WO2012010851A1 - Capillary electrophoresis of carbohydrates - Google Patents

Abstract

This invention relates to the capillary electrophoresis of carbohydrates using sets of mobility standards that comprise two or more standard compounds comprising a primary amine group, such as amino acids and peptides, which are labelled with a fluorophore and have different ratios of mass to charge. These standards allow accurate comparison of the mobilities of saccharides in different separations and may, for example, be useful in identifying or characterising multiple saccharides in a sample.

Description

Capillary Electrophoresis of Carbohydrates

This invention relates to the analysis of carbohydrates by capillary electrophoresis.

Various methods are available currently for the analysis of carbohydrate structures. Polyacrylamide gel electrophoresis (PAGE) is often used to analyse carbohydrate structures or distinguish or separate carbohydrate substances (Jackson P., (1990) Biochem. J, 270,705-713; Jackson, P. 1992 Anal. Biochem. 196, 238-244; US5104508; US5340453; US5472582; US6294667). Carbohydrate substances are labelled with a fluorophore and the labelled substances are then subjected to electrophoretic separation in a polyacrylamide gel. The fluorophore typically comprises a primary amino group which reacts with the aldehyde or ketose group of reducing saccharides through reductive amination to form a covalent bond. The fluorophores used in this labelling method include 8-aminonaphthalene-l , 3 , 6-trisulphonic acid (ANTS) and 2- aminoacridone (AMAC) . These fluorophores facilitate the

electrophoretic separation of neutral or charged reducing

saccharides. PAGE may be useful in distinguishing or identifying the reducing saccharides in carbohydrate substances.

Fluorophores may also be used for the detection and or visualisation of the labelled saccharides in the gel matrix after separation by PAGE. Visualisation may be effected by the naked eye or by other means, by way of example, photography or electronic imaging. This method is known commonly as Fluorophore Assisted Carbohydrate

Electrophoresis (FACE™ ; Glyko Inc) or Polyacrylamide Gel

Electrophoresis of Fluorophore labelled Saccharides (PAGEFS)

Goubet et al. Plant J. (2009) 60, 527-38) Barton CJ et al Planta. 2006 Jun; 224 ( 1 ): 163-74 ; Brown DM et al Plant J. 2009 Feb; 57 ( 4 ): 32- 46; and Brown DM et al . Plant J. 2007 Dec; 52 ( 6 ): 1154-68 report the application of the FACE method to the analysis of the carbohydrate structures of plant cell walls. This method is known as

Polysaccharide Analysis using Carbohydrate gel Electrophoresis ( PACE ) .

Labelled reducing saccharides have been separated by capillary electrophoresis (CE) (Guttman A. Nature. 1996 Apr ; 380 ( 6573 ) : 61-2 ; Guttman A, et al Electrophoresis. 1996 Feb; 17 (2) : 12-7; Guttman A et al Anal Biochem. 1996 Jan 15; 233 (2) : 234-42; Guttman A et al

Electrophoresis. 1995 Oct; 16 (10) : 1906-11; Khandurina J et al

Electrophoresis. 2004 Oct ; 25 ( 18-19) : 3117-21. -aminopyrene-1 , 3, 6- trisulfonic acid (APTS) is used as a fluorophore for labelling the reducing saccharides. The labelling method is in principle reductive amination as disclosed in the FACE method. The fluorophore enables the detection and or visualisation of the substances during and/or after the electrophoresis. The fluorophores APTS and ANTS both carry negative ionic charges and both can be used for the electrophoresis of both neutral and charged reducing saccharides.

Capillary electrophoresis (CE) DNA sequencers have been used for the separation of reducing saccharides that have been labelled with the fluorophore APTS by reductive amination (Lee KJ et al Biochem

Biophys Res Commun. 2009 Mar 6; 380 (2 ) : 223-9; Callewaert N et al Glycobiology. 2001 Apr; 11 ( 4 ) : 275-81 ) .

CE-DNA sequencers have advantages over other methods for saccharide analysis: they are more sensitive than PACE and can analyse large numbers of samples rapidly. However, a significant drawback is the variability in the mobility of a fluorophore labelled reducing saccharide in different capillaries, even when electrophoresed simultaneously using identical electrophoretic conditions

(Khandurina J et al Electrophoresis. 2004 Oct; 25 (18-19) : 3122-27) . This variability creates uncertainty in the identification of saccharides using the CE mobility data and makes the comparison of separation profiles from different samples difficult.

Oligonucleotide standards have been proposed for electrophoretic analysis of carbohydrate structures (WO01/92890; EP2112506) .

However, oligonucleotide standards are unstable and have

insufficient electrophoretic mobility to cover the full range of mobilities required for analysis of carbohydrates or for the resolution of small saccharides. Furthermore, because they increase in minimum increments of lbp, oligonucleotide standards are often insufficiently flexible for use as standards to resolve specific saccharides in a separation.

This invention relates to the development of electrophoretic mobility standards that facilitate the analysis of carbohydrates by capillary electrophoresis. These methods allow accurate comparison of the electrophoretic mobilities of fluorophore labelled

saccharides in different separations and may be useful in

identifying or characterising multiple saccharides in a sample.

An aspect of invention provides a method of analysing saccharides in a sample comprising:

(i) providing a sample comprising one or more saccharides,

(ii) labelling the saccharides in the sample with a first fluorophore,

(iii) providing a set of fluorescent mobility standards; said set comprising two or more standard compounds, each standard compound comprising a primary amine group and being labelled with a second fluorophore, wherein the standard compounds have different ratios of mass to charge and the first and second fluorophore emit fluorescence at different wavelengths,

(iv) mixing the set of standards and the labelled sample, (v) separating the mixture by capillary electrophoresis to produce a separation profile of the amount of fluorescence from the first and second fluorophores relative to electrophoretic mobility, wherein fluorescence from the first fluorophore is indicative of saccharides in the sample and fluorescence from the second fluorophore is indicative of the standards; and,

(vi) determining from the mobilities of the first and second fluorophores in the profile, the mobility of one or more saccharide in the sample relative to the mobility of one or more of said set o standards .

A method may further comprise producing the set of fluorescent mobility standards by;

(a) providing two or more standard compounds comprising a primary amine group, said standard compounds having different ratios of mass to charge, and;

(b) labelling said compounds with a second fluorophore, wherein the first and second fluorophore emit fluorescence at different wavelengths .

One or more saccharides in the sample may be detected, identified and/or characterised from their mobility in the separation profile relative to the mobility of one, two or more standards from the set of standards.

A CE separation profile plots the amount of fluorescence detected at a detection wavelength by a detector against time. Fluorescently labelled molecules which move faster under capillary electrophoresis and therefore display high electrophoretic mobility are detected first followed by progressively slower molecules which display lower mobilities. Separation profiles may be expressed graphically, numerically or in any other convenient form. Fluorescently labelled molecules are detected by detecting presence of a peak in the amount of fluorescence detected at the detection wavelength at a particular time; or in some embodiments, the presence of two or more peaks in the amount of fluorescence detected at the detection wavelength at particular times. The separation profile may therefore display the mobilities of multiple peaks of fluorescence which each represent fluorescently labelled molecules in the sample.

Saccharides labelled with the first fluorophore are detected by the presence of peaks in the amount of fluorescence which is detected at a first detection wavelength at particular times in the separation and standards labelled with the second fluorophore are detected by the presence of a peak in the amount of fluorescence which is detected at a second detection wavelength at particular times in the separatio .

The first fluorophore is used to label saccharides in the sample, so fluorescence from the first fluorophore in the separation profile is due to saccharides in the sample. Fluorescence from the first fluorophore may be detected at the first detection wavelength i.e. upon excitation; the first fluorophore emits light at the first detection wavelength which is then detected. Preferably, the second fluorophore does not emit at the first detection wavelength, so fluorescence at the first detection wavelength in the separation profile is all or substantially all produced by the first

fluorophore. A peak of fluorescence from the first fluorophore in the separation profile (i.e. an increased amount of fluorescence at a particular mobility relative to higher and lower mobilities) is indicative of the presence of a labelled saccharide in the sample which has that mobility. The mobilities of different labelled saccharides in the sample may therefore be determined from the separation profile. The amount of a saccharide in the sample may be quantified from the size of the fluorescent peak in the separation profile. For example, the amount of a first saccharide relative to a second saccharide may be determined from the relative sizes of the fluorescent peaks.

In some embodiments, predetermined quantities of one, two, three, four or more known saccharides may be added to the sample before labelling with the first fluorophore. These known saccharides may be used as quantitation markers. For example, a different quantity of each known saccharide may be added, so that a simple standard curve of their fluorescent peak volumes versus the quantity added to the sample can be plotted. Quantities of other saccharides in the sample may be determined from this standard curve.

