WO2001092890A1 - Procede d'analyse de glucides presents en quantites picomolaires - Google Patents

Procede d'analyse de glucides presents en quantites picomolaires Download PDF

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WO2001092890A1
WO2001092890A1 PCT/EP2001/006042 EP0106042W WO0192890A1 WO 2001092890 A1 WO2001092890 A1 WO 2001092890A1 EP 0106042 W EP0106042 W EP 0106042W WO 0192890 A1 WO0192890 A1 WO 0192890A1
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carbohydrates
glycans
analysis
carbohydrate
glycan
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PCT/EP2001/006042
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Nico Luc Marc Callewaert
Roland Henry Contreras
Francis Stephaan Jan Molemans
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Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw
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Publication of WO2001092890A1 publication Critical patent/WO2001092890A1/fr

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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
    • G01N33/66—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving blood sugars, e.g. galactose

Definitions

  • the present invention relates to a miniaturized method to analyse carbohydrates that are present in less than picomole amounts in a sample. More particularly, the present invention relates to the fluorescent labelling of carbohydrates, the efficient separation of the labelling reagent from the labelled carbohydrates and subsequent electrophoretic separation for the analysis of the carbohydrates.
  • Protein-linked carbohydrates can be analysed using a variety of high-resolution techniques, such as high performance liquid chromatography (HPLC) 8 , capillary electrophoresis (CE)9 and mass spectrometry (MS) 10 , especially matrix-assisted laser desorption/ionization time-of-flight (MALDI-TOF) MS.
  • HPLC high performance liquid chromatography
  • CE capillary electrophoresis
  • MS mass spectrometry
  • MALDI-TOF matrix-assisted laser desorption/ionization time-of-flight
  • US patent 5,569,366 describes the use of APTS (9-aminopyrene-1 ,4,6-trisulfonic acid) to derivatize carbohydrates and after separation by capillary electrophoresis picomole amounts of carbohydrates can be detected.
  • APTS 9-aminopyrene-1 ,4,6-trisulfonic acid
  • a large amount of glycoconjugate for example a glycoprotein
  • the present invention relates to a novel, miniaturized, electrophoretic methodology.
  • the efficient removal of APTS, after derivatizing the carbohydrates leads to the unexpected finding that samples comprising less than picomole amounts of carbohydrates can be analysed.
  • the present invention aims at providing an identification method for the analysis of carbohydrates.
  • the invention aims at providing an extremely sensitive method for the analysis of underivatized carbohydrates which are present in less than 1 picomole in a sample.
  • the invention further aims at providing a method comprising derivatizing carbohydrates with a fluorescent label, removing the not reacted portion of said fluorescent label, electrophoretically separating said derivatized carbohydrates and detecting said derivatized carbohydrates.
  • the invention further aims at providing an essential clean-up step for the removal of the not reacted portion of the fluorescent label. Said clean-up step is an essential feature of this invention and may comprise the use of a gel filtration and/or gel permeation resin and /or a size-selective membrane.
  • the invention further aims at providing a device for the detection of less than 1 picomole of underivatized carbohydrates present in a sample.
  • the current invention further aims at providing a method comprising a DNA-sequencer.
  • the invention thus provides a sensitive method for sequencing free carbohydrates or carbohydrates derived from glycoconjugates present in a sample.
  • the invention also aims at providing diagnostic tools for the analysis of carbohydrate structures in specific disease processes and for the identification of specific carbohydrate structures derived from recombinant glycoproteins.
  • the invention aims at providing a sensitive analysis method for carbohydrate structures wherein said carbohydrates are bound to other biomolecules or are derived from organisms.
  • the glycoprotein is deglycosylated using PNGase F after binding to the Immobilon-P membrane at the bottom of a well in a 96-well plate.
  • the deglycosylation mixture is split in two parts. One part is treated with acetic acid in order for full conversion to reducing carbohydrates to occur.
  • the mixture is loaded on a AG-50-WX8 microcolumn in a well of a filterplate.
  • the eluate is loaded on a MALDI-TOF-MS target and subjected to MALDI-TOF mass spectrometry.
  • the other half of the deglycsolation mixture is evaporated to dryness at the bottom of a well of a tapered-96-well plate.
  • the glycans present in the pellets are then derivatized with APTS.
  • the derivatisation is stopped by the addition of water and the mixture is loaded on a Sephadex G10 microcolumn in a well of a filterplate.
  • the eluate is concentrated by vacuum evaporation and loaded on a lane of a sequencing gel after addition of internal standard and formamide.
  • final analysis can be preceded by digestion of the analytes with exoglycosidase arrays.
  • Detector response curve shows the linear relationship between the amount of starting glycan and the fluorescence response detected by the ABI 377 detection system over at least three orders of magnitude (from low femtomole to picomole amounts).
  • This figure illustrates the use of the disclosed methodology in profiling and sequencing of a standard mixture of triantennary trisialylated N-glycans.
  • 2 pmole total carbohydrate was used as the starting material and after APTS-derivatisation and cleanup using Sephadex G10 microcolumns, the sample was divided in six parts, each containing about 300 fmole of total labelled carbohydrate. 1 part was used to give the native profile (as shown in panel 2). Notice the isomeric resolution obtained.
  • This figure illustrates the described principles in the analysis of the N-glycans of human ⁇ i-acid glycoprotein.
  • the amount of starting material is 0,5 ⁇ g, which is equivalent with 10 pmole of said glycoprotein.
  • the protein was deglycosylated after binding to the PVDF membrane in a well of a 96-well plate, as described under Materials and Methods. After APTS derivatisation and cleanup using the Sephadex G10-packed multiscreen approach, the sample was split in 6 equal parts and the rest of the analysis was performed as described under Figure 3.
  • the peaks marked with an arrow on the figure are corresponding to analytes present in a mixture of rhodamine- labelled 6-,18-,30-,42-meric oligonucleotides.
  • the observed profiles are entirely consistent with the N-glycan structures reported in reference 19 and our profiles give accurate quantitative information on the analytes, which was not possible using MALDI-TOF-MS. This exemplifies the complementarity of
  • Figure 5 Sequencing of the N-glycans present on serum glycoproteins.
  • GU glucose units.
