  • Measuring galactosylation can be, for example, measuring a ratio between an amount of GO glycans and an amount of Gl glycans in the glycans.
  • GO denotes glycans having no galactose
  • Gl denotes glycans comprising exactly one galactose.
  • Glycans can be released from a body fluid or body tissue of the patient without purifying the glycoproteins, without treating the glycans with exoglycosidase and without exposing the body fluid or the body tissue to hydrazinolysis. Glycans can be released, for example, using one of the described above methods. Analysis of glycans in this embodiment can be carried out using any of the mentioned above techniques but preferably by HPLC, mass spectrometry or a combination thereof. Upon the glycans release, glycans can be labeled with fluorescent or radioactive label as discussed in other embodiments of the invention.
  • the body fluid can be, for example, whole serum, blood plasma, synovial fluid, urine, seminal fluid, or saliva.
  • This study is used to demonstrate that a direct measurement of glycans released from whole serum can be used as marker for rheumatoid arthritis without IgG purification by correlating GO/triple-Gl ratio from undigested glycans released from whole serum with the amount of GO glycans as a percentage of the total glycans released from purified IgG. Selection of patient sample.
  • Control patient serum was pooled discarded clinical material from individuals undergoing routine employee health screening.
  • RA patients were selected based on a combination of physician global activity score, rheumatoid factor seropositivity and active joint count.
  • IgG purification :
  • IgG was isolated from whole serum via affinity chromatography employing protein-G sepharose as described in 'Antibodies: A laboratory manual", Cold Spring Harbor Laboratory, Cold Spring Harbor, 1988, and P.L. Ey et. al. "Isolation of pure IgG;, IgG 2a andIgG 2b immunoglobulins from mouse serum using protein A- Sepharose ", Molecular Immunology, vol. 15, pp. 429, 1978, both incorporated hereby by reference in their entirety. Briefly, lOO ⁇ l of whole serum was diluted with 300 ⁇ l of 10OmM Tris pH 8.0 and allowed to pass over a ImI column of protein-G sepharose beads (Amersham Biosciences).
  • IgG presence in eluted fractions was confirmed via 10% polyacryl amide gel electrophoresis (PAGE) under reducing conditions (as described, e.g., in Laemmli, "Cleavage of structural proteins during the assembly of the head of bacteriophage TY', Nature, : 227, 680-685, 1970, incorporated hereby by reference in its entirety) and via western blot (Current Protocols in Immunology. John Wiley and Sons, 1994, incorporated hereby by reference in its entirety) utilizing horseradish-peroxidase conjugated donkey-anti- human IgG (Jackson Immunochemicals) and visualized with Western Lightning Chemiluminescence Reagent Plus (Perkin Elmer). Quantitative depletion of serum IgG in column flow through material was confirmed via western blot analysis. Glycans release:
  • Glycans were released from purified IgG by running the reduced and alkylated sample on sodium-dodecyl sulphate polyacryl amide gel electrophoresis (SDS- PAGE), cutting out the heavy chain and digesting with peptide N-glycosidase F (PNGaseF) as described in Kuster, B., Wheeler, S. F., Hunter, A. P., Dwek, R. A., and Harvey, D. J. (1997). Sequencing of N-linked oligosaccharides directly from protein gels: in-gel deglycosylation followed by matrix-assisted laser desorption/ionization mass spectrometry and normal-phase high-performance liquid chromatography.
  • PNGaseF peptide N-glycosidase F
  • Figure 1 shows SDS-PAGE and NP-HPLC profiles from samples GBRAl and GBRAl 3.
  • insets (a) and (b) of figure 1 provide SDS-PAGE gel pictures of the purified IgGs from the respective samples separated into heavy (H) and light (L) chain bands.
  • Insets (c) and (d) of figure 1 provide NP-HPLC profiles for heavy and light chain glycans released from the gel bands shown in (a) and (b) and not subjected to digestion with sialidase and fucosidase. Since no glycans were detected on the light chain, only the heavy chain was required for analysis.
  • Figure 2 illustrates the details of (a) the measurement of the GO/triple-Gl ratio directly from undigested glycans released from purified IgG and (b) the 'classic' measurement of the ratio GO glycans to the total glycans released from purified IgG and digested with sialidase and fucosidase.
  • Figure 2 shows NP-HPLC profiles from the sample GBRAl 5. Each peak corresponds to certain glycan(s). The peaks in each profile are integrated to give the area under the curve for each peak.
  • the ratio GO/triple-Gl is actually the peak area of FcA2G0 divided by the peak area of FcA2Gl[6]+FcA2Gl[3]+FcA2BGl[6] +FcA2BGl[3] (which elutes as a triplet).
  • the area under the peaks corresponding to the GO peaks is divided by the total area under all the peaks in the profile and expressed as a percentage.
  • Figure 3 illustrates NP-HPLC profiles of control sample and the sample GBRAl 5.
  • insets (a) and (d) show glycans released from whole sera of the respective samples
  • insets (b) and (e) show undigested heavy chain glycans released from respective purified IgGs
  • insets (c) and (f) show heavy chain glycans released from respective purified IgGs and digested with sialidase and fucosidase.
  • Table 1 lists the ratios of the GO to triple-Gl peak from whole serum and purified IgG from the same serum samples from 15 RA patients and one pooled control.

Abstract

A method for diagnosing and monitoring an autoimmune disease comprise releasing glycan of glycoproteins from samples of a body fluid without purifying the glycoproteins, and without exposing the body fluid to hydrazinolysis; and quantitatively analyzing the glycans. Another method of diagnosing and monitoring an autoimmune disease comprise measuring diseased glycosylation profiles and one or more control glycosylation profiles, wherein the diseased glycosylation profiles are glycosylation profiles of glycans of glycoproteins from autoimmune disease patients and the one or more control glycosylation profiles are glycosylation profiles of glycans of glycoproteins from patients without the autoimmune disease; and comparing peak ratios in the diseased glycosylation profiles and in the one or more control glycosylation profiles and selecting a ration having a highest correlation with parameters of the autoimmune disease patients out of the peak ratios as a glycosylation marker of the autoimmune disease. A high throughput method for diagnosing and monitoring rheumatoid arthritis in a patient comprises releasing glycans of glycoproteins from a body fluid or a body tissue of the patient; and measuring a ratio between an amount of G0 glycans and an amount of G1 glycans in the glycans, wherein the G0 glycans and an amount of G1 glycans in the glycans, wherein the G0 glycans comprise no galactose and the G1 glycans comprise exactly one galactose.

Description

HIGH THROUGHPUT GLYCAN ANALYSIS FOR DIAGNOSING AND MONITORING RHEUMATOID ARTHRITIS AND OTHER
AUTOIMMUNE DISEASES
This application claims priority to US provisional patent application No. 60/674,722 to Dwek et. al. filed April 26, 2005, incorporated hereby by reference in its entirety.
BACKGROUND OF THE INVENTION
This invention is directed to diagnostic and monitoring methods for rheumatoid arthritis and other autoimmune diseases and, in particular, to diagnostic and monitoring methods for rheumatoid arthritis (RA) and other autoimmune diseases based on detailed glycosylation analysis of glycoprotein glycans.