The second fluorophore is used to label the standard compounds so fluorescence from the second fluorophore in the separation profile is due to the standards (and not the saccharides in the sample) . Fluorescence from the second fluorophore may be detected at the second detection wavelength. In some embodiments, the first

fluorophore produces little or no emission at the second detection wavelength, so fluorescence at this wavelength is all or

substantially all produced by the second fluorophore. A peak of fluorescence from the second fluorophore in the separation profile (i.e. an increased amount of fluorescence at a particular mobility relative to higher and lower mobilities) is indicative of a standard having that mobility. The mobilities of labelled standards may therefore be determined from the separation profile.

Each of standards in the set of standards used for a separation has the same fluorescence emission characteristics because they are all labelled with the same second fluorophore, but each possesses different electrophoretic characteristics■ because the standard compounds which are linked to. the second fluorophore possess different ratios of mass to charge. The mobility of a saccharide relative to standards may be determined using the methods described herein as a fractional (or standardised) mobility .

The fractional mobility of a saccharide may be determined relative to the mobilities of two of the standards in the separation profile. Fractional mobility may be expressed as: (T_u-To)/ (Ti-To) where T_u is the mobility of the saccharide in a separation profile, Ti and T₀ are the mobilities of the first and second standards, respectively.

Preferably, the first standard has a greater mobility than the saccharide in the separation profile and the second standard has a lower mobility than the saccharide in the separation profile. The fractional mobility of the saccharide relative to the first and second standards will then be between 0 and 1.

Preferably, the first standard is a standard (i.e. a standard peak in the separation profile) whose mobility is higher than the saccharide of interest. The second standard is preferably any standard in the separation profile whose mobility is lower than the saccharide of interest. In some embodiments, the first standard may be the fastest standard in the set of standards (i.e. the highest mobility) and the second standard may be the slowest standard in the set of standards (i.e. the lowest mobility). In other embodiments, standards whose mobilities are close to the mobility of the

saccharide of interest may be selected to determine fractional mobility. For example, the first. and second standards may be the standards within the set of standards which display the closest mobilities to the saccharide of interest. In other embodiments, the mobility of the saccharide of interest may be coincident with the first or second standard in the separation profile or may have a higher or lower mobility than both of the first and the second standards. The fractional mobility of the saccharide relative to the first and second standards may then be <= 0 or >=1.

The choice of which standards in the set of standards are selected as the first and second standards for calculating fractional mobility depends on the circumstances and the preferences of the user .

A method may comprise the step of selecting a set of standard compounds which comprise a first standard compound having an electrophoretic mobility greater than the saccharide of interest in the sample having greatest mobility and a second standard compound having a mobility less than saccharide of interest in the sample having lowest mobility. For example, the mobility of the first standard compound may be up to 10% up to 5%, or up to 1% or less faster than the fastest saccharide of interest in the sample in a raw, unnormalised CE separation profile (i.e. a direct plot of the detected fluorescence from the first and second fluorophores over time) and the mobility of the second standard compound may be up to 10%, up to 5%, or up to 1% slower than the slowest saccharide of interest in the sample. In some embodiments, standard compounds with mobilities outside these ranges may be employed.

The electrophoretic mobility of a labelled standard compound may be determined from its mass/charge ratio. The mass/charge ratio of a compound labelled with a fluorophore may be determined using standard techniques. In^' some embodiments, the standards for a particular separation may be selected to include labelled standard compounds with mobilities which are close to the mobilities of known saccharides (when labelled with the first fluorophore) . These known saccharides may be known or suspected to be present in the sample. These standards may then be used to calculate fractional mobility. The use of standards with mobilities close to the saccharide of interest increases the accuracy and discriminatory power of the fractional mobility calculation .

A method may comprise the step of selecting a set of standard compounds which comprise a first standard compound which has an electrophoretic mobility which is greater than a saccharide of interest in the sample and a second standard compound which has a mobility which is less than the saccharide of interest. For example, the mobility of the first standard compound may be up to 10% up to 5%, or up to 1% faster than the saccharide of interest in the sample and the mobility of the second standard compound may be up to 10%, up to 5%, or up to 1% slower than the saccharide of interest in the sample in a raw, non-standardised CE separation profile (i.e. a direct plot of the detected fluorescence from the first and second fluorophores over time) . In some embodiments, standard compounds with mobilities outside these ranges may be employed .

The fractional mobility of a saccharide in the sample may be used to identify the saccharide. For example, the fractional mobility of an unidentified saccharide in the sample may be compared with the fractional mobilities of known saccharides or significant known peaks . relative to the same standards.

The fractional mobilities of known saccharides may be recorded in a database or may be determined from a reference separation profile of known saccharides with the first and second standards. The separation profile of a test sample may be compared to the separation profiles of one or more reference samples. This may useful, for example, in identifying one or more saccharides in the test sample, or otherwise analysing or obtaining information from the separation profile of the test sample.

A reference sample may contain one or more known saccharides.

Alternatively, a reference sample may contain saccharides which, although unidentified, produce a separation profile which may be compared with the separation profile of the test sample to provide information about the test sample. For example, the reference sample may be an enzyme digest in which not all of the saccharide products have been fully identified, but which produces a separation profile characteristic of the activity of the enzyme. Any differences in the separation profile of the test sample relative to the reference sample, such as missing or new peaks, may be indicative of

differences in enzyme activity.

The fractional mobilities of the saccharides in the one or more reference samples and the test samples may be determined and compared, in order to identify the saccharides in the test sample or otherwise characterise it.

Capillary electrophoresis may be carried out as described herein multiple times on the same sample to allow for statistical variation between individual separations. For example, the methods described herein may be carried out in a 96 well microtitre plate in which multiple wells, for example 4, 6, 12 or more wells, contain the same sample. Data from different separations of the same sample may be analysed using standard statistical techniques. The use of fractional mobilities eliminates, or reduces to insignificant levels, the variation in the electrophoretic

mobilities of saccharides between different separations. The fractional mobility of any saccharide may be compared with that of any other saccharide. Saccharides that have identical fractional mobilities may be distinguished from any saccharides, either in a single sample or in any other sample in which the same standards are co-electrophoresed with those saccharides under similar conditions. In addition, the fractional mobilities of known saccharides may be measured and recorded and used to identify unknown saccharides in samples .

The analysis of saccharides as described herein may be useful in a range of applications.

For example, it may be useful in the analyses of the structure of plant cell walls; comparison of plants with mutations of selected enzymes involved in plant cell wall synthesis; for characterising novel hydrolytic enzymes; or for monitoring industrial processes which involve the degradation of plant cell walls, such as

production of materials for biofuel production.

The analysis of saccharides as described herein may also be useful in profiling metabolites or extracellular glycans, such as

glycosaminoglycans , heparan sulphate, chondroitin sulphate,, keratan sulphate and hyaluronic acid, from humans and other animals. This may be useful, for example, in the diagnosis, prognosis and/or assessment of disease conditions, for example, diseases associated with the extracellular matrix.

The analysis of saccharides as described herein may also be useful in the analysis and quality control of therapeutic proteins expressed, for example, in cell culture, such as growth factors and antibodies. For example, the glycosylation of a therapeutic protein produced in an expression system may be determined and the effects of changes in the expression system on glycosylation assessed.

Any sample which contains one or more saccharides, for example monosaccharides or oligosaccharides, may be analysed as described herein. A suitable sample may be derived from any naturally

occurring or synthetic material.

In some embodiments, the sample may contain saccharide metabolites. Analysis of the metabolites as described herein may facilitate the characterisation and analysis of metabolic pathways.

The one or more saccharides in the sample may be pure (i.e. the sample may consist of saccharides), partially pure or impure. For example, the sample may comprise other carbohydrate or non- carbohydrate substances, in addition to the one or more saccharides.

The sample may be treated prior to analysis. For example, a sample may be treated to purify or partially purify the saccharides therein using conventional purification techniques.

In some embodiments, a sample may be treated to degrade or partially degrade polysaccharides in the sample and produce saccharides.

Polysaccharides which may be degraded include plant cell wall polysaccharides, such as cellulose, xylans, pectin, arabinoxylans , callose, hemicelluloses , starch, amylose, amylopectin, mannan, and galactomannan and non-plant polysaccharides, such as polysaccharides associated with glycoproteins, for example N-linked glycans, 0- linked glycans, and glycosaminoglycans . Non-plant polysaccharides may include glycogen, chitin and chrysolaminarin .

