ADVANCED METHODS FOR AUTOMATED HIGH-PERFORMANCE IDENTIFICATION OF CARBOHYDRATES AND CARBOHYDRATE MIXTURE COMPOSITION PATTERNS AND SYSTEMS THEREFORE AS WELL AS METHODS FOR CALIBRATION OF MULTI WAVELENGTH FLUORESCENCE DETECTION SYSTEMS THEREFORE, BASED ON NEW FLUORESCENT DYES
20220026434 · 2022-01-27
Inventors
- Erdmann RAPP (Magdeburg, DE)
- René HENNIG (Magdeburg, DE)
- Udo Reichl (Magdeburg, DE)
- Stefan HELL (Göttingen, DE)
- Vladimir BELOV (Göttingen, DE)
- Matthias BISCHOFF (Dortmund, DE)
- Dirk Meineke (Köln, DE)
- Laura THOMAS (Lüneburg, DE)
- Kirill KOLMAKOV (Magdeburg, DE)
- Gyuzel MITRONOVA (Göttingen, DE)
- Elizaveta SAVICHEVA (St. Petersburg, RU)
Cpc classification
G01N1/30
PHYSICS
C07F9/093
CHEMISTRY; METALLURGY
International classification
G01N1/30
PHYSICS
Abstract
The present invention relates to improved (simplified/easier, more robust and more reproducible) methods for identification of carbohydrates compositions, e.g. out of complex carbohydrate mixtures, as well as the determination of carbohydrate mixture composition patterns (e.g.: of glycosylation patterns) based on advanced internal standards to determine precise and highly reproducible migration and retention time indices using novel fluorescent dyes in combination with high performance separation technologies, like capillary (gel) electrophoresis (C(G)E) or (ultra)high performance liquid chromatography (U)HPLC with a highly sensitive detection like (laser induced) fluorescence detection. In a first aspect, the present invention relates to methods for an automated determination and/or identification of carbohydrates and/or carbohydrate mixture composition pattern profiling as well as a method for an automated carbohydrate mixture composition pattern profiling based on the use of at least a first and second fluorescent label for labelling the migration/retention time alignment standard and sample or different samples, respectively, whereby the at least one of that fluorescent dye is a compound as defined herein. Moreover, the present invention relates to a method for calibration of multi wavelength fluorescence detection systems as well as calibration systems or calibration standards and new compounds suitable for calibration are described. The present invention relates further to a kit or system for determining or identifying carbohydrate mixture composition patterns as well as a kit or system for determining and/or identifying carbohydrate mixture composition pattern. Further, a carbohydrate dye conjugate comprising the dye as defined herein for use in a method according to the present invention is provided. The dyes employed for forming the carbohydrate dye conjugate have formula A or B below:
Claims
1. A method for an automated determination and/or identification of carbohydrates and/or carbohydrate mixture composition pattern profiling comprising the steps of: a) obtaining a sample containing at least one carbohydrate; b) labelling said carbohydrate(s) with a first fluorescent label; c) providing a standard of known composition labelled with a second fluorescent label; d) determining the migration/retention time(s) of said carbohydrate(s) and the standard of known composition using electrokinetic/chromatographic separation techniques combined with fluorescence or laser induced fluorescence detection; e) aligning the migration/retention time(s) to migration/retention time indice(s) based on given standard migration/retention time indice(s) of the standard; f) comparing these migration/retention time indice(s) of the carbohydrate(s) with standard migration/retention time indice(s) from a database; g) identifying or determining the carbohydrate(s) and/or the carbohydrate mixture composition pattern, wherein the standard composition is added to the sample containing the unknown carbohydrate and/or carbohydrate mixture composition, the first fluorescent label and the second fluorescent label are different and wherein the first fluorescent label or the second fluorescent label is a fluorescent dye, preferably having multiple ionizable and/or negatively charged groups which is selected from the group consisting of compounds of the following general Formula A and B: ##STR00032## wherein R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5 are independent from each other and may represent: H, CH.sub.3, C.sub.2H.sub.5, a straight or branched C.sub.3-C.sub.12, preferably C.sub.3-C.sub.6, alkyl or perfluoroalkyl group, a phosphonylated alkyl group (CH.sub.2).sub.mP(O)(OH).sub.2, where m=1-12, preferably 2-6, with a straight or branched alkyl chain, (CH.sub.2).sub.nCOOH, where n=1-12, preferably 1-5, or (CH.sub.2).sub.nCOOR.sup.6, where n=1-12, preferably 1-5, and R.sup.6 may be alkyl, in particular C.sub.1-C.sub.6, CH.sub.2CN, benzyl, fluorene-9-yl, polyhalogenoalkyl, polyhalogenophenyl, e.g. tetra- or pentafluorophenyl, pentachlorophenyl, 2- and 4-nitrophenyl, N-succinimidyl, sulfo-N-succinimidyl, 1-oxybenzotriazolyl, or other potentially nucleophile-reactive leaving groups, alkyl sulfonate ((CH.sub.2).sub.nSO.sub.3H) or alkyl sulfate ((CH.sub.2).sub.nOSO.sub.3H) where n=1-12, preferably 1-5, and the alkyl chain in any (CH.sub.2).sub.n may be straight or branched; a hydroxyalkyl group (CH.sub.2).sub.mOH or thioalkyl group (CH.sub.2).sub.mSH, where m=1-12, preferably 2-6, with a straight or branched alkyl chain, a phosphorylated hydroxyalkyl group (CH.sub.2).sub.mOP(O)(OH).sub.2, where m=1-12, preferably 2-6, with a straight or branched alkyl chain; one of R.sup.1 or R.sup.2 groups may be a carbonate or carbamate derivative of (CH.sub.2).sub.mOCOOR.sup.7 or COOR.sup.7, where m=1-12 and R.sup.7=methyl, ethyl, tert-butyl, benzyl, fluoren-9-yl, CH.sub.2CN, N-succinimidyl, sulfo-N-succinimidyl, 1-oxybenzotriazolyl, phenyl, substituted phenyl group, e.g., 2- or 4-nitrophenyl, pentachlorophenyl, penta-fluorophenyl, 2,3,5,6-tetrafluorophenyl, 2-pyridyl, 4-pyridyl, pyrimid-4-yl; (CH.sub.2).sub.mNR.sup.aR.sup.b, where m=1-12, preferably 2-6, with a straight or branched alkyl chain; R.sup.a, R.sup.b are independent from each other and represent hydrogen and/or C.sub.1-C.sub.4 alkyl groups, a hydroxyalkyl group (CH.sub.2).sub.mOH, where m=2-6, with a straight or branched alkyl chain, a phosphorylated hydroxyalkyl group (CH.sub.2).sub.mOP(O)(OH).sub.2, where m=1-12, preferably 2-6, with a straight or branched alkyl chain; an alkyl azide (CH.sub.2).sub.mN.sub.3, where m=m=1-12, preferably 2-6, with a straight or branched alkyl chain; R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5 may contain a terminal alkyloxyamino group (CH.sub.2).sub.mONH.sub.2, where m=1-12, preferably 2-6, with a straight or branched alkyl chain, that can include one or multiple alkylamino (CH.sub.2).sub.mNH or alkylamido (CH.sub.2).sub.mCONH groups in all possible combinations with m=0-12; (CH.sub.2).sub.nCONHR.sup.B, with n=1-12, preferably 1-5; R.sup.8=H, C.sub.1-C.sub.6 alkyl, (CH.sub.2).sub.mN.sub.3, or (CH.sub.2).sub.m—N-maleimido, (CH.sub.2).sub.m—NH—COCH.sub.2X (X=Br or I), with m=1-12, preferably 2-6, and with straight or branched alkyl chains in (CH.sub.2).sub.n, (CH.sub.2).sub.m and R.sup.8; a primary amino group, preferably as R.sup.1, R.sup.2, or R.sup.3, which forms aryl hydrazines; a hydroxy group, preferably as R.sup.2 or R.sup.3, which forms aryl hydroxylamines; further, one of the residues R.sup.1, R.sup.2, R.sup.3, R.sup.4, R.sup.5 may represent CH.sub.2—C.sub.6H.sub.4—NH.sub.2, COC.sub.6H.sub.4—NH.sub.2, CONHC.sub.6H.sub.4—NH.sub.2 or CSNHC.sub.6H.sub.4—NH.sub.2 with C.sub.6H.sub.4 being a 1,2-, 1,3- or 1,4-phenylene, COC.sub.5H.sub.3N—NH.sub.2 or CH.sub.2—C.sub.5H.sub.3N—NH.sub.2, with C.sub.5H.sub.3N being pyridin-2,4-diyl, pyridin-2,5-diyl, pyridin-2,6-diyl, or pyridin-3,5-diyl; additionally, R.sup.2-R.sup.3 (R.sup.4-R.sup.5) may form a four-, five, six-, or seven-membered cycle, or a four-, five, six-, or seven-membered cycle with or without a primary amino group NH.sub.2, secondary amino group NHR.sup.a, where R.sup.a=C.sub.1-C.sub.6 alkyl, a hydroxyl group OH, or a phosphorylated hydroxyl group —OP(O)(OH).sub.2 attached to one of the carbon atoms in this cycle; optionally R.sup.2-R.sup.3 (R.sup.4-R.sup.5) may form a four-, five, six-, or seven-membered heterocycle with an additional 1-3 heteroatoms such as O, N or S included into this heterocycle; further, R.sup.1 may represent an unsubstituted phenyl group, a phenyl group with one or several electron-donor substituents chosen from the set of OH, SH, NH.sub.2, NHR.sup.a, NR.sup.aR.sup.b, R.sup.aO, R.sup.aS, where R.sup.a and R.sup.b are independent from each other and may be C.sub.1-C.sub.6 alkyl groups with straight or branched carbon chains, a phenyl group with one or several electron-acceptors chosen from the set of NO.sub.2, CN, COH, COOH, CH═CHCN, CH═C(CN).sub.2, SO.sub.2R.sup.a, COR.sup.a, COOR.sup.a, CH═CHCOR.sup.a, CH═CHCOOR.sup.a, CONHR.sup.a, SO.sub.2NR.sup.aR.sup.b, CONR.sup.aR.sup.b, where R.sup.a and R.sup.b are independent from each other and may be H, or C.sub.1-C.sub.6 alkyl group(s) with straight or branched carbon chains; or R.sup.1 may represent a heteroaromatic group; with the proviso that in all compounds of Formula A above at least two, preferably at least 3, 4, 5 or 6 negatively charged groups are present under basic conditions, i.e. 7<pH<14, and these negatively charged groups represent at least partially deprotonated residues of ionizable groups selected from the following: SH, COOH, a sulfonic acid residue SO.sub.3H, a primary phosphate group OP(O)(OH).sub.2, a secondary phosphate group OP(O)(OH)R.sup.a, where R.sup.a=C.sub.1-C.sub.4 alkyl or substituted C.sub.1-C.sub.4 alkyl, a primary phosphonate group P(O)(OH).sub.2, a secondary phosphonate group P(O)(OH)R.sup.a, where R.sup.a=C.sub.1-C.sub.4 alkyl or substituted C.sub.1-C.sub.4 alkyl; and compounds of Formula A can exist and can be used as salts, solvates and hydrates, preferably as salts with alkaline metal cations including Na.sup.+, Li.sup.+, K.sup.+ and organic ammonium or organic phosphonium cations; ##STR00033## wherein R.sup.1 and/or R.sup.2 are independent from each other and may represent: H, CH.sub.3, C.sub.2H.sub.5, a linear or branched C.sub.3-C.sub.12 alkyl or perfluoroalkyl group, or a substituted C.sub.2-C.sub.612 alkyl group; in particular, (CH.sub.2).sub.nCOOR.sup.3, where n=1-12, preferably 1-5, R.sup.3 may be H, alkyl, in particular C.sub.1-C.sub.6, CH.sub.2CN, benzyl, fluorene-9-yl, polyhalogenoalkyl, polyhalogenophenyl, e.g. tetra- or pentafluorophenyl, pentachlorophenyl, 2- and 4-nitrophenyl, N-succinimidyl, sulfo-N-succinimidyl, 1-oxybenzotriazolyl, or other potentially nucleophile-reactive leaving groups, and the alkyl chain in (CH.sub.2).sub.n may be straight or branched; and R.sup.1-R.sup.2 may form a four-, five, six-, or seven-membered non-aromatic carbocycle with an additional primary amino group NH.sub.2, secondary amino group NHR.sup.a, where R.sup.a=C.sub.1-C.sub.6 alkyl, or hydroxyl group OH attached to one of the carbon atoms in this cycle; optionally R.sup.1-R.sup.2 may form a four-, five, six-, or seven-membered non-aromatic heterocycle with an additional heteroatom such as O, N or S included into this heterocycle; a hydroxyalkyl group (CH.sub.2).sub.mOH, where m=1-12, preferably 2-6, with a straight or branched alkyl chain; one of R.sup.1 or R.sup.2 groups may be a carbonate or carbamate derivative (CH.sub.2).sub.mOCOOR.sup.4 or COOR.sup.4, where m=1-12 and R.sup.4=methyl, ethyl, 2-chloroethyl, N-succinimidyl, sulfo-N-succinimidyl, 1-oxybenzotriazolyl, a phenyl group or substituted phenyl group, e.g., 2- and 4-nitrophenyl, pentachlorophenyl, pentafluorophenyl, 2,3,5,6-tetrafluoro-phenyl, 2-pyridyl, or 4-pyridyl; (CH.sub.2).sub.mNR.sup.aR.sup.b, where m=1-12, preferably 2-6, with a straight or branched alkyl chain; R.sup.a, R.sup.b are independent from each other and may be H, or optionally substituted C.sub.1-C.sub.4 alkyl group(s), in particular, one of R.sup.1 or R.sup.2 groups may be an alkyl azide group (CH.sub.2).sub.mN.sub.3 with m=2-6 and a straight or branched alkyl chain; one of R.sup.1 or R.sup.2 may be (CH.sub.2).sub.nSO.sub.2NR.sup.5NH.sub.2 with n=1-12, while the substituent R.sup.5 can be represented by H, alkyl, hydroxyalkyl or perfluoroalkyl groups C.sub.1-C.sub.12; one of R.sup.1 or R.sup.2 groups may be a primary amino group to form aryl hydrazines Ar—NR.sup.6NH.sub.2 where Ar is the entire pyrene residue in Formula B and R.sup.6=H or alkyl; one of R.sup.1 or R.sup.2 groups may be a hydroxy group to form aryl hydroxylamines Ar—NR.sup.7OH where Ar is the entire pyrene residue in Formula B and R.sup.7=H or alkyl; one of R.sup.1 or R.sup.2 groups may contain a terminal alkyloxyamino group (CH.sub.2).sub.nONH.sub.2 with n=1-12, which can be linked via one or multiple alkylamino (CH.sub.2).sub.mNH, alkylamido (CH.sub.2).sub.mCONH, alkyl ether or ester group(s) in all possible combinations with m=0-12; one of R.sup.1 or R.sup.2 groups may be CO(CH.sub.2).sub.nCOOR.sup.B, with n=1-5 and a straight or branched alkyl chain (CH.sub.2).sub.n and with R.sup.8 selected from H, straight or branched C.sub.1-C.sub.6 alkyl, CH.sub.2CN, 2- and 4-nitrophenyl, 