RA is generally considered a systemic inflammatory disease in which an immune response by the adaptive immune system translates into an attack on the diarthrodial joints (synovium, cartilage, and bone with attendant joint destruction) and less frequently on other anatomic sites. There exists substantial evidence implicating the adaptive immune system — lymphocytes — in RA pathogenesis. Histologically, T- cells account for a portion of the mononuclear infiltrate in the synovial sublining, see Van Boxel, J. A., and S. A. Paget. Predominantly T-cell infiltrate in rheumatoid synovial membranes. New England Journal of Medicine 293:517, 1975. Genetically, the strong HLA-DR association localizing to small regions of the DRBl * 040 land *0404 alleles (Wordsworth, B. P., et. al. HLA-DR4 subtype frequencies in rheumatoid arthritis indicate that DRBl is the major susceptibility locus within the HLA class II region. Proceedings of the National Academy of Sciences of the United States of America 86:10049, 1989; and Ronningen, K. S., et. al. Rheumatoid arthritis may be primarily associated with HLA-DR4 molecules sharing a particular sequence at residues 67-74. Tissue Antigens 36:235, 1990) implies involvement of CD4+ T lymphocytes. There is also experimental evidence implicating B-lymphocyte and IgG involvement in RA pathogenesis. A growing list of autoantibodies associated with RA (reviewed in van Boekel, M. A., et. al. Autoantibody systems in rheumatoid arthritis: specificity, sensitivity and diagnostic value. Arthritis Res 4:87, 2002.) including serologic reactivity to keratin (anti-keratin antibodies (AKA)) (Young, B. J. et. al. "Anti-keratin antibodies in rheumatoid arthritis", Br Med J 2:97, 1997), Sa (Despres, N. et. al. "The Sa system: a novel antigen-antibody system specific for rheumatoid arthritis", J Rheumatol 21:1027, 1994), BiP (Blass, S., Novel 68 kDa autoantigen detected by rheumatoid arthritis specific antibodies. Ann Rheum Dis 54:355, 1995), RA33 (Hassfeld, W., G. Steiner, K. Hartmuth, G. Kolarz, O. Scherak, W. Graninger, N. Thumb, and J. S. Smolen. Demonstration of a new antinuclear antibody (anti-RA33) that is highly specific for rheumatoid arthritis. Arthritis Rheum 32:1515, 1989), glucose-6-phosphate isomerase (GPI) (Schaller, M., et. al. Autoantibodies to GPI in rheumatoid arthritis: linkage between an animal model and human disease. Nat Immunol 2: 746, 2001 ; Kassahn, D., C. et. al. Few human autoimmune sera detect GPI. Nat Immunol 3:411, 2002; Schubert, D. et. al. Autoantibodies to GPI and creatine kinase in RA. Nat Immunol 3:411; discussion 412, 2002) and anti-perinuclear factor (APF or anti-fillagrin) (Nienhuis, L. F., and E. A. Mandema. A new serum factor in patients with rheumatoid arthritis. The antiperinuclear factor. Annals of Rheumatic Disease 23:302, 1964). Additionally, the frequent presence of rheumatoid factor in patients with RA and the recent demonstration that B-lymphocyte ablative therapy is an effective RA therapeutic (Edwards, J. C, et. al. Efficacy of B-cell-targeted therapy with rituximab in patients with rheumatoid arthritis. N Engl J Med 350:2572, 2004) points to dysregulation of the humoral adaptive immune response in these patients. Furthermore, as in the case for T-lymphocytes, B-cells are frequently found in the synovial mononuclear infiltrate in RA. With discrete differences, these lymphocytes can organize into aggregates similar to those found in lymph nodes and Peyer's patches (Rooney, M., A. et.al. The immunohistologic features of synovitis, disease activity and in vitro IgM rheumatoid factor synthesis by blood mononuclear cells in rheumatoid arthritis. Journal of Rheumatology 16:459, 1989). Taken together, these findings implicate autoimmunity involving T-lymphocytes, B-lymphocytes and IgG in the pathogenesis of RA.
A clear correlation between RA and the percentage of the galactosylation on N-glycans released from purified immunoglobulin G (IgG) has been established in Parekh et ah, see "Association of Rheumatoid Arthritis and Primary Osteoarthritis with Changes in the Glycosylation Pattern of Total Serum IgG, "Nature, 316, pp. 452- 457, 1985, incorporated hereby by reference in its entirety. In addition, the specific activity of galactosyltransferase towards asialo-agalacto IgG was found to be reduced to 50-60% of control levels in adult RA, see Parekh et. al. "Galactosylation of IgG Associated Oligosaccharides Is Reduced in Patients with Adult and Juvenile Onset Rheumatoid Arthritis and Is Related to Disease Activity" , Lancet, No. 8592, vol. 1, pp. 966-969, 1988, incorporated hereby by reference in its entirety. Various glycosylation changes were also identified for other autoimmune diseases. For example, IgG glycosylation profiling distinguishes between a range of rheumatic diseases, see Watson, M., Rudd, P.M., Bland, M., Dwek, R.A. and Axford, J.S, Sugar Printing Rheumatic Diseases. A Potential Method for Disease Differentiation Using Immunoglobulin G Oligosaccharides. Arthritis and Rheumatism, vol. 42(8), pp. 1682- 1690, 1999, incorporated hereby by reference in its entirety.
The relationship established between rheumatoid arthritis and the galactosylation on N-glycans from purified IgG led to a so-called 'classic' diagnostic method for rheumatoid arthritis, see Parekh, et. al. Nature, 316, pp. 452-457, 1985. The 'classic' diagnostic method is described also, for example, in US patent No. 4,659,659 "Diagnostic Method for Diseases Having an Arthritic Component' to Dwek et. al. issued on April 21, 1987, incorporated hereby by reference in its entirety. In the 'classic' diagnostic method, analyzed glycans are released from purified glycoproteins, e.g. immunoglobulin G (IgG) of serum or other body fluid. Methods for diagnosing and monitoring diseases based on mass-spectrometric measuring of glycosylation profiles of glycans released from purified glycoproteins are also disclosed in US patent application publication "Glycan Markers for Diagnosing and Monitoring Disease" No. 2004/0147033 to Shriver et. al. published on July 29, 2004.
Sample preparations in the classic diagnostic method for RA and methods of US patent application publication No. 2004/0147033 require purifying glycoproteins. This step can be lengthy in time and can require large amounts of serum or other body fluid, thus, making the "classical" method incompatible with a high throughput diagnostics and monitoring methods. Overcoming this problem, Butler et. al. demonstrated that a glycosylation analysis can be performed on glycans released directly from whole serum glycoproteins without glycoprotein purification, see Butler, M., Quelhas, D., Critchley, A. J., Carchon, H., Hebestreit, H. F., Hibbert, R. G., Vilarinho, L., Teles, E., Matthijs, G., Schollen, E., Argibay, P., Harvey, D. J., Dwek, R. A., Jaeken, J. and Rudd, P. M. (2003). "Detailed glycan analysis of serum glycoproteins of patients with congenital disorders of glycosylation indicates the specific defective glycan processing step and provides an insight into pathogenesis." Glycobiology 13: 601-22, incorporated hereby by reference in its entirety. Although Butler et. al. eliminated the step of glycoprotein purification, the glycan profiles and analysis were flawed because hydrazinolysis was used to release the glycans. Using hydrazinolysis for glycan release results in the desialylation of the significant proportion of the glycans and the introduction of a number of artifacts such as a loss of iV-acetyl and iV-glycolyl groups from the amino sugar residues (which can be subsequently re iV-acetylated and this can result in both under and over acetylation), as well as loss of O-acetyl substitutions in sialic acids. Callewaert et. al. used capillary electrophoresis for analysis of glycans released from a total serum of patients, see Callewaert et. al. Electrophoresis, 2004, 25, 3128-3131. However, the Callewaert et. al. were able to identify only the major desialylated structures. Thus, it is highly desirable to develop a method of diagnosing and monitoring of rheumatoid arthritis and other autoimmune diseases based on a detailed glycosylation analysis of glycans of glycoproteins released from a body fluid or a body tissue which would not require glycoprotein purification and the use of hydrazinolysis for the release of glycans.