Suitable polysaccarides may be derived from plants, fungi, animals, bacteria, viruses and algae. A sample containing one or more polysaccharides may be treated enzymatically, for example using a hydrolytic enzyme, chemically, for example by alkaline extraction, or mechanically, for example by milling or grinding, to produce saccharides, which can then be labelled as described above. Hydrolytic enzymes include exo- and endo-glycosidases , for example xylanases and xylosidase. Suitable treatments are known in the art. Samples which contain one or more polysaccharides may include samples of glycosylated polypeptides expressed in eukaryotic expression systems. The glycosylated polypeptides may be treated with one or more hydrolytic enzymes, such as glycosidases , to degrade the glycosyl moieties and liberate saccharides, such as mono- or oligosaccharides for labelling and analysis, as described herein. The extent and nature of the glycosylation of the

polypeptide may be determined from analysis of the mono- or oligosaccharides . Samples which contain one or more polysaccharides may include samples of plant material containing plant cell wall

polysaccharides, such as cellulose and hemicelluloses . The plant material may be treated to degrade the polysaccharides and liberate saccharides, such as mono- or oligosaccharides for labelling and analysis, as described herein. Suitable treatments include treatments with hydrolytic enzymes such as exo- and endo- glycosidases, for example xylanases and xylosidase.

Plant material may include any material from a plant which includes plant cell walls, for example, a seed, stalk, leaf, trunk, stem or any other tissue. The material may be processed or partly processed before treatment to degrade polysaccharides. Material from any type of plant may be employed, including biofuel plants, such as wheat, corn (maize) , switchgrass, sugar beet, sugar cane, rapeseed, palm oil, miscanthus, willow and jatropha.

In some embodiments, the effect of the treatment on the

polysaccharides in the sample may be well-characterised. Analysis of the saccharides in the sample produced by such a treatment may provide information about the polysaccharide. This may be useful for example, in analysing the structures of polysaccharides, such as plant cell wall polysaccharides and the glycosyl moieties of proteins. In other embodiments, the effect of the treatment on the polysaccharides may be uncharacterised . Analysis of the saccharides in the sample produced by such a treatment may provide information about the effect of the treatment. Typically, a sample for analysis as described herein contains one or more different saccharides.

Suitable saccharides for analysis include mono- and

oligosaccharides, for example straight chain or branched saccharides containing up to 10, up to 15 , up to 20, up to 30, up to 40, up to 50, up to 60 or more monomeric units.

Monosaccharides may include xylose, arabinose, fucose, rhamnose, glucose, galactose, mannose, glucuronic acid, and galacturonic acid

Oligosaccharides may include branched and straight chain oligomers of the above monosaccharides, including xylan and mannose, which may further comprise one or more side chain substitutions by arabinose, glucuronic acid, methylated derivatives of glucuronic acid and other moieties. Monosaccharides naturally occur in a wide variety of polymeric combinations both in straight chain and branched

structures and may also be linked covalently to other substances such as lignin. Oligosaccharides may include di-, tri-, tetra-, penta-, hexa-, nona- or deca-saccharides .

Saccharides may also comprise modifications and/or substitutions, for example methylation, sulfonylation, phosphoprylation,

carboxylation and/or amination.

Preferably, the saccharides in the sample are reducing saccharides i.e. they contain an aldehyde or ketose group.

In the methods described herein, saccharides in the sample are labelled with the first fluorophore.

Fluorophores are molecules which absorb light energy at a first wavelength and then emit the energy as light at a second wavelength. Many fluorophores are known in the art and any suitable fluorophore may be used as a first fluorophore. A fluorophore with any

convenient wavelength of excitation may be used as the first fluorophore. For example the wavelength of excitation of the first fluorophore may be in the UV, visible or IR range.

In some embodiments, it may be convenient to employ a CE-DNA sequencer. In such embodiments, a first fluorophore may be selected whose wavelength of excitation is the wavelength of the lasers found in the DNA sequencer, for example 488 and/or 515 nm.

The first fluorophore typically possesses zero net charge (e.g.

AMAC) , one or more negative charges (e.g. ANTS and APTS), or one or more positive charges (e.g. 6-aminoquinoline or fluorophores disclosed in US6294667) such that saccharides labelled with the first fluorophore migrate through the capillary electrophoresis. Neutral fluorophores, such as AMAC, may, for example, be useful in analysing acidic saccharides, such as glucuronic acid and

galacturonic acid. Suitable first fluorophores are well known in the art and include 8 - aminopyrene-1, 3, 6-trisulfonic acid (APTS) (Sharrett et al (2009) Org Biomol Chem. 2009 Apr 7 ; 7 (7 ) : 1 61-70) . APTS has Xex = 428 nm, Xem = 500 nm.

Saccharides in the sample may be labelled with the first fluorophore by any convenient method. In some preferred embodiments, the first fluorophore comprises a primary amino group which reacts with reducing saccharides by reductive amination to form a covalent bond.

In some embodiments, the sample may be purified or partially purified after labelling with the first fluorophore to remove excess unbound fluorophore, which may obscure the separation profile during capillary electrophoresis, for example in regions where fast running (neutral and acidic) monosaccharides occur.

The standards which make up the set of standards are selected to provide a set of fluorescence peaks that may be detected and visualised in different separation profiles. The profile of peaks displayed by the set of standards will be similar in different separation profiles. The standard which corresponds to each peak in the separation profile is easily recognised and identified by virtue of its fluorescence characteristics and its electrophoretic mobility relative to other standards in the set.

Each fluorescent standard in the set comprises a standard compound which is attached to a second fluorophore. This combination of components provides great flexibility in the range of

electrophoretic mobilities that can be attained by the set of standards, without affecting the fluorescent characteristics of the standards . The two or more standard compounds for the set of standards are selected such that the set includes labelled standards with two or more different mobilities. Standard compounds which have different electrophoretic mobilities may be selected, for example, on the basis of their ratios of mass to charge (M/Z) . The mass to charge ratio of any compound may be readily determined by the skilled person from the known mass and the charge at the pH of the separation.

A standard in the set may migrate as a single species which produces a single peak in the CE separation profile; or may migrate as multiple species with different mobilities which produces multiple peaks in the separation profile. For example, the CE separation profile of an individual fluorescent standard may consist of one, two, three or more peaks. In some embodiments, multiple peaks may be caused by impurities in the standard or artefacts of the coupling reaction of the standard compound to fluorophore. Preferably, the CE separation profile for a standard compound consists of a single major peak. The mobility of any peak which is produced by a standard may be selected for use in determining the fractional mobility of a saccharide in the sample relative to that standard. Preferably, the greatest single peak caused by the standard is selected. Peaks from two different standards (a first and a second standard) from the set of standards may be selected for use in determining the fractional mobility of saccharides in the sample. As described above, in preferred embodiments, the selected peak of a first standard has a greater mobility than the saccharide of interest in the sample and the selected peak of a second standard has a lesser mobility than the saccharide of interest in the sample. In other embodiments, the selected peaks of the first and second standards both have either greater or lesser mobility than the saccharide of interest (such that the fractional mobility is >1 or <0, or the selected peaks of one of the first and second standards is coincident with the saccharide of interest.

Preferably, the set of standards includes standards with a range of different mobilities. In general, as the number of standards within the set of standards increases, the accuracy of the alignment of all the peaks in the separation profile increases, especially when computer software is used to align the peaks in a profile. For example, a set of standards may comprise 2, 3, 4, 5, 6, 7, 8, 9, 10 or more individual labelled amino acids or peptides.

For example, the separation profile of the set of standards may contain one or more peaks, preferably two or more. For example, a separation profile may contain 2 to 12 peaks, preferably 6 to 10 peaks although more are possible. This allows the most appropriate standards to be selected for determining the fractional mobility of the saccharides in the sample.

The choice of molecules for the set of standards may depend on the likely mobilities of the saccharides in the sample.

For example, the mass/charge (M/Z) ratios of a set of fluorescently labelled standards may range from 50 to 700, 100 to 650 or 150 to 600.

As described above, the set of standards may include a standard with a lower mobility than any of the labelled saccharides in the sample and a standard with a greater mobility than any of the labelled saccharides in the sample. The mobilities of the saccharides in the sample may be standardised relative to these standards (i.e. the^' highest and lowest mobility standards may be used as the first and second standards for determining fractional mobility, as described above) . In some embodiments, the set of standards includes a first standard which has a greater mobility than a saccharide of interest in the sample and a second standard which has a lower mobility than the saccharide of interest. The first and second standards may be used for determining fractional mobility, as described above.

Suitable first and second standards may be selected for each saccharide in a separation profile. For example, the closest standard with a greater mobility may be selected as a first standard and the closest standard with a lower mobility may be selected as a second standard. Preferably, the standard compounds in the set are selected such that suitable first and second standards with mobilities close to each saccharide in the sample or suspected of being in the sample are present.