2,3,5,6-tetrafluorophenyl, pentachlorophenyl, pentafluoro-phenyl, N-succinimidyl, sulfo-N-succinimidyl, 1-oxybenzotriazolyl; further, one of R.sup.1 or R.sup.2 may be (CH.sub.2).sub.nCONHR.sup.9, with n=1-5 and R.sup.9=H, C.sub.1-C.sub.6 alkyl, (CH.sub.2).sub.mN.sub.3, (CH.sub.2).sub.m—N-maleimido, (CH.sub.2).sub.m—NHCOCH.sub.2X (X=Br or I), where m=2-6 and with straight or branched alkyl chains in (CH.sub.2).sub.n and R.sup.9; or one of R.sup.1 or R.sup.2 may represent CH.sub.2—C.sub.6H.sub.4—NH.sub.2, COC.sub.6H.sub.4—NH.sub.2, CONHC.sub.6H.sub.4—NH.sub.2 or CSNHC.sub.6H.sub.4—NH.sub.2 with C.sub.6H.sub.4 being a 1,2-, 1,3- or 1,4-phenylene, COC.sub.5H.sub.3N—NH.sub.2 or CH.sub.2—C.sub.5H.sub.3N—NH.sub.2, with C.sub.5H.sub.3N being pyridin-2,4-diyl, pyridin-2,5-diyl, pyridin-2,6-diyl, or pyridin-3,5-diyl; or one of R.sup.1 or R.sup.2 may be an alkyl azide (CH)N.sub.3 or alkine, in particular propargyl; the linker L comprises at least one carbon atom and may comprise alkyl, heteroalkyl, in particular alkyloxy such as CH.sub.2OCH.sub.2, CH.sub.2CH.sub.2O CH.sub.2CH.sub.2OCH.sub.2, alkylamino or dialkylamino, particularly diethanolamine or N-methyl (alkyl) monoethanolamine moieties such as N(CH.sub.3)CH.sub.2CH.sub.2O— and N(CH.sub.2CH.sub.2O—).sub.2, perfluoroalkyl, like single or multiple difluoromethyl (CF.sub.2), alkene or alkyne moieties in any combinations, at any occurrence, linear or branched, with the length ranging from C.sub.1 to C.sub.12; the linker L may also include a carbonyl (CH.sub.2CO, CF.sub.2CO) moiety, also as part of an amide group; the linker L may also comprise or contain a residue of 1,3,5-triazine, thus providing two attachment points for group X; X denotes a solubilizing and/or ionizable anion-providing moiety, in particular consisting of or including a moiety selected from the group comprising hydroxyalkyl (CH.sub.2).sub.nOH, thioalkyl ((CH.sub.2).sub.nSH), carboxy alkyl ((CH.sub.2).sub.nCO.sub.2H), alkyl sulfonate ((CH.sub.2).sub.nSO.sub.3H), alkyl sulfate ((CH.sub.2).sub.nOSO.sub.3H), alkyl phosphate ((CH.sub.2).sub.nOP(O)(OH).sub.2) or phosphonate ((CH.sub.2).sub.nP(O)(OH).sub.2), wherein n is an integer ranging from 0 to 12, or an analogon thereof wherein one or more of the CH.sub.2 groups are replaced by CF.sub.2, further, the anion-providing moieties may be linked by means of non-aromatic O, N and S-containing heterocycles, e. g., piperazines, pipecolines, or, alternatively, one of the groups X may bear any of the moieties listed above for groups R.sup.1 and R.sup.2, also with any type of linkage listed for group L, and independently from other substituents; Compounds of Formula B can exist and can be used as salts, solvates and hydrates, preferably as salts with alkaline metal cations including Na.sup.+, Li.sup.+, K.sup.+ and organic ammonium. With the proviso that in all compounds represented by Formula B three or six negatively charged groups are present in the residues X of Formula B under basic conditions, i.e. 7<pH<14, and these negatively charged groups represent at least partially deprotonated residues of ionizable groups selected from the following: SH, COOH, SO.sub.3H, OP(O)(OH).sub.2, OP(O)(OH)R.sup.a, where R.sup.a=C.sub.1-C.sub.4 alkyl or substituted C.sub.1-C.sub.4 alkyl, P(O)(OH).sub.2, P(O)(OH)R.sup.a, where R.sup.a=C.sub.1-C.sub.4 alkyl or substituted C.sub.1-C.sub.4 alkyl is provided; and compounds of Formula B can exist and can be used as salts, solvates and hydrates, preferably as salts with alkaline metal cations including Na.sup.+, Li.sup.+, K.sup.+ and organic ammonium or organic phosphonium cations;
2. The method according to claim 1 wherein the standard of known composition is a standard base pair ladder and/or a known carbohydrate mixture composition.
3. A method for an automated carbohydrate mixture composition pattern profiling comprising the steps of a) providing a first sample containing a first carbohydrate mixture composition; b) labelling of said carbohydrate mixture composition with a first fluorescent label; c) providing a second sample containing a second carbohydrate mixture composition labelled with a second fluorescent label which may be added optionally to said first sample; d) generating electropherograms/chromatograms of the carbohydrate mixture composition of said sample using electrokinetic/chromatographic separation techniques combined with fluorescence or laser induced fluorescence detection; e) analyzing the identity and/or differences between the carbohydrate mixture composition pattern profiles of the first and the second sample, wherein the first fluorescent label of the first sample is different to the second fluorescent label of the second sample and wherein at least one of the first fluorescent label and the second fluorescent label is a fluorescent dye as defined in claim 1.
4. A method for an automated carbohydrate mixture composition pattern profiling according to claim 3 comprising the steps of a) providing a sample containing a first carbohydrate mixture composition; b) labelling of said carbohydrate mixture composition with a first fluorescent label; c) providing a second sample labelled with a second fluorescent label containing a second carbohydrate mixture composition to be compared with; d) generating electropherograms/chromatograms of the carbohydrate mixture composition of the first and second sample using electrokinetic/chromatographic separation techniques combined with fluorescence or laser induced fluorescence detection; e) comparing the standard migration/retention time indices calculated from the obtained electropherogram/chromatogram of the first sample and the second sample; f) analyzing the identify and/or differences between the carbohydrate mixture composition pattern profiles of the first and second sample, wherein standard migration/retention time indices of the carbohydrates present in the sample are calculated based on internal standards of known composition labelled with a third fluorescent label and wherein one of the first or the second fluorescent label is a fluorescent dye as defined in claim 1.
5. The method according to claim 1 whereby at least two orthogonal standards are added to the sample and orthogonal cross-alignment is performed based on the given standard migration/retention time indices of the at least two orthogonal standards.
6. The method according to claim 1 wherein the sample contains a mixture of carbohydrates.
7. The method according to claim 1 wherein the sample is an extraction of glycans and the method allows for the identification of a glycosylation pattern profile.