SUMMARY OF THE INVENTION
One embodiment of the invention is a method for diagnosing and monitoring an autoimmune disease comprising releasing glycans of glycoproteins from samples of a body fluid without purifying the glycoproteins, and without exposing the body fluid to hydrazinolysis; and quantitatively analyzing the glycans.
The method may further be used to improve therapy for an autoimmune disease by establishing optimal dosage for an existing therapeutic agent used to treat the autoimmune disease. In this method, the glycosylation profile during treatment of an autoimmune disease patient is monitored to assess whether different dosages of a therapeutic agent change the glycosylation profile so that it moves closer to the glycosylation profile of a normal individual.
The method may further be used to screen for new therapeutic agents by generating a candidate agent to be assessed for therapeutic activity in the treatment of an autoimmune disease and determining whether the candidate agent changes the glycosylation profile in an autoimmune disease patient so that it moves closer to the glycosylation profile of a normal individual, hi this regard, combinatorial chemistry may be used to rapidly generate candidate agents for screening in the method of the present invention to determine therapeutic activity in the treatment of an autoimmune disease.
Another embodiment of the invention is a method of diagnosing and monitoring an autoimmune disease comprising measuring diseased glycosylation profiles and one or more control glycosylation profiles, wherein the diseased glycosylation profiles are glycosylation profiles of glycans of glycoproteins from autoimmune disease patients and the one or more control glycosylation profiles are glycosylation profiles of glycans of glycoproteins from patients without the autoimmune disease; and comparing peak ratios in the diseased glycosylation profiles and in the one or more control glycosylation profiles and selecting a ratio having a highest correlation with parameters of the autoimmune disease patients out of the peak ratios as a glycosylation marker of the autoimmune disease.
Yet another embodiment of the invention is a high throughput method for diagnosing and monitoring rheumatoid arthritis in a patient comprising releasing glycans of glycoproteins from a body fluid or a body tissue of the patient; and measuring a ratio between an amount of GO glycans and an amount of Gl glycans in the glycans.
BRIEF DESCRIPTION OF THE DRAWINGS
FIGURE 1 shows sodium dodecyl sulphate polyacryl amide gel electrophoresis (SDS-PAGE) and normal phase high performance liquid chromatography (NP-HPLC) profiles of glycans released from purified immunoglobulin G (IgG) of samples GBRA13 and GBRAl.
FIGURE 2 shows NP-HPLC profiles of glycans released from purified IgG of sample GBRAl 5.
FIGURE 3 shows NP-HPLC profiles of control and sample GBRAl 5.
FIGURE 4 shows a correlation between GO/tripleGl versus GO as a percentage of total purified IgG glycans for purified IgG glycans.
FIGURE 5 shows a correlation between GO/tripleGl from serum versus purified IgG.
FIGURE 6 shows a correlation between GO/tripleGl for glycans released from whole serum and GO as a percentage of total glycans released from purified IgGs.
FIGURE 7 shows GO/tripleGl ratios in glycans released from whole serum using polyvinyldene fluoride (PVDF) membranes (serum PVDF) and in glycans released from purified IgG heavy chain gel bands (purified IgG heavy chain gel band).
DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS
Unless otherwise indicated, "a" or "an" means "one or more".
The present invention is directed to diagnostic and monitoring methods for autoimmune diseases and, in particular, to diagnostic and monitoring methods for autoimmune diseases based on detailed glycosylation analysis of glycans of glycoproteins.
This application incorporates by reference in their entirety US provisional patent application No. 60/674,724 "An automated glycofmgerprinting strategy" to Dwek et. al. filed April 26, 2005, and US provisional patent application No. 60/674,723 "Glycosylation markers for cancer diagnostics and monitoring" to Dwek et. al. filed April 26, 2005.
One embodiment of the invention is a method for diagnosing and monitoring an autoimmune disease comprising releasing glycans of glycoproteins from samples of body fluid without purifying the glycoproteins and without exposing the body fluid to hydrazinolysis; and quantitatively analyzing the glycans. The method of the invention can be also used for prognosticating and predicting response to specific therapies in a patient of the autoimmune disease. The autoimmune disease can be, for example, rheumatoid arthritis, osteoarthritis, juvenile chronic arthritis, systematic lupus erythematosus, Sjogren's syndrome, ankylosing spondylitis, psoriatic arthritis, multiple sclerosis, inflammatory bowel disease, graft-vs-host disease or scleroderma. The methodology of the invention can be also applied to other diseases associated with glycosylation changes, for example, to congenital disorders of glycosylation and cancers.
Releasing glycans.
Glycans can be released from a sample of a body fluid or a body tissue, such as a sample of whole serum, blood plasma, urine, seminal fluid, seminal plasma, feces or saliva. The released glycans can be N-glycans or O-glycans. In some embodiments, releasing a glycan pool of glycoproteins from a sample of a body fluid or a body tissue can be carried out without purifying the glycoproteins. In other words, the released glycans are glycans of all or substantially all of the glycoproteins present in the sample of a body fluid or a body tissue rather than of one or more purified and isolated glycoproteins. In some embodiments, substantially all of the glycoproteins can mean all the glycoproteins that are recovered, yet in some embodiments substantially all of the glycoproteins can mean all the glycoproteins except those that are specifically removed. Releasing glycans can be carried out without exposing a sample of a body fluid or a body tissue to hydrazinolysis. In some embodiments, releasing glycans can be carried out from a very small sample of a body fluid. In some embodiments, samples of a body fluid can be less than 100 microliters, yet preferably less than 50 microliters, yet more preferably less than 20 microliters, yet more preferably less than 10 microliters, yet most preferably less than 5 microliters. The present methods of releasing can be optimized to work with body fluid samples of less than 1 microliters. In some embodiments, releasing glycans can comprise releasing glycans from total glycoproteins of a body fluid or a body tissue in solution. Yet in some embodiments, releasing glycans can comprise immobilizing total glycoproteins of a body fluid or a body tissue, for example, on protein binding membrane or in a gel. IProtein binding membrane can be any protein binding membrane, for example, polyvinyldene fluoride (PVDF) membrane, nylon membrane or Polytetrafiuoroethylene (PTFE) membrane. In some embodiments, releasing glycans can further comprise releasing glycans from the total glycoproteins immobilized on the protein binding membrane or in the gel. When released glycans are iV-linked glycans, releasing glycans from the immobilized glycoproteins can be carried out using enzymatic release with, for example, peptide N glycosidase F. When the glycoproteins are immobilized in the gel, releasing glycans can comprise separating the gel into a plurality of bands and selecting one or more bands from the plurality of bands from which the glycans are subsequently released (in gel band method). In some embodiments, releasing glycans from the gel can be carried out from the total gel, i.e. without separating gel into the bands. In some embodiments, releasing glycans is carried out by chemical release methods, such as /^-elimination or ammonia-based /^-elimination, which can be used for releasing JV-linked or O-linked glycans from glycoproteins in solution or from glycoproteins immobilized on protein binding membrane. For using the methods of this invention in a high throughput format, it may be preferred to release a glycan pool from total glycoproteins immobilized in a gel or on a protein binding membrane as it can allow to use smaller samples of body fluid or body tissue.