The standard compounds for use as standards contain a primary amino group .

Any compound that contains a primary amine group which is capable of reacting with the fluorophore may be used as a standard compound. This may include, for example, simple primary amines which can be coupled to a charged fluorophore and compounds with a primary amine group and a negatively charged group but which are not common naturally occurring amino acids, such as 2-aminoethyl hydrogen sulphate

Suitable standard compounds include amino acids, peptides, secondary amines; sulfhydryl containing compounds, including peptides with a terminal cysteine group; and amino group containing aromatic compounds, such as benzoic acid or anthranilic acid.

Suitable standard compounds may not interfere with the fluorescence detection of the labelled saccharides, for example by emitting fluorescence at the same wavelength as the first fluorophore. Preferably, each standard compound contains only one primary amine. However, in other embodiments, a standard compound may contain multiple primary amines.

Preferred standard compounds include amino acids, and peptides, for example di-, tri-, tetra-, penta-, hexa-, nona- or deca-peptides . In some embodiments, the set of standards may consist of amino acids and/or peptides labelled with the second fluorophore. Amino acids may be naturally occurring or non-naturally occurring amino acids.

Preferably, a mixture of different standard compounds is used to produce the set of standards, for example a mixture of different amino acids, peptides and/or other compounds containing primary amines. The choice of standard compounds depends on the different mass/charge ratios required for the set of standards for a

particular application.

Preferably, standard compounds for use as standards are water- soluble after labelling with the second fluorophore.

Suitable molecules include amino adipic acid, aspartic acid (D), aspartyl-aspartic acid (Asp-Asp) (DD) , aspartyl-aspartyl-aspartic acid (Asp-Asp-Asp) (DDD) ; aspartyl-aspartyl-aspartyl-aspartic acid (Asp-Asp-Asp-Asp) (DDDD) ; cysteic acid; glutamic acid (E) ; glutamyl- glutamic acid (Glu-Glu) (EE); glycine (Gly) (G) ; glycyl-glycyl (Gly- Gly) (GG) ; glycyl-glycyl-glycine (Gly-Gly-Gly ) (GGG) ; Glycyl-glycyl- glycyl-glycine (Gly-Gly-Gly-Gl ) (GGGG) ; Glycyl -glycyl-glycyl- glycyl-glycine (Gly-Gly-Gly-Gly-Gly) (GGGGG) ; Glycyl-glycyl-glycyl- glycyl-glycyl-glycine (Gly-Gly-Gly-Gly-Gly-Gly ) (GGGGGG) ; 2- aminoethyl hydrogen sulphate; 2-aminoethyl hydrogen sulphate; L- cysteinesulfinic acid; L-homocysteic acid; L-homocysteine sulfinic acid; S-carboxymethyl-L-cysteine; DL-2-methylglutamic acid hemihydrate; DL-threo-B-methylaspatic acid; L-2-aminoadipic acid; taurine; and hypotaurine.

Other naturally occurring or non-naturally occurring amino acids and peptides may also be used as standard compounds for producing the set of standards.

Other suitable standard compounds will be readily apparent to the skilled person.

The choice of labelled standard compounds in the set of standards is determined by the mobilities of the saccharides of interest in the sample . Amino acids or peptides may be selected as standard compounds such that the mobility ^' standards have small differences in mobilities. This may allow more accurate standardisation between separate electrophoretic separations. For example, amino acids or peptides may be selected which generate mobility standards having a

difference in mass charge ratio of at least 0.1, at least 0.5, or at least 1, and 100 or less, 50 or less, 40 or less, 30 or less, 20 or less, 15 or less, 10 or less, or 5 or less.

Multiple standard compounds are labelled with the second fluorophore to produce the set of standards.

Preferably, the individual standard compounds are labelled with the second fluorophore separately and then mixed to produce the set of standards. Conveniently, the choice and amount of each labelled standard in the mixture may be selected to optimise the positions and heights of the peaks produced by the set of standards in the separation profile. Suitable second fluorophores possess either zero net charge (e.g. DY480xl) or alternatively, one or more positive or negative charges (for example DY481xl), such that the labelled standard possesses migrates through the capillary electrophoresis.

Second fluorophores with a negative net charge may be useful in labelling negative and neutral standard compounds. Second

fluorophores with a zero net charge on the fluorophore may be useful in labelling acidic standard compounds, such as amino acids and peptides.

The labelled standard compounds may possess an overall negative charge and migrate through the capillary electrophoresis towards the anode .

In other embodiments, the labelled standard compounds may possess an overall positive charge and migrate through the capillary

electrophoresis towards the cathode. This may be useful in discriminating positively charged

fluorescently labelled saccharides, for example, negative or uncharged saccharides which are labelled with a first fluorophore having a positive charge (US6294667) In some embodiments, saccharides may be discriminated as described herein by analysing both mobility towards the cathode and mobility towards the anode.

The excitation wavelength of the second fluorophore may be the same or different to the excitation wavelength of the first fluorophore. In embodiments in which a CE-DNA sequencer is employed, it may be convenient for the excitation wavelength of the second fluorophore to correspond to the wavelength of the lasers in the DNA sequencer. For example, the excitation wavelength of the second fluorophore may be 488 nm and/or 515 nm.

The emission wavelength of the second fluorophore is different to the emission wavelength of the first fluorophore i.e. the first fluorophore emits at a first detection wavelength and the second fluorophore emits at a second detection wavelength. Preferably, the emission wavelength of the second fluorophore is sufficiently different from the emission wavelength of the first fluorophore that fluorescent emission from the second fluorophore does not

significantly interfere with the detection of fluorescent emission from the first fluorophore. In other words, the second fluorophore produces no or substantially no fluorescent emission at the first detection wavelength.

This allows fluorescent emission from the two fluorophores to be distinguished in separation profiles and prevents fluorescence from the mobility standards from interfering with the detection or quantitation of saccharides in the sample. The presence of standards labelled with the second fluorophore in the separation does therefore have any significant effect on the detection, mobility or quantitation of saccharide in the sample.

In some preferred embodiments, the first fluorophore produces no or substantially no fluorescent emission at the second detection wavelength,, such that fluorescent emission from the first

fluorophore does not significantly interfere with the detection of fluorescent emission from the second fluorophore.

To avoid interference between the fluorescent emissions of the sample and the standards, the second fluorophore preferably has a different (e.g. a higher or lower) Stoke' s shift than the first fluorophore. This causes the peak of fluorescent emission from the second fluorophore to occur at a different (e.g. longer or shorter) wavelength than the peak of fluorescent emission from the first fluorophore, even when the excitation wavelengths are the same or similar. For example, the first fluorophore may display a Stokes shift of <100nm and the second fluorophore may display a Stokes shift of >100nm or vice versa. This allows a first detection wavelength to be selected at which the first fluorophore emits fluorescence but the second fluorophore produces no fluorescence or substantially no fluorescence. Preferably it also allows the selection of a second detection wavelength to be selected at which the second fluorophore emits fluorescence but the first fluorophore produces no fluorescence or substantially no fluorescence.

In some embodiments, the second fluorophore may absorb light at about 515nm and emit at about 650nm, but other wavelengths are equally possible, depending on the wavelengths of the excitation light and detectors which are used.

The standard compounds, such as peptides and amino acids, when labelled with the second fluorophore are preferably soluble in the electrophoresis buffer and do not affect the separation of labelled saccharides in the sample.

Suitable second fluorophores include DY-480XL; DY481XL, DY-485XL; DY-510XL; DY-520XL; DY-521XL; DY-530; DY-547; DY-548; DY-549; DY- 550; DY-554; DY-555; and DY-560 (Dyomics, Gmbh) . In some preferred embodiments, the second fluorophore is DY481xl (Dyomics, Gmbh) .

Suitable methods for labelling compounds with fluorophores are well- known in the art. .

Preferably, the second fluorophore is reactive with the primary amino group of each standard compound. For example, preferred second fluorophores comprise a reactive group which forms covalent bounds with primary amino groups. Conveniently, the second fluorophore may comprise a N-hydroxysuccinimide ester group ( -NHS ) which forms a covalent bond with the standard compound via the primary amino group. For example, the second fluorophore may be an NHS-ester derivative of DY481xl. Suitable NHS-ester derivatives of fluorophores are commercially available and/or may be synthesised using standard synthetic routes. In some embodiments, the labelled standard compounds may be purified or partially purified after labelling with the second fluorophore to remove excess unbound fluorophore.