8. The method according to claim 1 wherein the glycosylation pattern of a glycoprotein is identified.
9. The method according to claim wherein the components of the carbohydrate mixture are determined quantitatively.
10. A method for calibration of a multi wavelength fluorescence detection system, in particular, a capillary-gel electrophoresis system, with acridone and/or pyrene based fluorescent dyes which may optionally be present as conjugates with a substrate moiety including carbohydrates, whereby the method includes the detection of at least one of the compounds according to Formula A or B as defined in claim 1 together with additional fluorescent dyes and their carbohydrate conjugates emitting at different wavelengths, preferably including at least one of the compounds: APTS, 6-R, 8-H, 15, 19, 20, 23 or 23b, as shown in the following scheme: ##STR00034## ##STR00035##
11. The method according to claim 10 wherein the acridone and/or pyrene based dyes, which may optionally be present as conjugates with a substrate moiety including carbohydrates, include the combination of APTS, 6-H, 19 and 20, or APTS, 6-Me, 19 and 20, or 15, 6-Me, 19 and 20, or APTS, 15, 19 and 20, or APTS, 15, 6-Me and 20, or APTS, 8-H, 6-Me and 19, or APTS, 8-H, 6-Me and 20, or APTS, 8-H, 19 and 20, or APTS, 23, 19 and 20, or APTS, 15, 6-Me and 19, or APTS, 23, 6-Me and 19, or APTS, 23, 6-Me and 20, or 23, 6-Me, 19 and 20, or APTS, 8-H, 6-Me, 20 and 19, or APTS, 15, 6-Me, 20 and 19, or APTS, 23, 6-Me, 20 and 19, or APTS, 8-H, 6-H, 20 and 19, or APTS, 15, 6-H, 20 and 19, or APTS, 23, 6-H, 20 and 19.
12. The method according to claim 1 wherein the fluorescent dye of Formula B is a dye having the following Formula C with n=0-12 ##STR00036## wherein R.sup.1 and/or R.sup.2 are independent from each other and may represent: H, CH.sub.3, C.sub.2H.sub.5, a straight or branched C.sub.3-C.sub.12, preferably C.sub.3-C.sub.6, alkyl group, or a substituted C.sub.2-C.sub.12, preferably C.sub.2-C.sub.6, alkyl group; in particular, (CH.sub.2).sub.nCOOR.sup.3, where n=1-12, preferably 1-5, R.sup.3 may be H, CH.sub.2CN, 2- and 4-nitrophenyl, 2,3,5,6-tetrafluorophenyl, pentachlorophenyl, pentafluorophenyl, N-succinimidyl, sulfo-N-succinimidyl, 1-oxybenzotriazolyl and the alkyl chain in (CH.sub.2).sub.n may be straight or branched; and R.sup.1-R.sup.2 may form a four-, five, six-, or seven-membered non-aromatic carbocycle with an additional primary amino group NH.sub.2, secondary amino group NHR.sup.a, where R.sup.a=C.sub.1-C.sub.6 alkyl, or hydroxyl group OH attached to one of the carbon atoms in this cycle; optionally R.sup.1-R.sup.2 may form a four-, five, six-, or seven-membered non-aromatic heterocycle with an additional heteroatom such as 0, N or S included into this heterocycle; a hydroxyalkyl group (CH.sub.2).sub.mOH, where m=1-12, preferably 2-6, with a straight or branched alkyl chain; one of R.sup.1 or R.sup.2 groups may be a carbonate or carbamate derivative (CH.sub.2).sub.mOCOOR.sup.4 or COOR.sup.4, where m=1-12 and R.sup.4=methyl, ethyl, 2-chloroethyl, N-succinimidyl, sulfo-N-succinimidyl, 1-oxybenzotriazolyl a phenyl group or substituted phenyl group, e.g., 2- and 4-nitrophenyl, pentachlorophenyl, pentafluorophenyl, 2,3,5,6-tetrafluoro-phenyl, 2-pyridyl, or 4-pyridyl; (CH.sub.2).sub.mNR.sup.aR.sup.b, where m=1-12, preferably 2-6, with a straight or branched alkyl chain; R.sup.a, R.sup.b are independent from each other and may be H, or optionally substituted C.sub.1-C.sub.4 alkyl group(s), in particular, one of R.sup.1 or R.sup.2 groups may be an alkyl azide group (CH.sub.2).sub.mN.sub.3 with m=2-6 and a straight or branched alkyl chain; one of R.sup.1 or R.sup.2 groups may be (CH.sub.2).sub.nCOOR.sup.5, with n=1-5 and a straight or branched alkyl chain (CH.sub.2).sub.n and with R.sup.5 selected from H, straight or branched C.sub.1-C.sub.6 alkyl, CH.sub.2CN, 2- and 4-nitrophenyl, 2,3,5,6-tetrafluorophenyl, pentachlorophenyl, pentafluoro-phenyl, sulfo-N-succinimidyl, N-succinimidyl or 1-oxybenzotriazolyl; further, one of R.sup.1 or R.sup.2 may be (CH.sub.2).sub.bCONHR.sup.6, with n=1-12, preferably 1-5, and R.sup.6=H, C.sub.1-C.sub.6 alkyl, (CH.sub.2).sub.mN.sub.3, (CH.sub.2).sub.m—N-maleimido, (CH.sub.2).sub.m—NHCOCH.sub.2X (X=Br or I), where m=2-6 and with straight or branched alkyl chains in (CH.sub.2).sub.n and R.sup.6; or one of R.sup.1 or R.sup.2 may represent CH.sub.2—C.sub.6H.sub.4—NH.sub.2, COC.sub.6H.sub.4—NH.sub.2, CONHC.sub.6H.sub.4—NH.sub.2 or CSNHC.sub.6H.sub.4—NH.sub.2 with C.sub.6H.sub.4 being a 1,2-, 1,3- or 1,4-phenylene, COC.sub.5H.sub.3N—NH.sub.2 or CH.sub.2—C.sub.5H.sub.3N—NH.sub.2, with C.sub.5H.sub.3N being pyridin-2,4-diyl, pyridin-2,5-diyl, pyridin-2,6-diyl, or pyridin-3,5-diyl; one of R.sup.1 or R.sup.2 groups may be a primary amino group to form aryl hydrazines Ar—NR.sup.6NH.sub.2 where Ar is the entire pyrene residue in Formula C and R.sup.7=H or alkyl; one of R.sup.1 or R.sup.2 groups may be a hydroxy group to form aryl hydroxylamines Ar—NR.sup.8OH where Ar is the entire pyrene residue in Formula C and R.sup.7=H or alkyl; one of R.sup.1 or R.sup.2 groups may contain a terminal alkyloxyamino group (CH.sub.2).sub.nONH.sub.2 with n=1-12, which can be linked via one or multiple alkylamino (CH.sub.2).sub.mNH, alkylamido (CH.sub.2).sub.mCONH, alkyl ether or alkyl ester group(s) in all possible combinations with m=0-12; the (CH.sub.2).sub.n—CH.sub.2 linker, with n=1-5, between the SO.sub.2 fragment and the residue X in Formula B may represent a straight-chain, branched or cyclic group having 2-6 carbon atoms; X=SH, COOH, SO.sub.3H, OP(O)(OH).sub.2, OP(O)(OH)R.sup.a, where R.sup.a=optionally substituted C.sub.1-C.sub.4 alkyl, P(O)(OH).sub.2, P(O)(OH)R.sup.a, where R.sup.a=optionally substituted C.sub.1-C.sub.4 alkyl; with the proviso that in all compounds represented by Formula C three or six negatively charged groups are present in the residues X of Formula B under basic conditions, i.e. 7<pH<14, and these negatively charged groups represent at least partially deprotonated residues of ionizable groups selected from the following: SH, COOH, SO.sub.3H, OP(O)(OH).sub.2, OP(O)(OH)R.sup.a, where R.sup.a=C.sub.1-C.sub.4 alkyl or substituted C.sub.1-C.sub.4 alkyl, P(O)(OH).sub.2, P(O)(OH)R.sup.a, where R.sup.a=C.sub.1-C.sub.4 alkyl or substituted C.sub.1-C.sub.4 alkyl; and compounds of Formula C can exist and can be used as salts, solvates and hydrates, preferably as salts with alkaline metal cations including Na.sup.+, Li.sup.+, K.sup.+ and organic ammonium or organic phosphonium cations.