The details of some of the release methods and their applicability to both N- glycans and O-glycans are discussed below, however, it should be understood that the present invention is not limited to the discussed below release methods.
In-gel-band: This method can be used for N-glycan release from single glycopeptides in sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS PAGE) gel bands and is based on the method described in Kuster, B., Wheeler, S. F., Hunter, A. P., Dwek, R. A. and Harvey, D. J. (1997) "Sequencing of N-linked oligosaccharides directly from protein gels: in-gel deglycosylation followed by matrix-assisted laser desorption/ionization mass spectrometry and normal-phase high- performance liquid chromatography." Anal-Biochem 250: 82-101, incorporated hereby by reference in its entirety. Samples can be reduced and alkylated by adding 4μl of 5X sample buffer (5X sample buffer: 0.04g Bromophenol blue, 0.625ml 0.5M Tris (6g for 100ml) adjusted to pH 6.6 with HCl, ImI 10%SDS, 0.5ml glycerol, in 2.875ml water), 2μl of 0.5M dithiothreitol (DTT) and water to make up to 20μl in total, incubated at 7O0C for lOmin, then alkylated by addition of 2μl of 10OmM iodoacetamide and incubated for 30 min in the dark at room temperature. Samples can be then separated on SDS-PAGE gels after which the proteins are stained with Coomassie brilliant blue, the band of interest is excised and destained. Subsequently, the gel band can be cut into lmm pieces and frozen for 2 hours or more (this can help break down the gel matrix). This gel band can be then washed alternatively with ImI of acetonitrile then ImI of digestion buffer (2OmM NaHCO3 pH 7), which can be repeated twice before the gel plug can be then dried. PNGase F buffer solution (30μl of 100 U/ml) is added (this is enough for 10- 15mm3 gel), more enzyme solution is added if larger gel bands can be used. The PNGaseF and gel pieces can be incubated overnight at 370C. The supernatant can be recovered along with 3 x 200 μl water washes (with sonication with gel pieces for 30 mins each) followed by an acetonitrile wash (to squeeze out the gel), another water wash and a final acetonitrile wash. Samples can be desalted using, for example, 50 μl of activated AG-SO(H+), filtered through a 0.45 μm LH Millipore filter and dried down for fluorescent labeling.
In-gel-block: To avoid the problems with clean up of samples following solution phase enzymatic glycan release an in-gel-block release from protein mixtures can be used. Briefly, the whole protein mixture (e.g. serum or plasma) can be reduced and alkylated as in the In-gel oligosaccharide release described above, then set into 15% SDS-gel mixture but without bromophenol blue. A total volume of gel of 185 μl can be used (initially set into a 48 well plate, then removed for cutting up) with 300 μl of 100 U/ml of PNGaseF. The washing procedures can be similar to those used for in-gel-band release. This procedure can be more suitable for automated glycan release than in-solution PNGaseF release, and can be the preferred method for high throughput glycan analysis. This system can be easily further modified to work with smaller amounts of gel set into a 96 well plate. Enzymatic release ofN-glycansfrom PVDF membranes
The glycoproteins in reduced and denatured serum samples can be attached to a hydrophobic PVDF membrane in a 96 well plate by simple filtration. The samples can be then washed to remove contaminates, incubated with PNGaseF to release the glycans based on the methods described in Papac, D. L, et. al. Glycobiology 8: 445- 54, 1998, and in Callewaert, N., et. al. Electrophoresis 25: 3128-31, 2004, both incorporated hereby by reference in their entirety. The iV-glycans can be then washed from the bound protein, collected and dried down ready for fluorescent labeling. N- glycans can be released in situ from the glycoproteins by incubation with PNGaseF and by chemical means. Chemical release of N- and 0-glycans
In contrast to the advantages that enzymatic release of N-glycans affords to N- glycan analysis, no enzymatic methodology currently exists for the release of structurally intact O-glycans. Chemical release by reductive β -elimination can require the concomitant reduction of the released oligosaccharides to their alditol derivatives (Amano, J. et. al. Methods Enzymol 179: 261-70, 1989) to prevent degradation (peeling). This reduction precludes the use of any post-release labeling so that detection is limited to mass spectrometry, pulsed amperometric detection and/or radioactivity.
Ammonia-based β-elimination can be used to release both N- and O-glycans by a modification of the classical β-elimination (Huang, Y. et. al. Analytical Chemistry 73: 6063-6069, 2001) which can be applied to glycoproteins in solution or on PVDF membranes. Ammonia-based β-elimination can be done from PVDF membranes. This strategy, can be optimized for high throughput, and can provide a powerful approach for releasing both N- and O-glycans in their correct molar proportions and in an open ring form suitable for post-release labeling.
Release of N- and O-glycans from protein binding PVDF membranes by ammonia based beta-elimination.
Samples of glycoprotein, mixtures of glycoproteins, whole serum or other body fluids are reduced and alkylated by adding 4μl of 5X sample buffer (5X sample buffer: 0.625ml 0.5M Tris (6g for 100ml) adjusted to pH 6.6 with HCl, ImI 10%SDS, 0.5ml glycerol, in 2.875ml water), 2μl of 0.5M dithiothreitol (DTT) and water to make up to 20 μl in total, incubated at 7O0C for lOmin, then alkylated by addition of 2μl of 10OmM iodoacetamide and incubated for 30 min in the dark at room temperature. Protein binding PVDF membranes (Durapore 13mm x 0.45 μm HVHP, Millipore) in Swinnex filter holders (Millipore) are pre-washed with 2 x 2.5 ml water using an all-polypropylene 2.5 ml syringe (Sigma), followed by a syringe full of air to remove most of the liquid from the membrane. The reduced and alkylated sample is then applied directly to the membrane and left to bind for 5 min before washing by pushing through 2 x 2.5 ml water slowly with a syringe, followed by a syringe full of air to remove most of the liquid from the membrane. The filter with the bound glycoprotein samples is then carefully removed from the filter holder and placed in a 1.5 ml screw capped polypropylene tube with a molded PTFE cap. 1 ml of ammonium carbonate saturated 29.2% aqueous ammonium hydroxide, plus lOOmg ammonium carbonate is added to the tube. This is incubated for 40 hours at 6O0C, then cooled in the fridge. The liquid is then transferred to a clean tube and evaporated to dryness. The released glycans are re-dissolved in water and re-dried until most of the salts are removed. 100 μl of 0.5M boric acid is added to the glycans and incubated at 370C for 30 min. The glycans are then dried under vacuum, ImI methanol added, re-dried, a further 1 ml methanol added and re-dried to remove the boric acid.
Quantitatively analyzing the glycans.
Labeling of glycans. Upon releasing the glycans can be labeled with, for example, a fluorescent label or a radioactive label. The fluorescent label can be, for example, 2- aminopyridine (2-AP), 2-aminobenzamide (2-AB), 2-aminoanthranilic acid (2-AA), 2-aminoacridone (AMAC) or 8-aminonaphthalene-l,3,6-trisulfonic acid (ANTS). Labeling of glycans with fluorescent labels is described, for example, by Bigge, J. C, et. al. "Nonselective and efficient fluorescent labeling of glycans using 2-amino benzamide and anthranilic acid." Anal Biochem 230: 229-38, 1995, incorporated hereby reference in its entirety, and Anumula, K. R. (2000). High-sensitivity and high-resolution methods for glycoprotein analysis. Analytical Biochemistry 283: 17- 26, incorporated by reference in its entirety. Fluorescent labels can label all glycans efficiently and non-selectively and can enable detection and quantification of glycans in the sub picomole range. The choice of fluorescent label depends on the separation technique used. For example, a charged label is specifically required for capillary electrophoresis. In particular, 2 AB label can be preferred for chromatographic, enzymatic and mass spectroscopic processes and analyses, while 2- AA label can be preferred for electrophoretic analyses. Unlabelled glycans can be also detected by, for example, mass spectrometry, however, fluorescent labelling may aid glycan ionisation, see e.g. Harvey, D. J. (1999). "Matrix-assisted laser desorption/ionization mass spectrometry of carbohydrates." Mass Spectrom Rev 18: 349-450.; Harvey, D. J. (2000). Electrospray mass spectrometry and fragmentation of N-linked carbohydrates derivatized at the reducing terminus. J Am Soc Mass Spectrom 11 : 900-915.