In some embodiments, the second fluorophore may be one of the standards in the set of standards, without attachment to a standard compound. For example, a second fluorophore comprising a carboxylic acid group may produce a standard peak on a separation profile. The second fluorophore comprising a carboxylic acid group may be added to the sample or may be produced during the labelling of the standard compounds. For example, during the labelling reaction of an NHS ester derivative of a second fluorophore such as DY481xl with the amine group of the standard compound, the NHS part is

substituted by the amine. However some of the fluorophore-NHS ester will also react with water (is hydrolysed) leaving a carboxylic group on the fluorophore but no standard compound. The NHS part is lost. This carboxylic group containing fluorophore may produce a single standard peak in a separation profile and may be used as one the set of standards. The methods described herein are especially suitable for the analysis of multiple samples of saccharides simultaneously, for example, in an automated high-throughput system. Suitable automated high throughput systems include capillary electrophoresis DNA sequencers. Suitable DNA sequencers are readily available and include the ABI 3730x1 (ABI Inc, CA USA) , MegaBACE500 7& 1000 (Amersham, NJ USA) , SCE2410 and 9610 ( SpectruMedix Corp, PA USA) or CEQ2000XL (Beckman Coulter Inc, CA USA) . CE-DNA sequencers may typically be used without modifying the buffer or separation conditions and typically contain 8, 16, 24, 48, or 96 separate capillaries that can be operated simultaneously, allowing up to 96 samples to be analysed in parallel.

Conventional DNA sequencers typically contain two excitation lasers which may, for example, emit at 488nm and 515nm; 488nm and 532nml or 650 nm and 750nm. Suitable first and second fluorophores which are compatible with these excitation wavelengths can be readily provided by the skilled person.

The mobility of a saccharide, for example an oligo- or monosaccharide may be determined relative to the standards, for example, as a fractional mobility. The fractional mobilities of saccharides may be compared between different electrophoretic separations.

The mobility of a saccharide relative to the standards may be determined by comparing the mobility of a saccharide with the mobilities of two standards in a separation profile. For example, a method may comprise;

identifying the mobilities of a first and a second standard in a CE separation profile; and;

calculating the mobilities of a saccharide in the sample relative to the mobilities of the first and second standards.

The mobilities of the first and second standards may identified by determining the peaks in the fluorescent emission of the second fluorophore which correspond to the labelled first and second standards. These peaks in fluorescent emission occur in the separation profile at the mobilities of the first and second standards. The peaks corresponding to the first and second

standards may be determined from the number of standards in the set of standards and their known mobilities or mass/charge ratios.

Preferably, the first and second standards are the standards in the set which are closest in mobility to the saccharide of interest.

The mobility of a saccharide of interest in the sample may be identified by identifying a peak in the fluorescent emission of the first fluorophore which corresponds to the saccharide of interest. This peak in fluorescent emission occurs in the separation profile at the mobility of the saccharide of interest. The mobility of the saccharide of interest relative to the

mobilities of the first and second standards (e.g. the fractional mobility) may then be determined as described above.

This mobility may be used to identify the saccharide of interest. For example, the fractional mobility of the saccharide relative to known standards may be compared with fractional mobilities of known saccharides relative to the same standards.

Fractional mobilities of known saccharides can be measured relative to the set of standards and recorded as references. The fractional mobilities of unidentified saccharides in a sample relative to the set of standards may be compared to these reference fractional mobilities in order to identify the unidentified saccharide. In order to reduce the amount of data handling required, it preferred that the analysis of data from electrophoretic separations is automated. For example, software may be used to calculate fractional mobilities, compare separations, quantitate, distinguish and/or identify saccharides. For example, a computer may be adapted to identify peaks in the separation profile of the set of standards and the sample, determine the mobilities of the standards and sample saccharides from the peaks, and calculate the fractional mobilities of one or more saccharides in the separation profile relative to standards. The standards used to calculate the fractional mobilities may be selected by the operator from the set of standards. Fractional mobilities may be calculated for all mobilities in the separation profile or for the significant fluorescent peaks in the separation profile which result from saccharides in the sample.

Fractional mobilities reduce significantly the variation in peak mobilities between separate electrophoretic analyses, whether run simultaneously or at different times.

Fractional mobility of a saccharide may be calculated from any pair of standards, preferably the mobilities of standards with greater and lesser mobilities than the saccharide in the separation profile, most preferably from the mobilities of the standards which have the closest greater and lesser mobility to the saccharide.

The fastest and slowest standards may be used to calculate the fractional mobilities for a particular analysis. Alternatively, any pair of standards, which may have either adjacent or non-adjacent peaks in the profile, may be selected for the calculation of fractional mobility. For comparison of fractional mobilities from different separations, the same pair of standards must be used. The advantage of this method is when mobility markers that are closer together than the fastest and slowest in the whole profile then an increases the resolution of the time base is achieved to make more certain the identification of unknown peaks that are close together, that is, they have similar but not identical mobilities Fractional mobility data may be used to align the peaks of all standards in the separation profiles from different separations and/or the peaks in the separation profiles of any reference samples. This process makes the identification of fluorophore labelled saccharide peaks more accurate than using only a pair of mobility standards, and is of particular value when identifying saccharides with small differences in mobility. After alignment of the peaks in different samples using the mobility standards in the set, the fractional mobilities of the saccharides relative to the fastest and slowest standards in the separation can be calculated. A table of fractional mobilities of known saccharides can be determined practically. The software can then generate a list of identities of saccharides present in any sample and distinguish them from those peaks that represent components of the sample that cannot be identified as known saccharides.

Alternatively, a reference sample containing one or more known saccharides may be separated with the set of standards, to determine the fractional mobilities of the known saccharides relative to the standards .

Further aspects of the invention provide: (i) computer-readable code for analysing separation profiles as described herein, (ii) a computer program product carrying such computer-readable code, and (iii) a computer system configured to perform a method described herein.

The term "computer program product" includes any computer readable medium or media which can be read and accessed directly by a computer. Typical media include, but are not limited to: magnetic storage media such as floppy discs, hard disc storage medium and magnetic tape; optical storage media such as optical discs or CD- ROM; electrical storage media such as RAM and ROM; and hybrids of these categories such as magnetic/optical storage media.

A typical computer system comprises a central processing unit (CPU) , input means, output means and data storage means (such as RAM). A monitor or other image display is preferably provided. The computer system may be operably linked to a CE apparatus, such as a CE-DNA sequencer.

For example, a computer system may comprise a processor adapted to perform a method as described above. For example the processor may be adapted;

(a) identify peaks in the separation profiles of the standards and one or more samples,

(b) align the peaks in the separation profile of the standards from one or more samples and align the peaks in the separation profiles of the one or more standards from the alignment of the standards,

(c) determine the mobilities of the set of standards and the saccharides in the sample,

(d) select first and second standards with greater and lesser mobilities than a saccharide in the sample,

(e) calculate the fractional mobility of the saccharide relative to the first and second standards, and, optionally

^•(f) identify the saccharide in the sample from the fractional mobility . The fluorescent emission and mobility data may be entered into the processor automatically from the emission detector and a separation profile plotted. The profile may be displayed, for example on a monitor. The computer system may further comprise a memory device for storing data. Separation profiles and fractional mobilities may be stored on another or the same memory device, and/or may be sent to an output device or displayed on a monitor.

Another aspect of the invention provides a capillary electrophoresis device having a computer system as described above for analyzing data obtained by the capillary electrophoresis. The device may comprise an output signal detector. The output signal detector may, for example, separately detect fluorescent light emitted by the first and second fluorophores . For example, the detector may have a first channel for detecting emission from the first fluorophore at the first detection wavelength and a second channel for detecting emission from the second fluorophore at the second detection wavelength. Suitable capillary electrophoresis apparatus, including excitation lasers and detectors, is well-known in the art. Another aspect of the invention provides a kit for use in a method of analysing a sample of saccharides as described above, comprising; a second fluorophore for standard compounds, and;

two or more standard compounds having different ratios of mass to charge and comprising a primary amine group reactive with the second fluorophore.

In some embodiments, a kit may comprise;

a set of fluorescent mobility standards comprising two or more standard compounds having different ratios of mass to charge, each standard compound being covalently bound to a second fluorophore via a primary amine group.

A kit of the invention may further comprise a first fluorophore for labelling saccharides in the sample. Suitable first and second fluorophores and standard compounds are described above. The kit may include instructions for use in a method of analysing a sample of saccharides as described above.

A kit may include one or more other reagents required for the method, such as buffer solutions and other capillary electrophoresis reagents. A kit for use in analysing a sample of saccharides may include one or more articles and/or reagents for performance of the method, such as means for providing the test sample itself and sample handling containers (such components generally being

sterile) .