  • Panel 1 malto-oligosaccharide ladder. The number of glucose units of the 5-mer and the 10-mer is indicated above the figure.
  • Panels 2-7 profiles obtained after digestion of serum glycoprotein N-glycans with different exoglycosidase mixtures.
  • Panel 2 profile of the desialylated serum glycoprotein N-glycans.
  • Panel 3 sialidase + ⁇ -1 , 3/4/6- fucosidase.
  • Panel 4 sialidase + ⁇ -1 ,3/4-fucosidase.
  • Panel 5 sialidase + galactosidase.
  • Panel 6 sialidase + galactosidase + -1 ,3/4/6-fucosidase.
  • Panel 7 same as Panel 6, but with extra ⁇ -N-acetylhexosaminidase digestion.
  • Panels 8-10 provide the profiles obtained after the following digestions of the reference glycan of structure shown in Fig.8a.
  • Panel 8 sialidase
  • Panel 9 sialidase + galactosidase
  • Panel 10 sialidase + galactosidase + ⁇ -1 ,3/4/6-fucosidase.
  • Panel 11 shows the profile obtained from the reference glycan of structure shown in Fig.8b and Panel 12 the profile of the same glycan after galactosidase digest.
  • Panel 13 and 14 the profiles are shown of the reference glycan of structure shown in Fig.8c, with sialidase digestion and sialidase + galactosidase digestion, respectively.
  • CDGIb phosphomannose isomerase
  • CDGIc ⁇ -1 ,3- glucosyltransferase
  • CDGId ⁇ -1 ,3-mannosyltransferase
  • CDGIe dolichol-phosphate- mannose synthase
  • CDGIf the dolichol chaperone enzymatically equivalent to the enzyme deficient in Lee 35 CHO cells.
  • B1 , B2, and B3 show structures of reference glycans. Detailed description of the invention
  • the labelled saccharide can be electrophoretically separated and detected with a fluorescent detector.
  • Another approach to the separation and detection of carbohydrates involves the reductive amination of monosaccharides and oligosaccharides with 8-aminonaphthalene-1 ,3,6-trisulfonic acid (ANTS) followed by their electrophoretic separation and detection utilizing a CCD fluorescence imaging device.
  • ANTS labelled oligosaccharides have been separated and detected also using capillary electrophoresis techniques.
  • reagents which fluoresce and react with the reducing ends of saccharides or aminated saccharides include 5-aminonaphthalene-2- sulfonate (ANA) 3-(4-carboxybenzoyl)-2-quinolinecarboxaldehyde (CBQCA), 4- aminobenzonitrile (ABN).
  • ANA 5-aminonaphthalene-2- sulfonate
  • CBQCA 3-(4-carboxybenzoyl)-2-quinolinecarboxaldehyde
  • ABSN 4- aminobenzonitrile
  • a widespread apparatus for the high resolution electrophoretic separation and fluorometric detection of biomolecules is the Applied Biosystems series 377 DNA-sequencer. These instruments use 12, 36 or 48 cm polyacrylamide-based gels as the separation matrix and contain an Ar-laser to excite the fluorescence of the analytes.
  • APTS 8-amino-1 ,3,6-pyrenetrisulfonic acid 1 3
  • APTS 8-amino-1 ,3,6-pyrenetrisulfonic acid 1 3
  • the present invention essentially overcomes in an efficient way the problems encountered in downscaling (miniaturizing) the analysis of underivatized carbohydrates and glycoconjugates to the subpicomole level. These problems typically involve sample loss due to adsorption on container or resin walls and contamination of the sample by components present in potentially all of the reagents and vessels used in the process.
  • the invention provides an electrophoretic method to analyse underivatized carbohydrates present in less than 1 picomole amount in a sample.
  • the present electrophoretic method is advantageous in that it is possible to start with less than 1 picomole amount of underivatized carbohydrates, such as for example glycosylated proteins, present in a sample, thus effectively increasing the sensitivity of the overall analytical process one to two orders of magnitude as compared to the situation in the prior art.
  • the electrophoretic method comprises: (1) derivatizing the carbohydrates with a derivatizing agent, (2) removing the portion of the derivatizing agent which did not derivatize with said carbohydrate, (3) separating the derivatized carbohydrates and (4) the detection of the derivatized carbohydrates.
  • the carbohydrates can be enzymatically and/or chemically released from a glycoconjugate using a miniaturized procedure. After release, the carbohydrates are derivatized with APTS.
  • An essential feature of this invention is that the APTS label can be efficiently separated from the labelled carbohydrates in order not to interfere with the background of the detection due to overloading of the analytical separation system.
  • the present invention provides an improved analytical method for analyzing sugars and carbohydrates including mono-, oligo- and polysaccharides as well as sugar or carbohydrate containing compounds.
  • a 'glycoconjugate' means any compound containing a carbohydrate moiety.
  • 'carbohydrate' it is meant a collection of molecules which can be arbitrarily subdived into four major groups- monosaccharides, disaccharides, oligosaccharides, and polysaccharides and derivatives of the members of these four groups.
  • Polysaccharides (literally this term means many sugars) consist of more than six units of monosaccharides or derivatives thereof and represent most of the structural and energy-reserve carbohydrates found in nature. Large molecules that may consist of as many as 10,000 monosaccharide units or derivatives thereof linked together, polysaccharides vary considerably in size, in structural complexity, and in sugar content; several hundred distinct types have thus far been identified.
  • Cellulose the principal structural component of plants, is a complex polysaccharide comprising many glucose units linked together; it is the most common polysaccharide.
  • the starch found in plants and the glycogen found in animals also are complex glucose polysaccharides.
  • the words "glycan” and "carbohydrate” are interchangeably.
  • the carbohydrates can belong to any class of protein or lipid linked carbohydrates comprising asparagine- linked glycans, Ser/Thr-linked glycans, glycosaminoglycans or proteoglycan derived sugars, glycolipid-derived glycans and GPI-anchor derived carbohydrate species.
  • the processes of the present invention provide enhanced detectability of femtomole amounts of glycans.
  • the standard buffer used for DNA-sequencing gels was used throughout. The borate in this buffer forms complexes of different stability with different carbohydrate isomers increases the chance that isomers can be electrophoretically resolved (as exemplified in Figure 3).