Quantitatively analyzing released glycans.
Quantitatively analyzing the glycans can be carried out, for example, by chromatography, mass spectrometry, electrophoresis or a combination thereof. In particular, the chromatographic technique can be high performance anion exchange chromatography (HPAEC), weak ion exchange chromatography (WAX), gel permeation chromatography (GPC), high performance liquid chromatography (HPLC), normal phase high performance liquid chromatography (NP-HPLC), reverse phase HPLC (RP-HPLC), or porous graphite carbon HPLC (PGC-HPLC). The mass spectrometry technique can be, for example, matrix assisted laser desorption/ ionization time of flight mass spectrometry (MALDI-TOF-MS), electrospray ionization time of flight mass spectrometry (ESI-TOF-MS), or liquid chromatography mass spectrometry (LC-MS). The electrophoretic technique can be, for example, gel electrophoresis or capillary electrophoresis. The use of these separation techniques for analyzing glycans is described, for example, in the following publications:
1) Guile, G. R., Wong, S. Y. and Dwek, R. A. (1994). "Analytical and preparative separation of anionic oligosaccharides by weak anion-exchange high-performance liquid chromatography on an inert polymer column." Analytical Biochemistry 222: 231-5 for HPLC, incorporated hereby by reference in its entirety;
2) Butler, M., Quelhas, D., Critchley, A. J., Carchon, H., Hebestreit, H. F., Hibbert, R. G., Vilarinho, L., Teles, E., Matthijs, G., Schollen, E., Argibay, P., Harvey, D. J., Dwek, R. A., Jaeken, J. and Rudd, P. M. (2003). "Detailed glycan analysis of serum glycoproteins of patients with congenital disorders of glycosylation indicates the specific defective glycan processing step and provides an insight into pathogenesis." Glycobiology 13: 601-22, for MALDI-MS, NP-HPLC and ESI-liquid chromatography/MS, incorporated hereby by reference in its entirety;
3) Jackson, P., Pluskal, M. G. and Skea, W. (1994). "The use of polyacrylamide gel electrophoresis for the analysis of acidic glycans labeled with the fluorophore 2- aminoacridone." Electrophoresis 15: 896-902, for polyacrylamide gel electrophoresis (PAGE), incorporated hereby by reference in its entirety;
4) Hardy, M. R. and Townsend, R. R. (1994). "High-pH anion-exchange chromatography of glycoprotein-derived carbohydrates." Methods Enzymol 230: 208- 25., for HPAEC using pulsed amperometric detection (PAD), incorporated hereby by reference in its entirety; 5) Callewaert, N., Contreras, R., Mitnik-Gankin, L., Carey, L., Matsudaira, P. and Ehrlich, D. (2004). "Total serum protein N-glycome profiling on a capillary electrophoresis-microfluidics platform." Electrophoresis 25: 3128-31 for capillary electrophoresis, incorporated hereby by reference in its entirety;
6) Guile, G. R., Rudd, P. M., Wing, D. R., Prime, S. B. and Dwek, R. A. (1996). "A rapid high-resolution high-performance liquid chromatographic method for separating glycan mixtures and analyzing oligosaccharide profiles." Anal Biochem 240: 210-26, for HPLC, incorporated hereby by reference in its entirety;
7) Caesar, J. P., Jr., Sheeley, D. M. and Reinhold, V. N. (1990). "Femtomole oligosaccharide detection using a reducing-end derivative and chemical ionization mass spectrometry." Anal Biochem 191: 247-52, for LC-MS, incorporated hereby by reference in its entirety;
8) Mattu, T. S., Royle, L., Langridge, J., Wormald, M. R., Van den Steen, P. E., Van Damme, J., Opdenakker, G., Harvey, D. J., Dwek, R. A. and Rudd, P. M. (2000). "O- glycan analysis of natural human neutrophil gelatinase B using a combination of normal phase-HPLC and online tandem mass spectrometry: implications for the domain organization of the enzyme." Biochemistry 39: 15695-704, for NP-HPLC and MS, incorporated hereby by reference in its entirety;
9) Royle, L., Mattu, T. S., Hart, E., Langridge, J. I., Merry, A. H., Murphy, N., Harvey, D. J., Dwek, R. A. and Rudd, P. M. (2002). "An analytical and structural database provides a strategy for sequencing O-glycans from microgram quantities of glycoproteins." Anal Biochem 304: 70-90, for NP-HPLC and MS, incorporated hereby by reference in its entirety;
10) Anumula, K. R. and Du, P. (1999). "Characterization of carbohydrates using highly fluorescent 2- aminobenzoic acid tag following gel electrophoresis of glycoproteins." Anal Biochem 275: 236-42, for gel electrophoresis, incorporated hereby by reference in its entirety;
11) Huang, Y. and Mechref, Y. (2001). "Microscale nonreductive release of O-linked glycans for subsequent analysis through MALDI mass spectrometry and capillary electrophoresis." Analytical Chemistry 73: 6063-6069, for a combination of MALDI- MS and capillary electrophoresis, incorporated hereby by reference in its entirety; 12) Burlingame, A. L. (1996). "Characterization of protein glycosylation by mass spectrometry." Curr Opin Biotechnol 7: 4-10, for mass spectrometry, incorporated hereby by reference in its entirety;
13) Costello, C. E. (1999). "Bioanalytic applications of mass spectrometry." Curr Opin Biotechnol 10: 22-8, for mass spectrometry, incorporated hereby by reference in its entirety;
14) Davies, M. J. and Hounsell, E. F. (1996). "Comparison of separation modes of high-performance liquid chromatography for the analysis of glycoprotein- and proteoglycan-derived oligosaccharides." J Chromatogr A 720: 227-33, for HPLC, incorporated hereby by reference in its entirety;
15) El Rassi, Z. (1999). "Recent developments in capillary electrophoresis and capillary electrochromatography of carbohydrate species." Electrophoresis 20: 3134- 44, for capillary electrophoresis and capillary electrochromatography, incorporated hereby by reference in its entirety;
16) Kuster, B., Wheeler, S. F., Hunter, A. P., Dwek, R. A. and Harvey, D. J. (1997). "Sequencing of N-linked oligosaccharides directly from protein gels: in-gel deglycosylation followed by matrix-assisted laser desorption/ionization mass spectrometry and normal-phase high-performance liquid chromatography." Anal- Biochem 250: 82-101, for NP-HPLC and MALDI-MS, incorporated hereby by reference in its entirety;
17) Reinhold, V. N., Reinhold, B. B. and Chan, S. (1996). "Carbohydrate sequence analysis by electrospray ionization-mass spectrometry." Methods Enzymol 271: 377- 402, for ESI-MS, incorporated hereby by reference in its entirety;
18) Mattu, T. S., Pleass, R. J., Willis, A. C, Kilian, M., Wormald, M. R., Lellouch, A. C, Rudd, P. M., Woof, J. M. and Dwek, R. A. (1998). "The glycosylation and structure of human serum IgAl, Fab, and Fc regions and the role of N-glycosylation on Fc alpha receptor interactions." Journal of Biological Chemistry 273: 2260-72, for WAX and NP-HPLC, incorporated hereby by reference in its entirety;
19) Callewaert, N., Schollen, E., Vanhecke, A., Jaeken, J., Matthijs, G., and Contreras, R. (2003). Increased fucosylation and reduced branching of serum glycoprotein N-glycans in all known subtypes of congenital disorder of glycosylation I. Glycobiology 13: 367-375, incorporated hereby by reference in its entirety.