13. The method according to claim 1 wherein the fluorescent dye of Formula B is a dye having the following Formula D ##STR00037## wherein R.sup.1 and/or R.sup.2 are independent from each other and may represent H, CH.sub.3, C.sub.2H.sub.5, or a straight or branched, optionally substituted, C.sub.3-C.sub.12, preferably C.sub.3-C.sub.6, alkyl group; in particular, (CH.sub.2).sub.nCOOR.sup.4, where n=1-12, preferably 1-5, R.sup.4 may be H, CH.sub.2CN, 2- and 4-nitrophenyl, 2,3,5,6-tetrafluorophenyl, pentachlorophenyl, pentafluorophenyl, N-succinimidyl, sulfo-N-succinimidyl, 1-oxybenzotriazolyl and the alkyl chain in (CH.sub.2).sub.n may be straight or branched; and R.sup.1-R.sup.2 may form a four-, five, six-, or seven-membered non-aromatic carbocycle with an additional primary amino group NH.sub.2, secondary amino group NHR.sup.a, where R.sup.a=optionally substituted C.sub.1-C.sub.6 alkyl, or hydroxyl group OH attached to one of the carbon atoms in this cycle; or optionally R.sup.1-R.sup.2 may form a four-, five, six-, or seven-membered non-aromatic heterocycle with a heteroatom such as 0, N or S included into this heterocycle; R.sup.1 and/or R.sup.2 may further represent: a hydroxyalkyl group (CH.sub.2).sub.mOH, where m=1-12, preferably 2-6, with a straight or branched, optionally substituted alkyl chain; one of R.sup.1 or R.sup.2 groups may be a carbonate or carbamate derivative (CH.sub.2).sub.mOCOOR.sup.5 or COOR.sup.5, where m=1-12 and R.sup.5=methyl, ethyl, 2-chloroethyl, CH.sub.2CN, N-succinimidyl, sulfo-N-succinimidyl, 1-oxybenzotriazolyl, a phenyl group or substituted phenyl group, such as 2- and 4-nitrophenyl, pentachlorophenyl, pentafluoro-phenyl, 2,3,5,6-tetrafluorophenyl, 2-pyridyl, 4-pyridyl; (CH.sub.2).sub.mN.sub.3, m=1-12, preferably 2-6, with a straight or branched alkyl chain; (CH.sub.2).sub.nCONHR.sup.6, where n=1-12, preferably 1-5 and R.sup.6=H, substituted or unsubstituted C.sub.1-C.sub.6 alkyl, (CH.sub.2).sub.mN.sub.3, (CH.sub.2).sub.m—N-maleimido, (CH.sub.2)m-NHCOCH.sub.2Y (Y=Br, I) where m=1-12, preferably 2-6, with straight or branched alkyl chains in (CH.sub.2).sub.n and R.sup.6; one of R.sup.1 or R.sup.2 groups may be a primary amino group to form aryl hydrazines Ar—NR.sup.7NH.sub.2 where Ar is the entire pyrene residue in Formula D and R.sup.7=H or alkyl; one of R.sup.1 or R.sup.2 groups may be a hydroxy group to form aryl hydroxylamines Ar—NR.sup.8OH where Ar is the entire pyrene residue in Formula D and R.sup.8=H or alkyl; one of R.sup.1 or R.sup.2 groups may contain a terminal alkyloxyamino group (CH.sub.2).sub.nONH.sub.2 with n=1-12, which can be linked via one or multiple alkylamino (CH.sub.2).sub.mNH, alkylamido (CH.sub.2).sub.mCONH, alkyl ether or alkyl ester group(s) in all possible combinations with m=0-12; further, R.sup.1 or R.sup.2 may represent CH.sub.2—C.sub.6H.sub.4—NH.sub.2, COC.sub.6H.sub.4—NH.sub.2, CONHC.sub.6H.sub.4—NH.sub.2 or CSNHC.sub.6H.sub.4—NH.sub.2 with C.sub.6H.sub.4 being a 1,2-, 1,3- or 1,4-phenylene, COC.sub.5H.sub.3N—NH.sub.2 or CH.sub.2—C.sub.5H.sub.3N—NH.sub.2, with C.sub.5H.sub.3N being pyridin-2,4-diyl, pyridin-2,5-diyl, pyridin-2,6-diyl, or pyridin-3,5-diyl; R.sup.3=H, (CH.sub.2).sub.qCH.sub.2X, C.sub.2H.sub.5, a straight or branched C.sub.3-C.sub.6 alkyl group, C.sub.mH.sub.2mOR, where m=2-6, with a straight or branched alkan-diyl chain C.sub.mH.sub.2m, and R=H, CH.sub.3, C.sub.2H.sub.5, C.sub.3H.sub.7, CH.sub.3(CH.sub.2CH.sub.2O).sub.kCH.sub.2CH.sub.2; with k=1-12; while the (CH.sub.2).sub.qCH.sub.2 linker may represent a straight-chain, branched or cyclic group having 2-6 carbon atoms; in Formula D, the (CH.sub.2).sub.n—CH.sub.2 linker, with n=1-12, preferably 1-5, between the sulfonamide fragment SO.sub.2N and the residue X may represent a straight-chain, branched or cyclic group having 2-6 carbon atoms; X=SH, COOH, SO.sub.3H, OP(O)(OH).sub.2, OP(O)(OH)R.sup.a, where R.sup.a=substituted or unsubstituted C.sub.1-C.sub.4 alkyl, P(O)(OH).sub.2, P(O)(OH)R.sup.a, where R.sup.a=substituted or unsubstituted C.sub.1-C.sub.4 alkyl; with the proviso that in all compounds represented by Formula D three, six, nine or twelve negatively charged groups are present in the residues X of Formula C under basic conditions, i.e. 7<pH<14, and these negatively charged groups represent at least partially deprotonated residues of ionizable groups selected from the following: SH, COOH, SO.sub.3H, OP(O)(OH).sub.2, OP(O)(OH)R.sup.a, where R.sup.a=C.sub.1-C.sub.4 alkyl or substituted C.sub.1-C.sub.4 alkyl, P(O)(OH).sub.2, P(O)(OH)R.sup.a, where R.sup.a=C.sub.1-C.sub.4 alkyl or substituted C.sub.1-C.sub.4 alkyl; and compounds of Formula D can exist and can be used as salts, solvates and hydrates, preferably as salts with alkaline metal cations including Na.sup.+, Li.sup.+, K.sup.+ and organic ammonium or organic phosphonium cations.