Another aspect of the invention provides a method of producing a set of fluorescent mobility standards for use in CE electrophoresis of carbohydrates, for example in a method described above, comprising;

(a) providing two or more standard compounds comprising a primary amine group, said standard compounds having different ratios of mass to charge, and;

(b) labelling said compounds with a fluorophore.

Suitable standard compounds are described above. Suitable

fluorophores include second fluorophores as described above.

Various further aspects and embodiments of the present invention will be apparent to those skilled in the art in view of the present disclosure .

All documents mentioned in this specification are incorporated herein by reference in their entirety.

"and/or" where used herein is to be taken as specific disclosure of each of the two specified features or components with or without the other. For example "A and/or B" is to be taken as specific disclosure of each of (i) A, (ii) B and (iii) A and B, just as if each is set out individually herein. Unless context dictates otherwise, the descriptions and definitions of the features set out above are not limited to any particular aspect or embodiment of the invention and apply equally to all aspects and embodiments which are described. Certain aspects and embodiments of the invention will now be illustrated by way of example and with reference to the figures and tables described below.

Figure 1 shows the increase in electrophoretic mobility of a set of mobility standards with increasing ratio of mass/charge

Figure 2 shows a capillary electrophoresis separation profile from a sample of saccarides. Peaks are as follows; Ul Xylose; U2 Xyl-Xyl; U3 GlcAXyl4; U4 MeGlcAXyl4; U5 Maltotetraose ; U6 Maltopentaose; U7 Maltohexaose; U8 Maltoheptaose .

Figure 3 shows a capillary electrophoresis separation profile from a set of standards. Mobility standard Ml is <Asp-Asp-Asp-Asp-DY48 lxl> and has a greater mobility than the APTS labelled xylose (Ul) .

Figure 4 shows the capillary electrophoresis separation profiles of the sample and standards shown in figures 2 and 3.

Figure 5a shows the structure of the carboxylic acid form of the fluorophore DY481xl. In the N-hydroxysuccinimide ester derivative (NHS-ester), the carboxylic acid group is modified to from an NHS- ester. The excitation and emission spectrum of DY481xl is shown in figure 5b. Figure 6a shows the structure of APTS. The excitation and emission spectra of APTS (λ_βχ = 428 nm, X_era = 500 nm) , APTS-butylmethacrylate (APTS-BuMA) (λ_βχ = 463 nm, A_em = 516 nm) , and APTS-diethylene glycol monomethacrylate (APTS-DEGMA (λ_6χ = 462 nm, A_em = 513 nm) in 4 ^χ 10-6 M in pH 7.4 phosphate buffer are shown in figure 6b.

Figure 7 shows multiple CE profiles produced by an ABI 3730x1 DNA sequencer before alignment (raw data point time scale) . Dark grey profiles: APTS labelled saccharides detected in channel 1; light gray profiles: Mobility Standards detected in channel 5.

Figure 8 shows multiple CE profiles produced by an ABI 3730x1 DNA sequencer after alignment (fractional mobility scale). Dark grey profiles: APTS labelled saccharides detected in channel 1; light gray profiles: Mobility Standards detected in channel 5.

Figures 9a and 9b show capillary electrophoresis separation profiles of plant cell wall derived saccharides using a set of DNA mobility standards. The dark grey line indicates APTS-labelled saccharides. The light grey line indicates DNA mobility standards, with marker positions indicated by dots.

Figure 10 shows a comparison of fractional mobility data calculated using the mobility difference between the fastest and slowest mobility standards (dark grey) or with an 8 fold multiplication

(grey) and using the mobilities of the adjacent faster and slower standards for each individual saccharide (pink - light grey) . The X axis represents four different scales 1. Mobility marker integral values in order of their mobility; 2. Fractional mobility values; 3. Fractional mobility values x8; and 4. Multi-fractional values indexed to nearest faster marker.

Figures 11a to 11c show CE separation profiles of mono- and di- saccharides labelled with AMAC . Figure 11a shows arabinose (top left), fucose (top right), galactose (bottom left) and galacturonic acid (bottom right), all at ΙΟρΜ. Figure lib shows glucose (top left), glucuronic acid (top right), mannose (bottom left) and rhamnose (bottom right) , all at ΙΟρΜ. Figure 11c shows xylose (top left), cellobiose (top right), and mixture of all saccharides

(bottom left), all at ΙΟρΜ and water (bottom right).

Figure 12 shows the relationship between CE mobility and mass to charge ratios (M/z) of a set of amino acids and peptides labelled with the fluorophore dy481xl shown in Table 3.

Figure 13 shows the relationship between CE mobility and mass to charge ratios (M/z) of the sets of amino acids and peptides labelled with the fluorophore dy481xl shown in Tables 2 and 3.

Tables 1 to 3 show mobility and mass/charge data for examples of sets of standards.

Experiments

Methods

Production of the Mobility Standards

Selected amino acids and peptides as shown in table 1 were labelled with the reactive fluorophore dy481xl-NHS-ester to produce mobility standards according to the protocol set out below.

1. Weigh a suitable amount of standard compound: amino adipic acid, Aspartyl-aspartyl-aspartic acid (Asp-Asp-Asp) (DDD) , Aspartyl- aspartyl-aspartyl-aspartic acid (Asp-Asp-Asp-Asp) (DDDD) , Cysteic acid, Glutamyl-glutamic acid (Glu-Glu) (EE) Glycine (Gly) (G) and Glycyl-glycyl-glycine (Gly-Gly-Gly) (GGG) .

2. Dissolve each in individual separate solutions in 0.2M NaHC03 to give a concentration of each reactant of lOOmM and then dilute in 0.1M NaHC03 to give a reactant concentration of lOmM

3. Add 275pL dry dimethyl sulphoxide (DMSO) to the vial of dry DY481xl-NHS-ester (0.2mg; Dyomics Gmbh) and mix to dissolve. 4. Place 2.5 pL(25 nmol) of each reactant separately in a separate small microcentrifuge tube (0.5mL)

5. Add to each reactant solution 1.5 pL of DMSO and mix

6. Add l.OpL (lnmol) of DY481xl-NHS-ester solution in DMSO to each reactant solution and mix well

7. Centrifuge the tubes briefly to ensure the reaction mix is in the tip

8. Incubate at room temperature for 60min. Exclude light.

9. Shake during the incubation at maximum rate using a microplate shaker for approximately lOsec every 5min.

10. Dilute reaction mix with 0.5mL water (1 pL = 2pmol total dye) : (store frozen -80oC)

11. Dilute 10 pL from each sample in 390 pL water ; store the

solution at -20oC)

Mixing of the mobility standards

A suitable quantity of each mobility standard was analysed using a DNA sequencer (ABI ) . The peak heights of the mobility standards were observed in each profile using detector channel 5 of the DNA sequencer or whatever detector setting is appropriate to detect the fluorophore fluorescence. If necessary, adjust the detector of the DNA sequencer to detect the fluorescent light from the mobility standards. This fluorescence has a peak maximum of approximately 650nm.

5-50 microlitres were taken from each solution of fluorophore labelled standards and mix all together so that the peak height of every standard was approximately the same.

The solution of standards was diluted with water so that the peak heights were near the midpoint of the detection dynamic range of the DNA sequencer when a defined volume, preferably in the range 1-20 microlitres, was placed in the sample wells of the 96-well microtitre sample plate. The diluted solution was the working solution.

Fluorophore labelling of the saccharides with APTS

Saccharides were labelled with APTS as follows;

1. Weigh a suitable quantity of citric acid and dissolve in water to give a 1.2M solution

2. Weigh a suitable quantity of 8 aminopyrene-1 , 3 , 6-trisulfonic acid (APTS) and dissolve in 1.2M citric acid solution to give 20mM solution of APTS in 1.2M citric acid solution

3. Place the sample of saccharides that is to be analysed in a microcentrifuge tube, preferably a capacity of 0.5mL. If the saccharides are in solution then dry the sample in the tube using a centrifugal vacuum evaporator (CVE)

4. Weigh a suitable quantity of sodium cyanoborohydride and dissolve in water to give a 1.0M solution

5. Add 5 microlitres of the 20mM APTS solution to the dry

saccharide sample and mix well to dissolve the saccharides

6. Add 5 microlitres of 1.0M sodium borohydride solution to the APTS/saccharide solution and mix well. Seal the tube tightly

7. Incubate the sealed sample in the absence of light at 37oC for 16h

8. Dry the sample in a CVE

9. Dissolve the dry sample in water, preferably l.OmL. If

necessary dilute the solution further with water so that a sample of volume, preferably 5- lOOmicrolitres , contains sufficient analytes to be detected by the DNA sequencer with peak height within the dynamic range of the detector.