20) Block, T.M. Comunale, M.A., Lowman, M., Steel, L.F., Romano, P.R.,, Fimmel, C, Tennant, B.C. London, A.A. Evans, B. S. Blumberg, R.A. Dwek, T.S. Mattu and A. S. Mehta , "Use of targeted glycoproteomics to identify serum glycoproteins that correlate with liver cancer in woodchucks and humans". PNAS USA (2005) 102, 779- 784, incorporated hereby by reference in its entirety.
21) D. J. Harvey, Fragmentation of negative ions from carbohydrates: Part 1; Use of nitrate and other anionic adducts for the production of negative ion electrospray spectra from TV-linked carbohydrates, J. Am. Soc. Mass Spectrom., 2005, 16, 622-630, incorporated hereby by reference in its entirety.
22) D. J. Harvey, Fragmentation of negative ions from carbohydrates: Part 2, Fragmentation of high-mannose JV-linked glycans, J. Am. Soc. Mass Spectrom., 2005, 16, 631-646, incorporated hereby by reference in its entirety;
23) D. J. Harvey, Fragmentation of negative ions from carbohydrates: Part 3, Fragmentation of hybrid and complex TV-linked glycans, J. Am. Soc. Mass Spectrom., 2005, 16, 647-659, incorporated, hereby by reference in its entirety.
Quantitatively analyzing the glycans can be determining particular glycan structures present in the released glycans. The particular glycan structure can be detected down to subpicomolar levels. The results of quantitatively analyzing the glycans can be presented as glycosylation profiles. The glycosylation profiles can comprise a plurality of peaks corresponding to the glycan structures present in the released glycans.
In one embodiment of the invention, glycans of glycoproteins can be released from two groups of samples: 1) samples from a body fluid of patients diagnosed with the autoimmune disease and 2) control samples. Control samples can be samples of a body fluid of a single healthy patient or from a pool of healthy patients. Healthy in context of the previous sentence means not diagnosed with the autoimmune disease. Quantitatively analyzing the glycans in this embodiment comprises quantitatively analyzing glycans released from the control samples and quantitatively analyzing glycans released from the diseased samples. Quantitatively analyzing the glycans can further comprise comparing the glycan profiles of the diseased glycans and the control glycans to determine a glycosylation marker of the autoimmune disease. The described above experimental techniques can present the results of quantitatively analyzing the glycans as a plurality of peaks or as a spectrum. When the results of quantitatively analyzing of the glycans are presented as a plurality of peaks or as a spectrum, comparing the glycan profiles of the diseased glycans and the control glycans to determine a glycosylation marker of the autoimmune disease can comprise comparing peak ratios in the diseased glycan profiles and in the controlled glycan profiles. One or more of the peak ratios with the highest correlation with parameters of the diseased patents can be selected as the glycosylation marker of the autoimmune disease. The parameters of the autoimmune disease patients can be, for example, diagnosis age, sex, disease stage, disease activity, disease severity, disease prognosis, remission, response to therapy or medication. The glycosylation marker can be applied for diagnosing, monitoring, prognosticating the autoimmune disease or predicting a response to a specific therapy or medication in one or more new patients in a high throughput fashion. Applying the glycosylation marker to diagnosing and monitoring the autoimmune disease in new patients can be carried out, for example, by releasing glycans of glycoproteins from a body fluid of a new patient, quantitatively analyzing the glycans from the new patient and determining a relative level of the glycosylation marker in the glycosylation profile of the glycans from the new patient.
One embodiment of the present invention is a method of diagnosing and monitoring an autoimmune disease comprising measuring diseased glycosylation profiles and one or more control glycosylation profiles, wherein the diseased glycosylation profiles are glycosylation profiles of glycans of glycoproteins from autoimmune disease patients and the one or more control glycosylation profiles are glycosylation profiles of glycans of glycoproteins from patients without the autoimmune disease; and comparing peak ratios in the diseased glycosylation profiles and in the one or more control glycosylation profiles and selecting a ratio having a highest correlation with parameters of the autoimmune disease patients out of the peak ratios as a glycosylation marker of the autoimmune disease. Measuring the glycosylation profiles in this embodiment can be carried out by any of the described above methods. Most preferably, quantification of glycans is carried out by HPLC or by HPLC in combination with mass spectrometry. In one embodiment, both glycans from autoimmune disease patients and control glycans can be released without purifying the glycoproteins. Both glycans from autoimmune disease patients and control glycans can be also released from purified glycoproteins. For example, purified glycoproteins can be serum immunoglobulin G (IgG), serum immunoglobulin A (IgA), IgM, complement components, or inflammatory markers. Glycans from autoimmune disease patients and control glycans can be also released from a body fluid without purifying the glycoproteins. Glycans can be released by the described above techniques. The method of this embodiment can further comprise applying the glycosylation marker to diagnosing the autoimmune disease, monitoring the autoimmune disease, prognosticating the autoimmune disease, or. predicting a response to a therapy in one or more new patients.
Yet another embodiment of the present invention is a high throughput method for diagnosing and monitoring rheumatoid arthritis in a patient comprising releasing glycans of glycoproteins from a body fluid or a body tissue of the patient; and measuring galactosylation of the released glycans. Measuring galactosylation can be, for example, measuring a ratio between an amount of GO glycans and an amount of Gl glycans in the glycans. GO denotes glycans having no galactose and Gl denotes glycans comprising exactly one galactose. Glycans can be released from a body fluid or body tissue of the patient without purifying the glycoproteins, without treating the glycans with exoglycosidase and without exposing the body fluid or the body tissue to hydrazinolysis. Glycans can be released, for example, using one of the described above methods. Analysis of glycans in this embodiment can be carried out using any of the mentioned above techniques but preferably by HPLC, mass spectrometry or a combination thereof. Upon the glycans release, glycans can be labeled with fluorescent or radioactive label as discussed in other embodiments of the invention. The body fluid can be, for example, whole serum, blood plasma, synovial fluid, urine, seminal fluid, or saliva. The methodology for diagnosing and monitoring an autoimmune disease can be illustrated in more details by the following example, however, it should be understood that the present invention is not limited thereto.
The invention is further illustrated by, though in no way limited to, the following examples.
Example
The measurement of the GQ/triple-Gl ratio directly from undigested glycans released from whole serum was compared with the 'classic' measurement of the amount of GO glycans as a percentage of the total glycans released from purified IgG after sialidase and fucosidase digestion. It has been shown that GO released from purified IgG is disease(RA) specific marker that correlates with disease progression and that can be used as a prognostic indicator of the disease, see e.g. US patent No. 4,659,659 "Diagnostic Method for Diseases Having an Arthritic Component' to Dwek et. al. issued on April 21, 1987; Parekh et al., see "Association of Rheumatoid Arthritis and Primary Osteoarthritis with Changes in the Glycosylation Pattern of Total Serum IgG, "Nature, 316, pp. 452-457, 1985; and Parekh et. al. " Galactosylation of IgG Associated Oligosaccharides Is Reduced in Patients with Adult and Juvenile Onset Rheumatoid Arthritis and Is Related to Disease Activity ", Lancet, No. 8592, vol. 1, pp. 966-969, 1988. This study is used to demonstrate that a direct measurement of glycans released from whole serum can be used as marker for rheumatoid arthritis without IgG purification by correlating GO/triple-Gl ratio from undigested glycans released from whole serum with the amount of GO glycans as a percentage of the total glycans released from purified IgG. Selection of patient sample.