  • underivatized carbohydrates When referring to 'to analyse underivatized carbohydrates', carbohydrates can be bound or not to another molecule.
  • 1 picomole or less of underivatived carbohydrates can be analysed.
  • the latter can mean that a sample comprising carbohydrates contains 1 picomole or less total carbohydrates or a specific carbohydrate of interest is present in less than 1 picomole in said sample.
  • the latter means that in a non-limiting example if total blood serum is analysed for diagnosis (which normally comprises more than 1 picomole amounts of underivatized carbohydrates) of a specific disorder that a disease specific carbohydrate structure is often present in only 1 picomole or less of said carbohydrate and it can still be analysed.
  • said carbohydrate analysis can be efficiently carried out on 1 , 0.5, 0.1 , 0.01 or 0.001 picomole amounts of carbohydrates present in a sample.
  • higher amounts of free carbohydrates can be analysed with this method such as 2, 5, 10, 50, 100, 500, 1000, 10.000 picomole or even higher amounts.
  • sample' comprises, but is not limited to, a specific sugar or a mixture of sugars in solution, a blood sample, a cell extract, urine, sperm, micro-dissected cells, cerebrospinal fluid, industrial water and sewage.
  • the efficient separation analysis of the derivatized carbohydrates is carried out by an electrophoretic process and the standard DNA- sequencing equipment can be used.
  • the high sensitivity of the overall analytical process is obtained using known in the art deglycosylation procedures, a derivatisation step with APTS, a highly efficient and reproducible novel post- derivatisation cleanup step to remove the APTS involving Sephadex G10.
  • the novel post-derivatisation step described here in the invention is surprisingly efficient considering 1) the enormous excess of free label that is efficiently removed and 2) the small difference in molecular size between the free label and the labelled analytes.
  • this unexpected finding could be carried out by for example spin columns packed with other suitable size selective media known in the art as a gel filtration or gel permeation resin, or using a size-selective membrane. It should be said that removal of APTS by means of gel filtration has been described in the art but samples of 1 nanomole or more were applied to the resin.
  • this novel method can be made medium-throughput by performing all sample preparation steps (enzymatic deglycosylation with PNGaseF, desalting, derivatisation with APTS and post-derivatisation cleanup) in 96-well based plates. This integrated sample preparation scheme is compatible with capillary electrophoresis platforms already in use.
  • the present invention provides methods for the electrophoretic separation and detection of saccharides including simple sugars, oligosaccharides, carbohydrates which or free or bound to other molecules (glycoconjugate).
  • a wide range of analytical applications can take benefit from the present invention, including carbohydrate and glycoprotein sequencing, industrial sugar and carbohydrate analytical procedures, drug analysis, drug discovery, and diagnostic procedures.
  • carbohydrate and glycoprotein sequencing including carbohydrate and glycoprotein sequencing, industrial sugar and carbohydrate analytical procedures, drug analysis, drug discovery, and diagnostic procedures.
  • those skilled in the art will appreciate that the ability to readily analyse less than 1 picomole amount of underivatized carbohydrates by a method which gives complementary information to mass spectrometry is very desirable in the analysis of carbohydrate and glycoconjugate sequencing reaction products and the diagnostic analysis of biological samples.
  • the present invention provides a method for the analysis of glycoproteins in combination with a DNA-sequencer it is clear for the person skilled in the art that this method can be applied in connection with capillary electrophoresis systems adaptable to a laser induced fluorescence detector.
  • capillary electrophoresis systems adaptable to a laser induced fluorescence detector.
  • Such systems include the P/ACE series Capillary Electrophoresis Systems (Beckman Instruments, Inc., Fullerton, Calif.).
  • the processes described herein can be applied with any electrophoresis system which is adaptable with a laser induced fluorescence detector.
  • an analytical system that has the same overall sensitivity as MALDI- TOF-MS and gives complementary information such as resolution of isobars and accurate quantitation is of utmost importance in the field of glycan analysis.
  • the term "derivatized carbohydrate compounds” means carbohydrates which have been labelled with a fluorescing compound. In order for analytes to migrate under electrophoretic conditions they must carry a charge and since many carbohydrates are not charged, the fluorescing compounds are preferably charged. Fluorescing compounds such as 9-aminopyrene-1 ,4,6-trisulfonic acid (APTS) and 8- aminonaphthalene-1 ,3,6-trisulfonate (ANTS) are particularly suitable in the practice of the present invention. Regarding the detection of the derivatized carbohydrates, any detection method known in the art can be applied, preferably the detection is carried out with a laser such as a diode laser, a He/Cd laser or an argon-ion laser.
  • a laser such as a diode laser, a He/Cd laser or an argon-ion laser.
  • the invention provides a device for the detection of less than 1 picomole of underivatized carbohydrates comprising: (1) a derivatization chamber, (2) a clean-up apparatus wherein said clean-up occurs via gel filtration and/or gel permeation resin, in which the fluid flow is driven by centrifugational force or vacuum, which remove the excess fluorescent and/or spectrometric label, and (3) an electrophoretic apparatus such as a capillary electrophoresis and/or DNA-sequencing equipment for the separation and detection of derivatized carbohydrates.
  • the carbohydrate analysis method can be supplemented pre- electrophoretically with an internal standard mixture that is labelled with a chromophore or fluorophore that is different from the label attached to the carbohydrate analytes.
  • the internal standard allows for accurate and reproducible determination of the electrophoretic mobilities of the carbohydrate analytes by referencing these mobilities to the mobilities of the components in the internal standard mixture.
  • a rhodamine-labelled oligonucleotide standard GenescanTM 500 (Applied Biosystems, Foster City, CA, USA) or a mixture of rhodamine-labelled 6-,18-,30-,and 42-meric oligonucleotides may be added to the derivatised glycans before profiling.
  • the analysis method of this invention can be used to distinguish recombinant glycoproteins form their endogenous counterparts on the basis of differences in their protein-linked glycosylation as is exemplified herein.
  • the method of the present invention can be used for diagnosing diseases where at least one modification of one or more carbohydrates is implicated.
  • the analysis method of this invention can be used for sensitive diagnostic purposes for disorders such as carbohydrate storage disorders, carbohydrate-deficient glycoprotein syndromes, cancer, mood disorders, disorders in the biosynthesis of protein or lipid-linked glycans and more generally in any field involving changes in carbohydrate profiles.