10. Place a suitable volume, preferably in the range 5-100

microlitres, of each sample in a 96-well microtitre plate suitable for introducing the samples into the DNA sequencer The microtitre plate is supplied by the supplier of the DNA sequencer.

11. Add to each sample a suitable volume of mobility marker solution, preferably in the range 1-20 microlitres 12. Dry the samples in a CVE equipped to take 96-well mictrotitre plates

13. Dissolve the dry sample in 20 microlitres of formamide

14. Shake the sample in the absence of light at room temperature (21oC) in microplate shaker for 5min at maximum speed (typically

1400 rpm)

15 Analyse the dissolved sample using CE preferably a DNA

sequencer . Saccharide analysis using capillary electrophoresis

A 3730x1 CE-DNA sequencer (Applied Biosystems, Warrington UK) was employed .

The electrophoresis buffer was Applied Biosystems type POP-7™ Polymer for 3730/3730x1 DNA Analyzers (Applied Biosystems product number 4363929) and no changes were made.

The detector system of the DNA sequencer detects the fluorescence from the APTS labelled saccharides without interference from the fluorescence from the DY481xl labelled mobility standards.

The loading conditions were 4000v for 20 seconds when using an Applied Biosystems type 3730x1 DNA sequencer with the

electrophoresis buffer type POP-7™ Polymer for 3730/3730x1 DNA Analyzers (Applied Biosystems product number 4363929) . The separation conditions employed were the same as those used for the separation of oligo- and polynucleotides using the DNA sequencer. The total time for the separations was usually equivalent to 8000 data points but longer or shorter times may equally be used.

Separation profiles were viewed using either the software supplied by Applied Biosystems or any suitable alternative software, for example the PACER software. In the case of the 3730x1 sequencer, these files are in an . fsa format The data files produced by the DNA sequencer were in . fsa format. The data files for each analysis produced by CE in the DNA sequencer were processed by computer using "PACER" software. This programme can provide the following information to the operator:

1. Relative fluorescence detected and measured at defined wavelengths

2. Relative fluorescence detected and measured during the CE

separation of analytes at defined, virtually continuous time intervals .

3. Export of the fluorescence versus time data to other

appropriate programmes.

4. Display of fluorescence versus time data

5. Detection of peaks

6. Peak time

7. Peak area

8. Detection simultaneously of different fluorophores including the APTS used to label the saccharides and the DY481xl used to label the mobility standards

9. Temporal correlation of fluorescent profiles produced from any single sample by the CE of fluorescent substances that are electrophoresed in the same analysis run (co-electrophoresis) .

10. Standardisation of electrophoretic mobilities in any selected individual capillary separation profiles using the co- electrophoresed mobility standards.

11. Standardisation of electrophoretic mobilities in any selected group of capillary separation profiles using the co- electrophoresed mobility standards.

Calculation of fractional mobilities

The DNA Sequencer output was fluorescence detected versus time (datapoint): This is the raw data. In order to standardise the electrophoresis, the mobilities of the saccharides were compared with those of the mobility standards and a standardised mobility was calculated which was known as the

Fractional Mobility (FM) .

The FM is calculated as follows :-

1. The Mobility Standard with the greatest mobility (fastest) is detected and its raw datapoint determined at its maximum fluorescent peak height Let this value =T0

2. The Mobility Standard with the least mobility (slowest) is detected and its raw datapoint determined at its maximum fluorescent peak height Let this value =T1

3. The mobility of any one analyte peak (substance U) of interest is detected and its raw datapoint determined at its maximum fluorescent peak height Let this value =Tu

4 . The FM for substance U (FMu) = (Tu-TO) / (T1-T0)

5. This calculation was made for all points on the timescale.

The profile was then be presented as fluorescence versus the

Fractional Mobility

Any peak may be assigned a FM value. Comparison and alignment of profiles from different capillary analysis profiles may be performed using the standardised FMs .

Results

Selection of Standards

The migration time of a standard was selected by choosing a suitable amino acid or peptide that determines the mobility of the marker, after it has been coupled covalently to the fluorophore. The fluorophore is constant so this ensures that all the fluorescent mobility standards can be detected by the DNA sequencer without interfering with the separation or the detection of the APTS labelled saccharides that are detected in a separate channel. The migration times were found to be approximately proportional to the mass to charge ratio of the standard. Prediction cf the mobility is therefore possible from the mass to charge ratio. This assists the selection of peptide/ amino acid marker molecules prior to the coupling to the fluorophore.

The variation of mobility with mass to charge ratio of various dy481xl labelled amino acids and peptides is shown in Tables 1 to 3 and in Figures 1, 12 and 13.

This data shows that the mass/charge ratio of a fluorophore labelled standard compound, such as an amino acid or peptide, is indicative of the CE mobility. A mobility standard was formed by labelling the tetrapeptide, Asp- Asp-Asp-Asp with the fluorophore DY481xl. This standard has a mobility which is greater that that of the APTS-labelled xylose (a neutral monosaccharide) with the greatest mobility of those neutral saccharides that we have tested (figures 2 to 4) .

Commercially available DNA-based standards were found to be

insufficiently mobile to run faster than the significant

monosaccharide or some of the acidic oligosaccharides. For example, the separation of the oligosaccharides GlcUA(xyl)₃ and its

methylated form (Me ) GlcUA (xyl ) ₃ is important for some plant cell wall analyses but figures 9a and 9b show that these oligosaccharides are outside the range of DNA standards

Resolution of peaks: comparison of two methods

Figure .10 shows a comparison of fractional mobility data calculated in two different ways. Each point in the chart shows the average for 12 separate analyses for an individual saccharide peak. The range of x3 standard

deviation for each point is shown.

The green points are Fractional Mobilities calculated by using the mobility difference between the fastest and slowest mobility standards in the set as the basis for the calculation. Numerically the range is zero to unity.

The red points are a simple multiplication x8 of the green numbers in order to show more clearly the separation between adjacent peaks.

The pink points are calculated using the mobilities of the adjacent faster and slower standards for each individual saccharide. This is known as the Multi-Fractional Mobility. And the range is zero to unity between adjacent standards. In order to represent more clearly the relative positions of each point an integral value has been added which is the position of each faster standard in the list of standards .

The multi fractional method enables a much better discrimination between peaks. Peak discrimination can therefore be enhanced markedly by using standards that are adjacent to the saccharide peaks that are close together.

The methods provided herein provide flexibility for CE analysis of polysaccharides. It allows amino acids or peptides to be selected as standards that can migrate in positions close to those saccharide peaks that are close and difficult to discriminate, owing to the variability in the raw datapoint mobilities. This enables more accurate and discriminatory calculations of saccharide standardised (fractional) mobilities. In practice, software may be used to implement these calculations using all the peaks for the alignment of profiles. It is dependent on the positions of the standards for the accuracy of the saccharide peak discrimination. Standards that have been designed to migrate close to saccharide peaks enable more accurate peak alignment. This in turn enables the more certain identification of unknown

saccharide peaks by comparison of their normalised positions with those in a list of known saccharides. Resolution of Negative Saccharides

Monosaccharides (arabinose, fucose, galactose, galacturonic acid, glucose, glucuronic acid, mannose, rhamnose and xylose) and a di- saccharide (cellobiose) were labelled with AMAC and separated by capillary electrophoresis. At the pH of the electrophoresis, glucuronic acid and galacturonic acid are both negatively charged. The CE separations are shown in figures 11a to 11c.

Despite having the same molecular weight, glucuronic acid and galacturonic acid showed significant peaks with similar but distinct electrophoretic mobilities. This shows that AMAC may be used in HT- PACE to discriminate between neutral and negatively charged saccharides. AMAC labelled neutral saccharides do not appear in the profiles shown in figures 11a to 11c and only GalA (Fig 11a bottom right) and GlcA (Fig lib top right) show as peaks. All the other peaks in the profiles are artefacts.