14. The method according to claim 1 wherein R.sup.1 and/or R.sup.2 in formula B, or D represent: H, deuterium, alkyl or deutero-substituted substituted alkyl, wherein one, several or all H atoms of the alkyl group may be replaced by deuterium atoms, in particular alkyl or deutero-alkyl with 1-12 C atoms, preferably 1-6 C atoms, 4,6-dihalo-1,3,5-triazinyl (C.sub.3N.sub.3X.sub.2) where halogen X is preferably chlorine, 2-, 3- or 4-aminobenzoyl (COC.sub.6H.sub.4NH.sub.2), N-[(2-, N-[(3- or N-[(4-aminophenyl)ureido group (NHCONHC.sub.6H.sub.4NH.sub.2), N-[(2-, N-[(3- or N-[(4-aminophenyl)thioureido group (NHCSNHC.sub.6H.sub.4NH.sub.2 or linked carboxylic acid residues and their reactive esters of the general formulae (CH.sub.2).sub.m1COOR.sup.3, (CH.sub.2).sub.m1OCOOR.sup.3 (CH.sub.2).sub.n1COOR.sup.3 or (CO).sub.m1(CH.sub.2).sub.m2(CO).sub.n1(NH).sub.n2(CO).sub.n3(CH.sub.2).sub.n4COOR.sup.3 where the integers m1, m2 and n1, n2, n3, n4 independently range from 1 to 12 and from 0 to 12, respectively, with the chain (CH.sub.2).sub.m/n being straight, branched, saturated, unsaturated, partially or completely deuterated, and/or or included into a carbo- or heterocylcle containing N, O or S, whereas R.sup.3 is H, D or a nucleophile-reactive leaving group, preferably including but not limited to N-succinimidyl, sulfo-N-succinimidyl, 1-oxybenzotriazolyl, cyanomethyl, polyhalogenoalkyl, polyhalogenophenyl, e.g. tetra- or pentafluorophenyl, 2- or 4-nitrophenyl.
15. The method according to claim 1 wherein the compound of Formulae A to B is selected from: ##STR00038## or a compound of 7-R (R=H, Me), 13a, 13b, 16, 18, 23 and 23b ##STR00039## ##STR00040## or salts thereof.
16. A kit or system for determining and/or identifying carbohydrate mixture composition patterns comprising a data processing unit having a non-transient memory, said memory containing a database, said database containing aligned migration/retention times and/or aligned migration/retention time indices of carbohydrates, said migration/retention times and/or migration/retention time indices are obtained by an automated determination and/or identification of carbohydrates and/or identification of carbohydrates and/or carbohydrate mixture composition pattern profiling comprising the steps of: a) obtaining a sample containing at least one carbohydrate; b) labelling said carbohydrate(s) with a first fluorescent label; c) providing a standard of known composition labelled with a second fluorescent label; d) determining the migration/retention time(s) of said carbohydrate(s) and the standard of known composition using electrokinetic/chromatographic separation techniques combined with fluorescence or laser induced fluorescence detection; e) aligning the migration/retention time(s) to migration/retention time indice(s) based on given standard migration/retention time indice(s) of the standard; f) comparing these migration/retention time indice(s) of the carbohydrate(s) with standard migration/retention time indice(s) from a database; g) identifying or determining the carbohydrate(s) and/or the carbohydrate mixture composition pattern, wherein the standard composition is added to the sample containing the unknown carbohydrate mixture composition, the first fluorescent label and the second fluorescent label are different and wherein the first fluorescent label or the second fluorescent label is a fluorescent dye, preferably having multiple ionizable and/or negatively charged groups which is selected from the group consisting of compounds of the general Formulae A to B and a fluorescent dye as defined in claim 1.
17. A kit or system for an automated carbohydrate mixture composition pattern profiling comprising a data processing unit having a non-transient memory, said memory containing a database, said database containing aligned migration/retention times and/or aligned migration/retention time indices of carbohydrates, said migration/retention times and/or migration/retention time indices are obtained by an automated determination and/or identification of carbohydrates and/or identification of carbohydrates and/or carbohydrate mixture composition pattern profiling comprising the steps of a) providing a first sample containing an unknown carbohydrate mixture composition; b) labelling of said carbohydrate mixture composition with a first fluorescent label; c) adding a second sample having a known carbohydrate mixture composition pattern labelled with a second fluorescent label to said first sample; d) generating electropherograms/chromatograms of the carbohydrate mixture composition of said sample using electrokinetic/chromatographic separation techniques combined with fluorescence or laser induced fluorescence detection, like capillary gel electrophoresis-laser induced fluorescence; e) analyzing the identity and/or differences between the carbohydrate mixture composition pattern profiles of the first and the second sample, wherein the first fluorescent label of the first sample is different to the second fluorescent label of the second sample and wherein at least one of the first fluorescent label and the second fluorescent label is a fluorescent dye as defined in claim 1.
18. A kit or system according to claim 16 further comprising a capillary gel electrophoresis-laser induced fluorescence apparatus, in particular, wherein the capillary gel electrophoresis-laser induced fluorescence apparatus is a capillary DNA-sequencer.
19. A carbohydrate dye conjugate comprising fluorescent dyes as defined in and used in the method of claim 1.
20. The carbohydrate dye conjugate according to claim 19 wherein the dye is selected from the compounds of the formula below ##STR00041## ##STR00042## ##STR00043##
21. A kit or composition comprising one or more of the dyes as defined in and for use in the method of claim 1.
22. A calibration standard, like an oligosaccharide standard, including a fluorescence dye according to Formula A, B, C or D which may be conjugated with a carbohydrate, optionally further comprising at least one of compounds 19, 20.
23. A kit containing a calibration standard according to claim 22 and, optionally, instructions for use.
24. A compound having the Formula 20 ##STR00044##
25. A standard composition composed of compounds labelled with a fluorescence dye according to Formula A or B, in particular, of Formula C or D or different dyes of Formulae A to D.
26. The standard composition according to claim 25 being composed of carbohydrates labelled with a fluorescence dye according to Formula A or B, in particular, of Formula C or D or different dyes of Formulae A to D.
27. The standard composition according to claim 25 wherein the fluorescence dye is at least one dye selected from 6-H, 6-Me, 8-R, 15, 13a, 13b, 16, 18, 23 and 23b.
28. (canceled)
29. A kit or composition comprising one or more of the carbohydrate dye conjugates of claim 19.
Description
BRIEF DESCRIPTION OF THE FIGURES
[0211]
[0212]
[0213]
[0214]
[0215]
[0216]
[0217]
[0218]
[0219]
[0220]
[0221]
[0222]
[0223]
[0224]
[0225]
[0226]
[0227]
[0228] ” curve, 15 kV), 200 V/cm (“
” curve, 10 kV), or 100 V/cm (“-” curve, 5 kV).
[0229] ” curve), 30° C. (“
” curve), or 18° C. (“-” curve).
[0230] ” curve), 36 cm capillary array (“
” curve), or 22 cm capillary array (“-” curve).
[0231] ” curve), or glyXpop_fast (experimental polymer from glyXera GmbH; black “
” curve).
[0232]
[0233]
[0234]
[0235]
[0236]
[0237]
[0238]
[0239]
[0240]
[0241]
[0242]
GENERAL MATERIALS AND METHODS
Reductive Amination of Carbohydrates
[0243] For reductive amination of carbohydrates using the compounds of the present invention, for example the prior art protocol for fluorescent labeling of N-glycans with 8-aminopyrene-1,3,6-trisulfonic acid trisodium salt (APTS) and a reducing agent as published by Hennig R, Rapp E, et al in Methods Molecular Biology in 2015 was used with small adaptations.
[0244] The original protocol requires a moderately strong acid (e.g., citric acid as monohydrate; CA) and solvents—dimethyl sulfoxide (DMSO), acetonitrile (ACN) and water (H.sub.2O). Main steps include the preparation of 10-80 mM dye solution in 1.2-3.6 M aqueous CA (solution A) and borane based reducing agent solution in DMSO (solution B). Then it is necessary to mix three components of equal volumes (1-4 μL) of solutions A, B and the sample (free carbohydrates or the carbohydrate moiety of glycoconjugates after release) and incubate at 37° C. for 3-16 h. After completion of the reductive amination, ACN—water mixture (80:20, v/v) is added. For example, if 2 μL of solution A, 2 μL of solution B, and 2 μL of the analyte sample were used, then 50 μL of aq. ACN were added and mixed. This operation provides clear solutions which can be subjected to electrokinetic and/or chromatographic separation-based glycoanalysis.