  • the method of the invention can be used for the identification of carbohydrate structures of recombinant glycoproteins in biological fluids.
  • the method can be used to differentiate between endogenously made erythropoetin and recombinant erythropoetin by analysing the carbohydrate structures of erythropoetin.
  • the analysis method of this invention can be used for the identification and/or structural characterization of carbohydrates which are bound to other biomolecules.
  • biomolecules molecules comprising nucleic acids, proteins, other carbohydrates and lectins.
  • this invention can be used for the analysis of carbohydrates derived from organisms comprising prions, viruses, bacteria, fungi, mycoplasma and parasites.
  • parasites it is meant organisms such as for example protozoa and worms.
  • the analysis method of this invention can be used for obtaining the information on the structure of the carbohydrates (this generally refers to sequencing of carbohydrate structures) by combining the current analysis method with a procedure known to those skilled in the art as chemical and exoglycosidase sequencing and modifications thereof.
  • the internal standardisation principle introduced in this invention is implementable on the capillary electrophoresis systems that are equipped with multi-colour fluorescent detection systems.
  • the acrylamide percentage of the gel used here (12%) and the other electrophoresis conditions were optimized for maximum resolution of a malto- oligosaccharide reference mixture with degrees of polymerization of 4-25. This is the size range that is most relevant for N-glycan mixtures derived from mammalian and plant tissues.
  • the standard buffer used for DNA-sequencing gels was used throughout (see Experimental protocol).
  • the borate in this buffer forms complexes of different stability with different carbohydrate isomers, thus increasing the chance of electrophoretically resolving these isomers. It should be straightforward to optimize the electrophoresis parameters for other classes of protein-linked glycans, if necessary. As shown in Fig. 2, the detector-response curve is linear over more than three orders of magnitude and 1 fmol of labelled chitotetraose (test compound) can be detected with a signal to noise ratio of >3.
  • the 96-well based cleanup-procedure of APTS-derivatized glycans described here is also applicable for capillary electrophoresis of these compounds ⁇ and thus allows the utilization of the full potential sensitivity of this methodology (for example, as commercialized by Beckman Inc., Fullerton, CA, USA).
  • MALDI-TOF-MS of underivatized N-linked glycans is a well established technique 4 . 1 ⁇ - 1 9 a t about the same level of sensitivity as the DNA- sequencer-assisted methodology descirbed here.
  • the two techniques can give complementary information on the analytes.
  • the present methodology can be combined with carbohydrate release procedures described in the literature 4 allowing the reproducible release of the N-linked glycans from low picomole amounts of glycoproteins, and with sequencing procedures of the glycans under study with arrays of exoglycosidases, thus generating a wealth of structural information on the glycans from an amount of starting material, the analysis of which was previously only feasible with MALDI-TOF mass spectrometry.
  • MALDI-TOF MS does not resolve isobaric structures, which are very prevalent in the glycan field, and is not absolutely accurate in the quantification of the analytes.
  • the high sensitivity of the methodology gives access to the study of glycosylation-related research topics that were previously not feasible. For example, it is now feasible to study the glycosylation potential of rare cell populations in the bloodstream and in the nervous system and the changes in this potential during development or during disease progress.
  • binding carbohydrates are isolated from a complex pool of labelled glycans by affinity procedures with the lectin, followed by structural studies of the binding carbohydrates with exoglycosidase sequencing. This structural information can then be used in drug discovery processes aimed at inhibiting the lectin-carbohydrate interaction under study.
  • the method discussed here can be used for the detection of recombinant glycoproteins in vivo if the glycosylation of the recombinant glycoprotein differs from the glycosylation of its endogenous equivalent. For example, this is the case with recombinant erythropoietin, IFN-gamma and tissue plasminogen activator which are derived from CHO cells and their native counterpart. As the profiles obtained from endogenous and recombinant glycoproteins are generally different, the latter can be reliably detected with our invention. In the case of EPO, this can allow for a more reliable test to be developed to detect the inappropriate use of recombinant EPO, for example in sports competitions.
  • the carbohydrates structures of recombinant glycoproteins from mammalian non-human and non-chicken cell lines or organisms such as recombinant human EPO (rhuEPO) derived from Chinese Hamster Ovary (CHO) or Baby Hamster Kidney (BHK), contain varying amounts of 5-N- glycolylneuraminic acid.
  • this amount is 1-2% of total sialic acids present on the N-glycans of the protein (HOKKE CH, BERGWERFF AA, VANDEDEM GWK, KAMERLING JP, VLIEGENTHART JF.EUROPEAN JOURNAL OF BIOCHEMISTRY 228: (3) 981-1008 MAR 15 1995).
  • This particular glycosylation feature is undetectable on human blood plasma proteins (Muchmore et al. Am. J. Phys. Anthropol. 107, 187-198, 1998).
  • a sample such as blood or urine
  • this glycosylation analysis can be performed after a pre-purification of the analytes of interest.
  • erythropoietin present in the sample is enriched using affinity techniques involving anti-erythropoietin antibodies or recombinant erythropoeitin receptors (Zhan et al., Protein Engineering, 12: (6), 503-513, 1999) or preparations thereof, after which a glycosylation analysis is performed.
  • Another embodiment can consist of isolating the protein-linked carbohydrates from all or a fraction of the proteins present in the said sample, followed by enrichment of the 5-N- glycolylneuraminic acid-containing saccharides using lectins and preparations thereof specifically recognizing said glycosylation epitope.
  • Such lectins include those isolated from Morus alba (Ratanapo-Sunanta et al., Plant Science Shannon 139 (2), 141-148, 1998), Pila globosa (Swarnakar et al., Biochemical and Biophysical Research Communications 178 (1), 85-94, 1991), Scylla serrata (Mercy et al. Eur.J.Biochem 215 (3) 697-704, 1993) and Anadara granosa (Dam et al., Biochemical and Biophysical Research Communications 196 (1), 422-429, 1993).
  • this glycoprotein is injected intravenously or subcutaneously in varying doses, normally several times per week for an extended period.
  • a typical treatment may consist of subcutaneous injection of 100 IU rhuEPO per kg body weight three times per week.