Table 1

Mol. Wt. m/z Data

Marker Charge on derivative point

Mol. Wt. deriv derivative negative H02.019

L-Cysteinesulfinic acid CysfA 171.17 785 -3 262 1770

L-Homocysteic acid hCysA 183.18 797 -3 266 1790

L-Homocysteine sulfinic acid hCSA 167.18 781 -3 260 1820

S-Carboxymethyl-L-cysteine CMC 179.16 793 -3 264 1840

DL-2-Methylglutamic acid hemihydrate MGIu 170.16 784 -3 261 1850

DL-threo-B-methylaspatic acid Masp 147.13 761 -3 254 1850

L-2-Aminoadipic acid AAA 161.16 775 -3 258 1870

Taurine Tau 125.15 739 -2 370 3170^'

2-Aminoethyl hydrogen sulfate AEHS 141.15 755 -2 378 3220

Hypotaurine hyTau 109.15 723 -2 362 3235

DY481xl carboxylic acid 631 631 -2 316 2850

Table 2 Mol. Wt. Charge m/z

Set 1 Mol. Wt. marker on derivative Oatapoint arKer marker derivatised derivative negative H02.019

H-Asp-Asp-Asp-Asp-OH Asp4 DDDD 478.37 1091 -6 182 930

H-Asp-Asp-Asp-

OH Asp3 DDD 363.28 976 -5 195 1010

H-Asp-Asp-OH Asp2 DD 248.19 861 -4 215 1275

H-GIU-GIU-OH Glu2 EE 276.25 889 -4 222 1330

Cysteic acid CysA 187.17 800 -3 267 1735

H-Asp-

OH Asp1 D 133.1 746 -3 249 1820

H-GIU-

OH Glu1 E 147.13 760 -3 253 1825

H-Gly-

OH Gtyl G 75.07 688 -2 344 3125

H-Gly-Gly-OH Gly2 GG 132.12 745 -2 373 3470

H-Gly-Gly-Gly-OH Gly3 GGG 189.2 802 -2 401 3790

H-Gly-Gly-Gty-Gly-OH Gly4 GGGG 246.22 859 -2 430 4140

H-Gly-Gly-Gly-Gly-Gly-OH Gly5 GGGGG 303.3 916 -2 458 4470

H-Gly-Gly-Gly-Gly-Gly-Gly-

OH Gly6 GGGGGG 360 3 973 -2 487 4820

DY48l l carboxylic acid 630 -2 315 2785

Table 3

References

1. European Patent Application: EP 2 112 506 Al

2. US patent 7,638,024 B2

3. Khandurina, J., et al. Electrophoresis (2004) 25, 3122-3127 4. Andrew Carroll¹ and Chris Somerville, Annual Review of Plant

Biology (June 2009) Vol. 60, 165-182

5. Lee, K. J., et al. Biochemical and Biophysical Research

Communicatons (2009) 380, 223-29

6. Barton, C.J., et al. Planta (2005) 224, 163-174

7. Brown, D.M., et al. Plant J. (2007) 52, 1154-68

8. Brown, D.M., et al . Plant J. (2009) 57, 732-46

9. Goubet, F . , et al . Plant J. (2009) 60, 527-38

10. Jackson P., (1990) Biochem. J, 270, 705-713

11. Jackson, P. (1992) Anal. Biochem. 196, 238-244

12. Jackson, P., in A Laboratory Guide to Glycoconjugate Analysis (Birkhauser, 1997), ppll3-139

13. US patent 5, 104, 508

1 . US patent 5, 340, 53

15. US patent 5, 472, 582

16. US patent 6,294,667

Claims

Claims :

1. A method of analysing saccharides in a sample comprising:

(i) providing a sample comprising one or more saccharides, (ii) labelling the saccharides in the sample with a first fluorophore,

(iii) providing a set of fluorescent mobility standards; said set comprising two or more standard compounds, each standard compound comprising a primary amine group and being labelled with a second fluorophore, wherein the standard compounds have different ratios of mass to charge and the first and second fluorophore emit fluorescence at different wavelengths,

(iv) mixing the set of standards and the labelled sample,

(v) separating the mixture by capillary electrophoresis to produce a separation profile of the amount of fluorescence from the first and second fluorophores relative to electrophoretic mobility, wherein fluorescence from the first fluorophore is indicative of saccharides in the sample and fluorescence from the second fluorophore is indicative of the standards, and,

(vi) determining from the mobilities of the first and second fluorophores in the profile, the mobility of one or more saccharides in the sample relative to the mobility of one or more of said set of standards .

2. A method according to claim 1 comprising identifying one or more saccharides in the sample from its mobility relative to said standards .

3. A method according to claim 1 or claim 2 wherein the

fractional mobility of a saccharide in the sample is determined relative to the mobilities of a first standard and a second standard from the set of standard.

. A method according to claim 3 wherein the first standard has a higher mobility than the saccharide and the second standard has a lower mobility.

5. A method according to claim 4 wherein the first standard has a higher mobility than any of the saccharides in the sample and the second standard has a lower mobility than any of the saccharides in the sample.

6. A method according to claim 4 wherein the first standard is the standard within the set of standards which has the closest higher mobility to the saccharide and the second standard is the standard in the set of standards which has the closest lower mobility to the saccharide.

7. A method according to any one of the preceding claims

comprising

producing the set of fluorescent mobility standards by

(a) providing two or more standard compounds comprising a primary amine group, said standard compounds having different ratios of mass to charge, and;

(b) labelling said compounds with a second fluorophore, wherein the first and second fluorophore emit fluorescence at different wavelengths,

8. A method according to any one of the preceding claims wherein the standard compounds are selected to produce a set of standards with mobilities which are higher and lower than the saccharides in the sample.

9. A method according to any one of the preceding claims wherein the sample is provided by treating material comprising

polysaccharides to degrade polysaccharides therein and produce a sample comprising mono- or oligo-saccharides .

10. A method according to claim 9 wherein the material is plant material comprising plant cell wall polysaccharides.

11. A method according to claim 9 wherein the material comprises animal, fungal, bacterial, viral or algal polysaccharides.

12. A method according to any one of the preceding claims wherein the saccharides are mono- or oligosaccharides.

13. A method according to any one of the preceding claims wherein the saccharides are labelled with the first fluorophore by reductive amination .

14. A method according to any one of the preceding claims wherein the first fluorophore is aminopyrene-1, 3, 6-trisulfonic acid

(APTS) , 8-aminonaphthalene-l, 3, 6-trisulphonic acid (ANTS) or 2- aminoacridone (AMAC) .

15. A method according to any one of the preceding claims wherein one or more of the standard compounds are peptides or amino acids.

16. A method according to claim 15 wherein one or more of the standard compounds are poly(Gly), poly(Asp) and/or poly(Glu).

17. A method according to claim 15 or claim 16 wherein the one or more standard peptides include one or more of di, tri or

tetrapeptides .

18. A method according to claim 15 wherein the standard compounds are selected from the group consisting of amino adipic acid,

Aspartic acid (D), Aspartyl-aspartic acid (Asp-Asp) (DD), Aspartyl- aspartyl-aspartic acid (Asp-Asp-Asp) (DDD) ; Aspartyl-aspartyl- aspartyl-aspartic acid (Asp-Asp-Asp-Asp) (DDDD) ; cysteic acid; Glutamic acid (E) ; Glutamyl-glutamic acid (Glu-Glu) (EE); Glycine (Gly) (G); Glycyl-glycyl (Gly-Gly) (GG) ; Glycyl-glycyl-glycine (Gly- Gly-Gly) (GGG) ; Glycyl-glycyl-glycyl-glycine (Gly-Gly-Gly-Gly) (GGGG) ; Glycyl -glycyl-glycyl-glycyl-glycine (Gly-Gly-Gly-Gly-Gly) (GGGGG) ; Glycyl-glycyl-glycyl-glycyl-glycyl-glycine (Gly-Gly-Gly- Gly-Gly-Gly) (GGGGGG) ; 2-aminoethyl hydrogen sulphate; L- cysteinesulfinic acid; L-homocysteic acid; L-homocysteine sulfinic acid; S-carboxymethyl-L-cysteine; DL-2-methylglutamic acid

hemihydrate; DL-threo-B-methylaspatic acid; L-2-aminoadipic acid; taurine; and hypotaurine.

19. A method according to any one of the preceding claims wherein the fluorescence emitted by the second fluorophore does not

interfere with the fluorescence emitted by the first fluorophore.

20. A method according to claim 19 wherein the second fluorophore is DY481xl.

21. A method according to any one of the preceding claims wherein multiple samples are analysed simultaneously.

22. A method according to any one of the preceding claims wherein the mobility of one or more saccharides in the sample relative to two of said set of standards is determined by computer analysis.

23. A kit for use in a method of analysing a sample of saccharides according to any one of claims 1 to 22, comprising;

a second fluorophore for labelling standard compounds, and; two or more standard compounds having different ratios of mass to charge and comprising a primary amine group reactive with the second fluorophore.

24. A kit for use in a method of analysing a sample of saccharides according to any one of claims 1 to 22, comprising; a set of fluorescent mobility standards comprising two or more standard compounds having different ratios of mass to charge, each standard compound being covalently bound to a second

fluorophore via a primary amine group.

25. A kit according to claim 23 or claim 24 further comprising a first fluorophore for labelling saccharides in a sample.