Hydrazide Labeling
[0245] The hydrazide labeling, using the compounds of the present invention, was performed at 60° C.-80° C. for 1 h-6 h at pH 6-8. A 10-80 mM dye solution was mixed in equal volumes (1-4 μL) with the sample. After completion of the reaction 50 μL of an ACN—water mixture (80:20, v/v) were added. A dilution of the labeling mixture was subjected to electrokinetic and/or chromatographic separation-based glycoanalysis.
Reactive Carbamate Chemistry
[0246] The disuccinimidyl carbonate- or NHS ester-assisted labeling of glycosylamines with compounds of the present invention, was performed at room temperature for 10 60 min at slightly basic pH. Samples were purified by HILIC-SPE as published by Hennig R, Rapp E et al 2015. Purified sample was subjected to electrokinetic and/or chromatographic separation-based glycoanalysis.
Example 1—Selected Fluorescent Dyes with Large Negative Net Charges and Required Spectral Properties (See Also Scheme 13 and Table 1)
[0247] ##STR00030##
[0248] The red-emitting rhodamine dye with multiple ionizable groups of structure 20 was obtained by phosphorylation of the corresponding hydroxyl-substituted rhodamine precursor and isolated analogously to compound 19 (another phosphorylated rhodamine dye, see Schemes 6 and 11 above) previously described by K. Kolmakov, et al. in Chem. Eur. J. 2012, 18, 12986-12998 (see compound 7-H therein for the properties and the phosphorylation details). The hydroxyl-substituted precursor for compound 20 was synthesized according to K. Kolmakov, et al. (Chem. Eur. Journal, 2013, 20, 146-157; see compound 14-Et therein). The phosphorylation was followed by saponification of the ethyl ester group via a routine procedure, as described.
[0249] Purity and identity of compound 20 was confirmed by the following analytical data: .sup.1H NMR (400 MHz, DMSO-d.sub.6): δ=1.23 (s, 6H, CH.sub.3), 1.28 (s, 6H, CH.sub.3), 2.62 (s, 6H, NCH.sub.3), 4.21 (m, 4H, 2CH.sub.2), 5.70 (s, 2H), 6.76 (s, 2H), 7.16-7.30 (br. m, 4H), 8.55 (m, 1H), 8.36 (m, 1H) ppm. .sup.13C NMR (101 MHz, DMSO-d.sub.6): δ=29.1 (CH.sub.3), 34.2 (CH.sub.3), 95.8 (CH.sub.2), 118.2 (CH), 121.7 (C) 122.6 (C), 125.5 (CH), 127.3 (CH), 127.4 (CH), 128.0 (CH), 129.8 (CH), 133.9 (C), 136, (C), 155.0 (CO), 157.0 (CO) ppm.
[0250] .sup.1H NMR (400 MHz, CD.sub.3OD, 20 as a Et.sub.3N-salt): δ=1.12 (t, J=7 Hz, 9H, CH.sub.3CH.sub.2), 1.25 (t, J=7 Hz, 27H, CH.sub.3CH.sub.2), 1.52 (s, 6H, CH.sub.3), 1.53 (s, 6H, CH.sub.3), 3.11, 3.31 (m, 24H, CH.sub.3CH.sub.2), 3.18 (s, 6H, NCH.sub.3), 3.61 (m, 2H, CH.sub.2), 4.45 (m, 2H, CH.sub.2), 6.03 (s, 2H), 6.8 (s, 2H), 6.9 (s, 2H), 7.28 (d, J=8 Hz, 1H), 8.16 (d, J=8 Hz, 1H), 8.66 (m, 1H) ppm. .sup.31P NMR (161.9 MHz): δ=−0.2 (DMSO-d.sub.6) and 0.63 (CD.sub.3OD) ppm (s, OP(O)(OH).sub.2)).
[0251] HPLC: t.sub.R=3.9 min (Kinetex EVO C-18 column, with 0.02 M aq. Et.sub.3N (A) and 3% MeCN (B), isocratic flow 0.5 mL/min, detection at 254 nm). TLC: R.sub.f=0.25 (silica gel plates, MeCN/H.sub.2O 5:1+0.2% Et.sub.3N). HR-MS (ESI): calc. for C.sub.35H.sub.35N.sub.2O.sub.13P.sub.2.sup.− ([M-H].sup.−) 753.1614, found 753.1672. UV-VIS (PBS buffer, pH=7.4) λ.sub.max. abs.=582 nm, λ.sub.max. fl.=609 nm.
Example 2—Spectral Calibration of Multi-Wavelength Fluorescence Detection Systems to a Set of Four Acridone and Pyrene Based Fluorescent Dyes as Described Herein
[0252] For the current example the procedure is exemplarily shown for modified commercial DNA Genetic Analyzer 310, 3100, 3130(xl), 3730(xl) and 3500 (all manufactured by Applied Biosystems, now Thermo Scientific). But, depending on the mode of detection, the here presented re-calibration is also possible for instruments of other manufacturers. The used commercial Genetic Analyzer contains a multiplexed capillary gel electrophoresis (xCGE) unit with laser induced fluorescence detection (LIF), which can (depending on the instrument and operating software) simultaneously detect up to six different fluorescent signals in separate dye channels.
[0253] According to the manufacturer virtual filters of the instrument can be calibrated to various pre-defined dye sets like F, D (both: four detection windows) or G5 (five detection windows). As a default spectral calibration for the analysis of oligosaccharides the pre-defined dye set G5 is used [EP 2112506 B1, Ruhaak 2010, Reusch 2015, Feng 2017]. G5 is calibrated to the DS-33 Matrix Standard containing the dyes 6-Fam™ (recorded inside the 522 nm dye trace), VIC® (at 554 nm), NED™ (at 575 nm), PET® (at 595 nm) and LIZ® (at 655 nm). With this calibration APTS labeled oligosaccharides are recorded inside the 6-Fam™ dye trace (522 nm) and the alignment standard GeneScan 500 LIZ™ inside the LIZ® dye trace (655 nm). Unfortunately, using the G5 spectral calibration APTS produces a signal in all other dye traces, as shown in
[0254] To be able to the use an alignment standard, different from LIZ500 and to reduce the spectral cross-talk the xCGE-LIF instrument was exemplarily calibrated to a set of four dyes, including APTS and three new dyes of the current invention. Before spectral calibration all fluorescent dyes (respectively their oligosaccharide derivates) showed a fluorescent signal in multiple dye traces/channels (
[0255] After this spectral calibration of xCGE-LIF instrument the 6-H-labeled maltose ladder could be used for internal alignment of APTS labeled carbohydrates. Therefore the 6-H labeled maltose ladder was co-injected with APTS labeled carbohydrates, sensing the same sample background as the APTS labeled carbohydrates. As a side effect, the better fitting spectral calibration results in an increased signal intensity for 6-H labeled ladder (
[0256] A spectral calibration of multi-wavelength systems to a set of four fluorescent dyes is possible to big variation of herein invented dyes, as shown in Table 2.