  • 1 IU of rhuEPO is equivalent with about 10 ng of the glycoprotein, the dose corresponds to about 70 microgram.
  • Calculating a molecular weight of about 35 kDa this dose is equivalent to about 2 nmol of rhuEPO.
  • Further calculating a blood volume of 5 liter one obtains a value of 0,4 pmol per ml blood.
  • each rhuEPO molecule contains 3 N-glycans that can be removed from the protein using chemical or enzymatic reactions. One can thus expect a yield of 1 ,5 pmol of total N-glycans originating from the rhuEPO. As mentioned, about 1-2 % of these glycans bear at least one 5-N-glycolylneuraminic acid modification.
  • Recombinant human EPO (rhuEPO) was purchased from Roche (Cat. No. 1120166). 1 unit of this preparation equals about 10-15 ng protein. A dilution series of 5,2,1 ,0.5 and 0.1 units EPO was analysed. The entire procedure was as described in the current patent application except that 50 mM APTS was used in the derivatization procedure and that the cleanup over the microtiter-Sephadex G10 bed was carried out two consecutive times, with volume reduction of the first eluate using vacuum evaporation.
  • the tetraantennary glycan is the major peak in the profiles, whereas the N-acetyllactosamino-derivative is the second tallest peak.
  • the ratio between the two peaks is easily quantifiable with an amount of starting material down to 0.5 units (which corresponds with only 100 femtomole EPO), making this assay useful for purposes where distinction between endogenous and recombinant EPO is necessary, such as in detection of abuse of this protein in sports competitions or in monitoring the EPO source in treatment of patients with chronic kidney insufficiency.
  • CDG I Congenital Disorders of Glycosylation are a group of inherited disorders characterized by alterations in protein glycosylation.
  • CDG I is hallmarked by the absence of one or more N-glycan chains on serum glycoproteins.
  • CDG la Several subtypes have been described, of which the most common is CDG la, caused by mutations in the PMM2 gene, which result in deficiency in phosphomannomutase activity. This enzyme is essential for the synthesis of GDP-mannose, a substrate that is indispensable in the biosynthesis of N- linked glycans.
  • CDG I is commonly diagnosed using isoelectric focusing of serum proteins, followed by immunodetection of transferrine isoforms. This technique basically detects different sialoforms of transferrine and has been extensively described.
  • N-glycans do not give detailed structural information on the N-glycans present.
  • sialidase digest (Fig.1 , panel 2), it's size is estimated to be 9 monosaccharide units from comparison with the malto- oligosaccharide reference ladder.
  • sialidase/ ⁇ -1 ,4-galactosidase digest (Fig. 1 , panel 5)
  • this glycan loses two galactose residues and it further loses two GlcNAc residues upon digestion with ⁇ -N-acetylhexosaminidase (Fig.1 , panel 7).
  • the residual glycan migrates at the position of the Man 3 GlcNAc 2 core N-glycan structure.
  • Peak 2 represents the biantennary, bi- ⁇ -1 ,4- galactosylated core ⁇ -1 ,6-fucosylated glycan. This is corroborated by the exact comigration of a reference glycan of this structure with peak 2, both undigested and after ⁇ -1 ,4-galactosidase and ⁇ -1 ,4-galactosidase/bovine kidney fucosidase double digest (compare panel 2 and panel 8; panel 5 and panel 9; panel 6 and panel 10, respectively).
  • Peak 3 the glycan corresponding to peak 3 is about 2 monosaccharide units taller than glycan 1 , is not digestable by bovine kidney fucosidase and comigrates with a triantennary fully ⁇ -1 ,4-galactosylated reference glycan.
  • ⁇ -1 ,4- galactosidase removes 3 galactose residues from the glycan (shift of 3 glucose units between panel 2 and panel 6), after which the glycan is 1 monosaccharide unit taller than the remnant of glycan 1 , in accordance with the one extra GlcNAc residue that is expected for a triantennary glycan when compared to a biantennary structure.
  • Peak 4 this glycan is one glucose unit taller than the triantennary unfucosylated glycan of peak 3 and is sensitive to both bovine kidney (panel 3) and almond meal fucosidase (panel 4), after which digests the glycan is converted to peak 3.
  • the fucose residue present on this glycan is ⁇ -1 , 3/4 linked.
  • the glycan of peak 4 is a branch-fucosylated derivative of glycan 3.
  • the exact position of the branch fucose residue cannot be determined using enzyme digests.
  • a patient can get a score ranging from 0 to 3 with increasing deviation of the normal profile.
  • These scores are shown in Fig. 2.
  • An obvious biochemical parameter that could correlate with the 'glyco'-phenotype is the residual PMM enzymatic activity.
  • the residual PMM activity as measured in patient skin fibroblasts is indicated in Fig.2. It is apparent that there is a tendency for the PMM activity to be low when the serum N-glycan profile differs severely from the normal one, but the correlation is by no means perfect. However, the standard deviation for the PMM activity measurements is very large, which may hamper correlation analysis. Subsequently, we compared the obtained profile scores with the clinical presentation of the patients.
  • CDGIa The symptoms of CDGIa can vary widely from patient to patient from a severely debilitating disease to a relatively mild mental retardation. It has been observed before that the severity of the disease does not correlate well with residual PMM activity.
  • the clinical presentation of the CDGIa patients studied here is indicated in Fig.2 in terms of 'severe', 'moderate to severe', 'mild' and 'very mild'. The patients were classified in these categories by experienced clinicians of the European CDGI national reference centers. For two non-European patients, relatives of each other and both with a low score for their serum glycoprofiles, no clinical information could be obtained. From comparison of the profile scores with the clinical evaluation, it can be concluded that mild cases of the disease also have a low profile score.
  • RCM buffer 8M urea, 360 mM Tris, pH 8.6, 3.2 mM EDTA
  • APTS derivatization reaction and cleanup N-glycan derivatisation with 8-amino-1 ,3,6-pyrenetrisulfonic acid and removal of excess free label were as described recently. Briefly, the deglycosylation mixture was evaporated to dryness and a 1 ⁇ l 1 :1 mixture of 20 mM APTS (Molecular Probes, Eugene, CA, USA) in 1.2 M citric acid and 1 M NaCNBH 3 in DMSO was added. The derivatisation was allowed for 18h at 37°C. After this, the reaction was quenched by the addition of 10 ⁇ l of distilled (Dl) water.