Control patient serum was pooled discarded clinical material from individuals undergoing routine employee health screening. RA patients were selected based on a combination of physician global activity score, rheumatoid factor seropositivity and active joint count. IgG purification:
IgG was isolated from whole serum via affinity chromatography employing protein-G sepharose as described in 'Antibodies: A laboratory manual", Cold Spring Harbor Laboratory, Cold Spring Harbor, 1988, and P.L. Ey et. al. "Isolation of pure IgG;, IgG2a andIgG2b immunoglobulins from mouse serum using protein A- Sepharose ", Molecular Immunology, vol. 15, pp. 429, 1978, both incorporated hereby by reference in their entirety. Briefly, lOOμl of whole serum was diluted with 300μl of 10OmM Tris pH 8.0 and allowed to pass over a ImI column of protein-G sepharose beads (Amersham Biosciences). Bound material was washed with 15 column volumes of 10OmM Tris pH 8.0. IgG was eluted using 10OmM glycine pH 2.6 buffer directly into 1/10 volume IM Tris pH 8.0 and collected in ImI fractions. Protein content of eluted fractions was determined by 28OnM (UV) absorbance (Beckman Coulter Model DU640 spectrophotometer). Protein containing eluted fractions were pooled and dialyzed into phosphate buffered saline. IgG presence in eluted fractions was confirmed via 10% polyacryl amide gel electrophoresis (PAGE) under reducing conditions (as described, e.g., in Laemmli, "Cleavage of structural proteins during the assembly of the head of bacteriophage TY', Nature, : 227, 680-685, 1970, incorporated hereby by reference in its entirety) and via western blot (Current Protocols in Immunology. John Wiley and Sons, 1994, incorporated hereby by reference in its entirety) utilizing horseradish-peroxidase conjugated donkey-anti- human IgG (Jackson Immunochemicals) and visualized with Western Lightning Chemiluminescence Reagent Plus (Perkin Elmer). Quantitative depletion of serum IgG in column flow through material was confirmed via western blot analysis. Glycans release:
Glycans were released from purified IgG by running the reduced and alkylated sample on sodium-dodecyl sulphate polyacryl amide gel electrophoresis (SDS- PAGE), cutting out the heavy chain and digesting with peptide N-glycosidase F (PNGaseF) as described in Kuster, B., Wheeler, S. F., Hunter, A. P., Dwek, R. A., and Harvey, D. J. (1997). Sequencing of N-linked oligosaccharides directly from protein gels: in-gel deglycosylation followed by matrix-assisted laser desorption/ionization mass spectrometry and normal-phase high-performance liquid chromatography. Analytical Biochemistry 250: 82-101, incorporated hereby by reference in its entirety. Glycans were released with PNGaseF from 5 μl of whole sera after binding the reduced and alkylated serum to MultiScreenJP, 0.45μm hydrophobic, high protein binding polyvinylidene fluoride (PVDF) membranes in a 96 well plate format (Millipore, Bedford, MA, USA). Released glycans were labeled with 2AB fluorescent label (Ludger Ltd, Oxford, UK) as described in Bigge, J. C, Patel, T. P., Bruce, J. A., Goulding, P. N., Charles, S. M., andParekh, R. B. (1995). Nonselective and efficient fluorescent labeling ofglycans using 2-amino benzamide and anthranilic acid. Analytical Biochemistry ,230: 229-238, incorporated hereby by reference in its entirety, and run by normal phase high performance liquid chromatography (NP- HPLC) on a 4.6 x 250 mm TSK Amide-80 column (Anachem, Luton, UK) using a Waters 2695 separations module equipped with a Waters 2475 fluorescence detector (Waters, Milford, MA, USA) as described in Guile, G. R., Rudd, P. M., Wing, D. R., Prime, S. B., andDwek, R. A. (1996). A rapid high-resolution high-performance liquid chromatographic method for separating glycan mixtures and analyzing oligosaccharide profiles. Analytical Biochemistry 240: 210-226. Purified, 2AB labeled IgG heavy chain glycans were also digested with sialidase and fucosidase to reduce all the structures to GO, Gl or G2 +/- bisect, then run on NP-HPLC. [GO denotes no galactose; Gl, one galactose; and, G2 two galactose, all on biantennary complex N-glycans.] Statistical analysis.
All the data for glycan ratios are listed in Table 1. Figures 4, 5 and 6 are plots showing correlations between these data. The R2 values were obtained by linear regression analysis using Microsoft Excel. Experimental results.
Figure 1 shows SDS-PAGE and NP-HPLC profiles from samples GBRAl and GBRAl 3. In particular, insets (a) and (b) of figure 1 provide SDS-PAGE gel pictures of the purified IgGs from the respective samples separated into heavy (H) and light (L) chain bands. Insets (c) and (d) of figure 1 provide NP-HPLC profiles for heavy and light chain glycans released from the gel bands shown in (a) and (b) and not subjected to digestion with sialidase and fucosidase. Since no glycans were detected on the light chain, only the heavy chain was required for analysis.
Figure 2 illustrates the details of (a) the measurement of the GO/triple-Gl ratio directly from undigested glycans released from purified IgG and (b) the 'classic' measurement of the ratio GO glycans to the total glycans released from purified IgG and digested with sialidase and fucosidase. In particular, Figure 2 shows NP-HPLC profiles from the sample GBRAl 5. Each peak corresponds to certain glycan(s). The peaks in each profile are integrated to give the area under the curve for each peak. In the measurement of the GO/triple-Gl ratio, the area under the peaks corresponding to the GO glycans (left box of the inset (a) of figure 2) are divided by the area under the triplet of peaks corresponding to the Gl glycans (right box of the inset (a) of figure 2). As the vast majority of glycans found in these experiments were core fucosylated, only core fucosylated glycans were included in these measurements, i.e. the ratio GO/triple-Gl is actually the peak area of FcA2G0 divided by the peak area of FcA2Gl[6]+FcA2Gl[3]+FcA2BGl[6] +FcA2BGl[3] (which elutes as a triplet).
In the 'classic' measurement, the area under the peaks corresponding to the GO peaks is divided by the total area under all the peaks in the profile and expressed as a percentage.
Figure 3 illustrates NP-HPLC profiles of control sample and the sample GBRAl 5. Particularly, insets (a) and (d) show glycans released from whole sera of the respective samples, insets (b) and (e) show undigested heavy chain glycans released from respective purified IgGs, insets (c) and (f) show heavy chain glycans released from respective purified IgGs and digested with sialidase and fucosidase.