  • Dl distilled
  • Exoglycosidase digestions 1 ⁇ l batches of the cleaned-up derivatized N-glycans were transferred to 250 ⁇ l PCR tubes or tapered-well microtiter plates for treatment with exoglycosidase arrays. In this example all digestions were done by overnight incubation at 37°C in 10 ⁇ l 20 mM sodium acetate pH 5.0.
  • the enzymes used in this study are: Arthrobacter ureafaciens sialidase (2 U/ml, Boehringer, Mannheim, Germany); Diplococcus pneumoniae ⁇ -1 ,4-galactosidase (1 U/ml, Boehringer, Mannheim, Germany); Jack bean ⁇ -N-acetylhexosaminidase (30 U/ml, Glyko, Novato, CA, USA); Jack bean ⁇ -mannosidase (100 U/ml, Sigma Biochemicals, Bornem, Belgium); bovine epididymis ⁇ -fucosidase (Glyko, Novato, CA, USA) and almond fucosidase (Glyko, Novato, CA).
  • Prerunning was done at 3000 V for 1h.
  • the electrophoresis voltage during separation was 3500 V and data were collected for 3h (separation of glycans up to 15 glucose units in size).
  • Data analysis was performed using the Genescan 3.1 software (Applied Biosystems, Foster City, CA, USA).
  • Using the positions of the peaks of the internal ROX-oligonucleotide standard all lanes on the same gel were aligned with the lane containing the APTS-labelled malto- oligosaccharide standard. After this alignment, samples on different gels can be easily and reliably compared by aligning the positions of the malto-oligosaccharides present on both gels. For clarity and to allow black-and white reproduction of the figures presented in this contribution, the peaks corresponding to the ROX-labelled internal standards have been omitted after the alignment procedure.
  • the present methodology can be used for the follow-up of drug-induced glycosylation changes.
  • Another example could be the analysis of the effect of potential inhibitors of glycosyltransferases or glycosidases on the glycosylation profile displayed by tumor cells, both in vitro and in vivo.
  • the exquisite sensitivity of the disclosed methodology only requires small biopsies of tumor tissue to be made.
  • Glycosaminoglycans are a class of polysaccharides with an extremely complex structure and several important biological functions, some of which are influenced by the exact patterns of sulphation and the exact sequence of the glycan chain. Their role as specific ligands for proteins has recently become apparent and this binding often modulates the function of the bound protein. Structure elucidation of the GAG fragments which bind to specific proteins can be accomplished by mass spectrometry and by chromatographic and electrophoretic methodologies. In particular, the use of specific glycosidases is paramount to the exact sequence determination of the GAG fragments. This technology has been recently developed for heparin and heparan sulphate.
  • GAGs consist of a disaccharide repeat of glucosamine and hexuronic acid.
  • O-sulfation which occurs predominantly at C-2 of IdoA and of the glucosamine residues, but also rarely at C-2 of GlcA and C-3 of glucosamine, adds structural complexity to the chain. At each step only a fraction of potential substrates are modified, resulting in a very large structural diversity.
  • the labeled saccharide is separated from the excess free label by passing the sample through a bed of Sephadex G10 and eluting the glycans with 4 times 10 microliter of water.
  • Structure determination All chemical cleavage steps and enzymatic digests are performed as fully described in Turnbull, JE et al. (1999) Proc. Natl. Acad. Sci. USA 96, 2698.
  • Analysis due to the inherent charge of the GAG chains, separation on size is more important than in N-glycan analytical separations. Therefore, a higher gel concentration is generally more favorable and we use 12, 15 and 20% gels to achieve a good resolution. Sample preparation and all other electrophoresis conditions are as described for N-glycans herein.
  • glycoproteins present on glycoproteins
  • Purifying these proteins by classical chromatographic techniques such as hydrophobic interaction chromatography and ion exchange chromatography without disturbing the glycoform distribution is often difficult as the carbohydrate chains present on the protein can strongly influence the physical properties of the glycoprotein entity.
  • glycan structure strongly influences the purification behavior of a glycoprotein, it is of definite advantage to have a technology that can give the glycan structural information without having to go through purification optimization. Typically, one would like to immunopurify a small quantity of the protein under study and get the glycan structural information from there.
  • glycoprotein impurities will often still be present and could significantly alter the obtained structural information.
  • SDS-PAGE separation of the post-immunopurification mixture can be a powerful second purification step.
  • methods to obtain the glycans after SDS-PAGE separation are limited.
  • One successful approach is the in-gel digestion with PNGase F, after which the N-glycans are eluted from the gel and analysed.
  • this method makes use of Coomassie Brilliant Blue detection of the proteins and has not been successfully applied on the submicrogram scale.
  • the Protoprep II SDS-PAGE kit with meltable polyacrylamide formulation was obtained from National Diagnostics in beta-test version. The kit is now available commercially from this manufacturer. Sypro-Orange was obtained from Molecular Probes. Immobilon-P lined 96-well filterplates were purchased from Millipore. Recombinant human EPO was obtained from Roche. All other chemicals were analytical grade reagents from major suppliers.
  • Samples for SDS-PAGE were prepared according to well known procedures in the art. The gel casting and electrophoresis procedures were performed according to the directions of the manufacturer. After completion of the separation, the gel was stained with sypro-orange (diluted 1 :5000 in 7% acetic acid) for 60 minutes and rinsed in 7.5% acetic acid for 30 seconds. The protein bands were visualized using UV- transillumination, as is normally done for ethidiumbromide-stained agarose gels in DNA-analytical procedures. The bands of interest were cut out and the gel pieces transferred to clean pre-weighed eppendorf tubes.
  • the pH of the gel piece was adjusted using repetitive incubations at room temperature with a volume of the gel dissolution buffer sufficient for completely immersing the gel piece until the pH of the solution reached 9.
  • the gel was dissolved using three volumes of the dissolution buffer and incubated at 60°C until complete dissolution was observed.
  • the dissolved acrylamide was removed by acetonitrile precipitation. Batches of 100 ⁇ l acetonitrile were added to the sample until no further precipitation is observed. The resulting white-coloured mass was removed using centrifugation and the supernatant was recovered.
  • RCM buffer 8 M ureum, 360 mM Tris, pH 8,6 and 3,2 mM EDTA
  • the procedure involves binding of the protein to an Immobilon P membrane in a 96-well plate, washing and deglycosylation of PNGase F.
  • the glycans are derivatized with APTS (8-aminopyrene-1 ,3,6-trisulfonic acid) and the excess APTS is removed over a Sephadex G10 bed packed in another 96-well filterplate.
  • APTS 8-aminopyrene-1 ,3,6-trisulfonic acid
  • the labeled glycans are analysed on a 10% polyacrylamide gel on a standard Applied Biosystems 377A DNA-sequencer.
  • the detection sensitivity of this technology is about 3 fmol of labeled carbohydrate.
  • a dilution series of recombinant human EPO 1 unit of rhuEPO equals 10-15 ng of the protein.
  • the protocol was elaborated in detail by Papac et al. 4 . Briefly, the PVDF membrane at the bottom of the wells of a Multiscreen-IP plate (Millipore, Bedford, CA, USA) was wetted with 100 ⁇ l methanol, washed three times with 300 ⁇ l of water and once with 50 ⁇ l of RCM buffer (8M urea, 360 mM Tris, pH 8.6, 3.2 mM EDTA). The glycoprotein was loaded in the wells, containing 10 ⁇ l RCM buffer. Subsequently, additional RCM buffer was added to a minimal volume of 50 ⁇ l. Protein was bound to the membrane with a gentle vacuum. This step was followed by two wash steps with 50 ⁇ l RCM buffer.
  • the bound protein was then reduced by the addition of 50 ⁇ l of 0.1 M dithiothreitol in RCM buffer and incubation at 37°C for 1h.
  • the reducing solution was removed by vacuum and the wells were washed three times with 300 ⁇ l of water.
  • Carboxymethylation was performed by addition of 50 ⁇ l of 0.1 M iodoacetic acid in RCM buffer and incubation for 30 min at room temperature in the dark. After removal of this solution, three washes with 300 ⁇ l of water followed. The remaining protein binding capacity of the wells was blocked by incubation with 100 ⁇ l 1 % polyvinylpyrrolidone 360 in water at room temperature for 1 h.
  • microcolumns of about 300 ⁇ l packed resin are easily and reproducibly obtained.
  • the cation exchange resin removes the protein and salt present in the deglycosyation mixtures with sufficient efficiency to allow direct MALDI- TOF-MS as described elsewhere 4 (results not shown).
  • APTS derivatisation reaction We have found it unnecessary to remove the PNGase prior to derivatisation with APTS, as this practice does not lead to the appearance of contaminant peaks in the size range of 3-25 glucose units.
  • the deglycosylation mixture was evaporated to dryness at the bottom of the tapered well microtiterplate using a Savant vacuum centrifuge equipped for plates and a 1 ⁇ l 1 :1 mixture of 20 mM APTS (Molecular Probes, Eugene, CA, USA) in 30% acetic acid and 1M NaCNBH 3 in DMSO was added to each well. After carefull vortexing and short centrifugation of the plate, it was incubated up side down at 37 °C overnight, tightly wrapped in parafilm. The following morning, the reaction is quenched by the addition of 20 ⁇ l of water.
  • the gel contains 12% of a 19:1 mixture of acrylamide:bisacrylamide (Biorad, Hercules, CA, USA) and is made up in the standard DNA-sequencing buffer (89 mM Tris, 89 mM borate, 2.2 mM EDTA). Polymerization is catalyzed by the addition of 200 ⁇ l 10% ammoniumpersulfate solution in water and 20 ⁇ l TEMED. The gels were of the standard 36 cm well-to-read length throughout the study. Prerunning is done at 3000 V for 1h. After prerunning the gel, the wells are thoroughly rinsed with the sequencing buffer and 1.8 ⁇ l of the samples is loaded.
  • the electrophoresis voltage during separation is 4000 V and data are collected for 5h (separation of glycans up to 25 glucose units in size). Data analysis is performed using Genescan 3.1 software. We use the same fluorescence- overlap correction matrix as for DNA sequencing using BigDye dye terminators on our machine. The fluorescence of APTS-derivatised carbohydrates and rhodamine- labelled oligonucleotides is obviously readily resolved. References

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Abstract

L'invention concerne un procédé miniaturisé d'analyse des glucides qui sont présents en quantités proches de la picomole dans un échantillon. Cette invention concerne plus particulièrement le marquage fluorescent ou spectroscopique des glucides, la séparation efficace du réactif de marquage des glucides marqués et la séparation électrophorétique subséquente pour l'analyse desdits glucides.