Table 1 lists the ratios of the GO to triple-Gl peak from whole serum and purified IgG from the same serum samples from 15 RA patients and one pooled control. The 'classic' measurement of the amount of GO glycans as a percentage of the total glycans (G0+G1+G2) from purified IgG is also shown. Comparing the results of the two different measurements taken from purified IgG, a high correlation (R2=0.9649) is found, indicating that the ratio GO/triple-Gl is as a good measurement as the 'classic' measurement of the percentage of GO glycans in total glycan pool (Figure 4). Comparing the GO/triple-Gl ratio between purified IgG and whole serum glycans gives a correlation of R2=0.8785 (Figure 5), whilst comparing the GO/triple- Gl ratio from whole serum glycans with the percentage GO glycans from purified IgG gives a correlation of R2=0.8174 (Figure 6). Figure 7 is a histogram showing the GO/triple-Gl ratios for all serum and IgG samples. Table 1
Figure imgf000025_0001
Conclusion.
The use of the high throughput PVDF membrane 96 well plate format with only 5μl of whole serum being used to obtain glycans for a direct measurement of the G0/triple-Gl ratio has been demonstrated in place of the more lengthy procedure of measuring the percentage of GO glycans in the glycans released from purified IgG determined after exoglycosidase treatment, as an indicator of RA disease state. Thus, to monitor the RA disease state, one can efficiently reduce working hours from sample preparation to results by using the PVDF membrane method with whole serum as well as reducing the amount of material (serum) used.
Although the foregoing refers to particular preferred embodiments, it will be understood that the present invention is not so limited. It will occur to those of ordinary skill in the art that various modifications may be made to the disclosed embodiments and that such modifications are intended to be within the scope of the present invention.
AU of the publications, patent applications and patents cited in this specification are incorporated herein by reference in their entirety.

Claims

WHAT IS CLAIMED IS:
L A method for diagnosing and monitoring an autoimmune disease comprising releasing glycans of glycoproteins from samples of a body fluid without purifying the glycoproteins, and without exposing the body fluid to hydrazinolysis; quantitatively analyzing the glycans.
2. The method of claim 1 , wherein the body fluid is a whole serum, a blood plasma, a synovial fluid, urine, seminal fluid, or saliva.
3. The method of claim 1 , wherein the body fluid is a whole serum.
4. The method of claim 1, wherein releasing glycans comprises preparing a gel from the body fluid.
5. The method of claim 4, wherein the glycans are iV-glycans and releasing glycans further comprises releasing the N-glycans from the gel using PNGaseF enzyme.
6. The method of claim 1, wherein releasing glycans comprises attaching glycoproteins to polyvinyldene fluoride membranes.
7. The method of claim 6, wherein the glycans are N-glycans and releasing glycans further comprises incubating polyvinyldene fluoride membranes with PNGaseF enzyme.
8. The method of claim 6, wherein releasing glycans further comprises chemically releasing the glycans by /^-elimination.
9. The method of claim 6, wherein releasing glycans further comprises releasing the glycans by ammonia-based /7-elimination.
10. The method of claim 1, further comprising labeling the glycans before quantitatively analyzing the glycans with a radioactive label or a fluorescent label.
11. The method of claim 10, wherein the fluorescent label is 2- aminobenzamide.
12. The method of claim 1, wherein quantitatively analyzing the glycans comprises analyzing the glycans by chromatography, mass spectrometry or a combination thereof.
13. The method of claim 12, wherein the chromatography is high performance liquid chromatography.
14. The method of claim 12, wherein quantitatively analyzing the glycans further comprises obtaining glycosylation profiles of the glycans, wherein each of the glycosylation profiles corresponds to one of the samples and wherein each of the glycosylation profiles comprises a plurality of peaks.
15. The method of claim 14, wherein the samples comprise diseased samples and one or more control samples, wherein diseased samples are body fluid samples of autoimmune disease patients and control samples are body fluid samples of patients without the autoimmune disease, and wherein the glycosylation profiles comprise diseased glycosylation profiles corresponding to the diseased samples and one or more control glycosylation profiles corresponding to the one or more control samples.
16. The method of claim 15, wherein quantitatively analyzing the glycans comprises comparing peak ratios in the diseased glycosylation profiles and in the one or more control glycosylation profiles and selecting out of the peak ratios a glycosylation marker of the autoimmune disease, wherein the glycosylation marker is a ratio having a highest correlation with parameters of the autoimmune disease patients out of the peak ratios.
17. The method of claim 16, wherein the parameters of the autoimmune disease patients are diagnosis, age, sex, disease activity, disease prognosis, remission, response to a therapy or a combination thereof.
18. The method of 16, further comprising applying the glycosylation marker to diagnosing the autoimmune disease, monitoring the autoimmune disease, prognosticating the autoimmune disease, or predicting response to a therapy in one or more new patients.
19. The method of claim 1 , wherein the autoimmune disease is rheumatoid arthritis, osteoarthritis, juvenile chronic arthritis, systematic lupus erythematosus, Sjogren's syndrome, ankylosing spondylitis, psoriatic arthritis, multiple sclerosis, inflammatory bowel disease, graft-vs-host disease or scleroderma.
20. The method of claim 1, wherein the autoimmune disease is rheumatoid arthritis.
21. A method of diagnosing and monitoring an autoimmune disease comprising measuring diseased glycosylation profiles and one or more control glycosylation profiles, wherein the diseased glycosylation profiles are glycosylation profiles of glycans of glycoproteins from autoimmune disease patients and the one or more control glycosylation profiles are glycosylation profiles of glycans of glycoproteins from patients without the autoimmune disease and wherein measuring diseased glycosylation profiles and one or more control glycosylation profiles is carried out by HPLC or a combination of HPLC and mass spectrometry; comparing peak ratios in the diseased glycosylation profiles and in the one or more control glycosylation profiles and selecting a ratio having a highest correlation with parameters of the autoimmune disease patients out of the peak ratios as a glycosylation marker of the autoimmune disease.
22. The method of claim 21 , wherein the parameters of the autoimmune disease patients are diagnosis, age, sex, disease activity, disease prognosis, remission, response to a therapy or a combination thereof.
23. The method of 21 , further comprising applying the glycosylation marker to diagnosing the autoimmune disease, monitoring the autoimmune disease, 80 prognosticating the autoimmune disease, or predicting a response to a therapy in one
81 or more new patients.
82 24. The method of claim 21, wherein the autoimmune disease is
83 rheumatoid arthritis, osteoarthritis, juvenile chronic arthritis., systematic lupus
84 erythematosus, Sjogren's syndrome, ankylosing spondylitis, psoriatic arthritis,
85 multiple sclerosis, inflammatory bowel disease, graft-vs-host disease or scleroderma.
86 25. The method of claim 22, wherein the glycans are released without
87 purifying the glycoproteins.
88 26. The method of claim 22, wherein the glycans are released from
89 purified glycoproteins.
90 27. The method of claim 22, wherein the glycans are released from
91 purified serum IgG.
92 28. A high throughput method for diagnosing and monitoring rheumatoid
93 arthritis in a patient comprising
94 releasing glycans of glycoproteins from a body fluid or a body tissue of the
95 patient;
96 measuring a ratio between an amount of GO glycans and an amount of Gl
97 glycans in the glycans.
98 29. The method of claim 28, wherein the body fluid is a whole serum, a
99 blood plasma or a synovial fluid.
100 30. The method of claim 28, wherein measuring a ratio is carried out by
101 chromatography, mass spectrometry or a combination thereof.
102 31. The method of claim 28, wherein releasing glycans does not comprise
103 purifying the glycoproteins.
104 32. The method of claim 28, wherein releasing glycans does not comprise
105 treating the glycans with exoglycosidase.
106 33. The method of claim 28, wherein releasing glycans does not comprise
107 exposing the body fluid or the body tissue to hydrazinolysis.
108 34. The method of claim 28, wherein the glycoproteins are purified
109 glycoproteins.
110
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