PCT/EP2001/006042 2000-05-26 2001-05-25 Procede d'analyse de glucides presents en quantites picomolaires WO2001092890A1 (fr)

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Cited By (13)

* Cited by examiner, † Cited by third party
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WO2003040389A2 (fr) * 2001-11-07 2003-05-15 Florian Schweigert Procede de detection de substances endogenes et exogenes dans l'organisme
WO2003087833A2 (fr) * 2002-04-16 2003-10-23 Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw Marqueur de la cirrhose du foie
EP1468286A2 (fr) * 2001-12-18 2004-10-20 Serenex, Inc. Dispositif de centrifugation integre de capture de proteines par affinite
EP2112506A1 (fr) 2008-04-24 2009-10-28 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Procédé d'identification automatique de haut rendement de glucides et motifs de composition de mélange de glucides ainsi que les systèmes associés
US8000904B2 (en) 2004-04-15 2011-08-16 Momenta Pharmaceuticals, Inc. Methods and products related to the improved analysis of carbohydrates
WO2012010851A1 (fr) 2010-07-23 2012-01-26 Cambridge Enterprise Limited Electrophorèse capillaire des glucides
US8209132B2 (en) 2004-04-15 2012-06-26 Momenta Pharmaceuticals, Inc. Methods and products related to the improved analysis of carbohydrates
WO2013180819A1 (fr) * 2012-06-01 2013-12-05 Arizona Board Of Regents Acting For And On Behalf Of Arizona State University Système, procédé et dispositif d'analyse des glucides
US9395352B2 (en) 2007-04-06 2016-07-19 Arizona Board Of Regents On Behalf Of Arizona State University Devices and methods for target molecule characterization
US9593372B2 (en) 2008-10-06 2017-03-14 Arizona Board Of Regents On Behalf Of Arizona State University Nanopore based sequencer
CN106814121A (zh) * 2015-11-30 2017-06-09 中国科学院大连化学物理研究所 一种基于微流控芯片的血清糖谱分型方法
US10288599B2 (en) 2012-10-10 2019-05-14 Arizona Board Of Regents On Behalf Of Arizona State University Systems and devices for molecule sensing and method of manufacturing thereof
WO2020151799A1 (fr) 2019-01-21 2020-07-30 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Méthodes avancées d'identification haute performance automatisée de glucides et de motifs de composition de mélange de glucides et systèmes correspondants, ainsi que méthodes d'étalonnage de systèmes de détection de fluorescence à longueurs d'onde multiples correspondantes, fondées sur de nouveaux colorants fluorescents

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US5569366A (en) * 1995-01-27 1996-10-29 Beckman Instruments, Inc. Fluorescent labelled carbohydrates and their analysis
WO1999038012A1 (fr) * 1998-01-21 1999-07-29 The University Of Newcastle Research Associates Limited Detection de groupes phosphate ou hydrate de carbone sur les residus serine
US5964999A (en) * 1996-01-19 1999-10-12 Beckman Instruments, Inc. Methods for profiling oligosaccharides released from glycoproteins

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5569366A (en) * 1995-01-27 1996-10-29 Beckman Instruments, Inc. Fluorescent labelled carbohydrates and their analysis
US5964999A (en) * 1996-01-19 1999-10-12 Beckman Instruments, Inc. Methods for profiling oligosaccharides released from glycoproteins
WO1999038012A1 (fr) * 1998-01-21 1999-07-29 The University Of Newcastle Research Associates Limited Detection de groupes phosphate ou hydrate de carbone sur les residus serine

Cited By (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003040389A2 (fr) * 2001-11-07 2003-05-15 Florian Schweigert Procede de detection de substances endogenes et exogenes dans l'organisme
WO2003040389A3 (fr) * 2001-11-07 2004-03-11 Florian Schweigert Procede de detection de substances endogenes et exogenes dans l'organisme
EP1468286A2 (fr) * 2001-12-18 2004-10-20 Serenex, Inc. Dispositif de centrifugation integre de capture de proteines par affinite
EP1468286A4 (fr) * 2001-12-18 2008-08-20 Serenex Inc Dispositif de centrifugation integre de capture de proteines par affinite
WO2003087833A2 (fr) * 2002-04-16 2003-10-23 Vlaams Interuniversitair Instituut Voor Biotechnologie Vzw Marqueur de la cirrhose du foie
WO2003087833A3 (fr) * 2002-04-16 2004-05-06 Vlaams Interuniv Inst Biotech Marqueur de la cirrhose du foie
US7335512B2 (en) 2002-04-16 2008-02-26 Vlaams Interubiversitair Instituut Voor Biotechnologie Vzw Marker for measuring liver cirrhosis
CN1662817B (zh) * 2002-04-16 2012-06-27 福拉姆斯大学生物技术研究所 N-多糖在检测肝硬化和肝癌中的用途
US8209132B2 (en) 2004-04-15 2012-06-26 Momenta Pharmaceuticals, Inc. Methods and products related to the improved analysis of carbohydrates
US8000904B2 (en) 2004-04-15 2011-08-16 Momenta Pharmaceuticals, Inc. Methods and products related to the improved analysis of carbohydrates
US9395352B2 (en) 2007-04-06 2016-07-19 Arizona Board Of Regents On Behalf Of Arizona State University Devices and methods for target molecule characterization
US10330632B2 (en) 2007-04-06 2019-06-25 Arizona Board Of Regents On Behalf Of Arizona State University Devices and methods for target molecule characterization
EP2533039A1 (fr) 2008-04-24 2012-12-12 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Procédé dýidentification automatique de haut rendement de glucides et motifs de composition de mélange de glucides ainsi que les systèmes associés
EP2112506A1 (fr) 2008-04-24 2009-10-28 Max-Planck-Gesellschaft zur Förderung der Wissenschaften e.V. Procédé d'identification automatique de haut rendement de glucides et motifs de composition de mélange de glucides ainsi que les systèmes associés
US9593372B2 (en) 2008-10-06 2017-03-14 Arizona Board Of Regents On Behalf Of Arizona State University Nanopore based sequencer
US10442771B2 (en) 2008-10-06 2019-10-15 Arizona Board Of Regents On Behalf Of Arizona State University Trans-base tunnel reader for sequencing
WO2012010851A1 (fr) 2010-07-23 2012-01-26 Cambridge Enterprise Limited Electrophorèse capillaire des glucides
WO2013180819A1 (fr) * 2012-06-01 2013-12-05 Arizona Board Of Regents Acting For And On Behalf Of Arizona State University Système, procédé et dispositif d'analyse des glucides
US10288599B2 (en) 2012-10-10 2019-05-14 Arizona Board Of Regents On Behalf Of Arizona State University Systems and devices for molecule sensing and method of manufacturing thereof
US11137386B2 (en) 2012-10-10 2021-10-05 Arizona Board Of Regents, Acting For And On Behalf Of Arizona State University Systems and devices for molecule sensing and method of manufacturing thereof
CN106814121A (zh) * 2015-11-30 2017-06-09 中国科学院大连化学物理研究所 一种基于微流控芯片的血清糖谱分型方法
CN106814121B (zh) * 2015-11-30 2019-07-23 中国科学院大连化学物理研究所 一种基于微流控芯片的血清糖谱分型方法
WO2020151799A1 (fr) 2019-01-21 2020-07-30 MAX-PLANCK-Gesellschaft zur Förderung der Wissenschaften e.V. Méthodes avancées d'identification haute performance automatisée de glucides et de motifs de composition de mélange de glucides et systèmes correspondants, ainsi que méthodes d'étalonnage de systèmes de détection de fluorescence à longueurs d'onde multiples correspondantes, fondées sur de nouveaux colorants fluorescents

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