TABLE-US-00002 TABLE 2 Spectral calibration of multi-wavelength systems to a set of four dyes. Exemplarily the possibilities are shown for a four dye spectral calibration of a 3100, 3130, 3130xL, 3730, 3730xL, 3500 and 3500xL instrument. For a spectral calibration one fluorescence dye per trace needs to be taken, without doubling. E.g. to analyze APTS-labeled samples the spectral trace 522 nm is calibrated to an APTS-labeled carbohydrate (APTSz). Simultaneous the spectral trace 560 nm is calibrated to one of the following dye: 6-H, 6-Me, 6-H.sup.z, 6-Me.sup.z, 8-H, 8-H.sup.z, 15, 15.sup.z, 23, 23.sup.z; the spectral trace 575 nm to 20, 6-H, 6-Me, 6-H.sup.z or 6-Me.sup.z, the spectral trace 607 nm to 19 or 20. One possible spectral calibration is APTS.sup.z,15.sup.z, 6-Me.sup.z and 19. These spectral calibration enables the analysis of up to three samples (APTS-, 15-, and 6-Me-labeled in spectral trace 522 nm, 560 nm and 575 nm) together with a base pair based internal alignment standard (in spectral trace 607 nm). Spectral trace Possible fluorescence dye for calibration of spectral trace 522 nm APTS APTS.sup.z 15 15.sup.z 23 23.sup.z 560 nm 6-H 6-Me 6-H.sup.z 6-Me.sup.z 8-H 8-H.sup.z 15 15.sup.z 23 23.sup.z 575 nm 6-H 6-Me 6-H.sup.z 6-Me.sup.z 20 607 nm 19 20 Small selection of possible combinations for spectral calibration No. 1 No. 2 No. 3 No. 4 No. 5 No. 6 No. 7 No. 8 No. 9 No. 10 522 nm APTS.sup.z APTS.sup.z APTS.sup.z APTS.sup.z APTS.sup.z APTS.sup.z APTS.sup.z APTS.sup.z 23.sup.z 15.sup.z 560 nm 6-H.sup.z 6-Me.sup.z 15.sup.z 15.sup.z 23.sup.z 8-H.sup.z 15.sup.z 23.sup.z 6-Me.sup.z 6-Me.sup.z 575 nm 20 20 6-Me.sup.z 6-Me.sup.z 6-Me.sup.z 6-Me.sup.z 20 6-Me.sup.z 20 20 607 nm 19 19 19 20 19 19 19 19 19 19 Example FIG. 2 FIG. 28 for and spectral FIG. 3 calibration Index z = fluorescent dye-carbohydrate derivate .fwdarw. 4 e.g. APTS.sup.z could be APTS-labeled maltotetraose (see in FIGURE 2), or 15.sup.z could be 15-labeled maltotriose (used in FIGURE 4). But .sup.z can be any other carbohydrate, like an O-glycan, N-glycan, milk oligosaccharide, a homopolymer (e.g. maltose, starch, cellulose, dextran) or a heteropolymer (e.g. hemicellulose, arabinoxylan, glucosaminoglycan) build from pentoses and/or hexoses.
Example 3—Spectral Calibration of Multi-Wavelength Fluorescence Detection Systems to a Set of Five Acridone and Pyrene Based Fluorescent Dyes as Described Herein
[0257] For the current example the procedure is exemplarily shown for modified commercial DNA Genetic Analyzer 310, 3100, 3130(xl), 3730(xl) and 3500 (all manufactured by Applied Biosystems, now Thermo Scientific). But, depending on the mode of detection, the here presented re-calibration is also possible for instruments of other manufacturers. The used commercial Genetic Analyzer contains a multiplexed capillary gel electrophorese (xCGE) unit with laser induced fluorescence detection (LIF), which can (depending on the instrument and operating software) simultaneously detect up to six different fluorescent signal in separate dye channels.
[0258] The virtual filters of these instruments can be calibrated to various pre-defined dye sets like E5, G5 or D. Thereby, dye set E5 and G5 define five detection windows for five different fluorescent dyes, whereas dye set D defines four detection windows for four different fluorescent dyes. For the analysis of oligosaccharides the pre-defined dye set G5 is used, calibrated to the DS-33 Matrix Standard containing the dyes 6-Fam™ (recorded inside the 522 nm dye trace), VIC® (at 554 nm), NED™ (at 575 nm), PET® (at 595 nm) and LIZ® (at 655 nm) [EP 2112506 B1, Ruhaak 2010, Reusch 2015, Feng 2017]. Subsequently, light emitted by the APTS-labeled oligosaccharides is recorded inside the dye trace 522 nm (Fam™ dye trace) and light emitted by the alignment standard GeneScan 500 LIZ™ (LIZ500) is recorded inside the dye trace 655 nm. As the instrument is not specifically calibrated to the APTS dye, APTS-labeled oligosaccharides emitting light into several dye traces, as shown in
[0259] Exemplarily a spectral calibration of the xCGE-LIF instrument was performed to a set of five dyes, as shown in
[0260] Furthermore, the spectral calibration to the dye derivate 15.sup.a and 6-Me.sup.aenabled the simultaneous use of two different carbohydrate-based standards for the comparison of the alignment performance as shown in
[0261] A spectral calibration of multi-wavelength systems to a set of five fluorescent dyes is possible to big variation of herein invented dyes, as shown in Table 3.
TABLE-US-00003 TABLE 3 Spectral calibration of multi-wavelength systems to a set of five dyes. Exemplarily the possibilities are shown for a five dye spectral calibration of a 3100, 3130, 3130xL, 3730, 3730xL, 3500 and 3500xL instrument. For a spectral calibration one fluorescence dye per trace needs to be taken, without doubling. E.g. to analyze APTS-labeled samples the spectral trace 522 nm is calibrated to an APTS-labeled carbohydrate (APTS.sup.z). Simultaneous the spectral trace 554 nm is calibrated to one of the following dye: 8-H, 8-H.sup.z, 15, 15.sup.z, 23 or 23.sup.z; the spectral trace 575 nm to 6-H, 6-Me, 6-H.sup.z or 6-Me.sup.z, the spectral trace 595 nm to 20 and the spectral trace 655 nm 19. E.g. spectral calibration to APTS.sup.z,23.sup.z, 6-Me.sup.z, 20 and 19 enables the analysis of two samples (APTS-and 23-labeled in spectral trace 522 nm and 554) together with carbohydrate based alignment standard (6-Me-labeled in spectral trace 575 nm) and/or a base pair based internal alignment standard (in spectral trace 655 nm). Spectral trace Possible fluorescence dye for calibration of spectral trace 522 nm APTS APTS.sup.z 554 nm 8-H 8-H.sup.z 15 15.sup.z 23 23.sup.z 575 nm 6-H 6-Me 6-H.sup.z 6-Me.sup.z 595 nm 20 655 nm 19 Selection of possible combinations for spectral calibration No. 1 No. 2 No. 3 No. 4 522 nm APTS.sup.z APTS.sup.z APTS.sup.z APTS.sup.z 554 nm 8-H.sup.z 8-H.sup.z 23.sup.z 15.sup.z 575 nm 6-H.sup.z 6-Me.sup.z 6-Me.sup.z 6-Me.sup.z 595 nm 20 20 20 20 655 nm 19 19 19 19 Example FIG 15-20 FIG 4-8, FIG. 15, for spectral 28, 29 and 31 calibration Index z = fluorescent dye-carbohydrate derivate .fwdarw. 4 e.g. APTS.sup.z could be APTS-labeled maltotetraose (see in FIGURE 2), or 15.sup.z could be 15-labeled maltotriose (used in FIGURE 4). But .sup.z can be any other carbohydrate, like an O-glycan, N-glycan, milk oligosaccharide, a homopolymer (e.g. maltose, starch, cellulose, dextran) or a heteropolymer (e.g. hemicellulose, arabinoxylan, glucosaminoglycan) build from pentoses and/or hexoses.
Example 4—Utilizing Acridone Fluorescent Dye Derivates According to the Present Invention for the Internal Migration Time Alignment
[0262] The current example includes the use of modified commercial DNA Genetic Analyzer 310, 3100, 3130(xl), 3730(xl) and 3500 (all manufactured by Applied Biosystems, now Thermo Scientific). Nevertheless, the here presented carbohydrate-based alignment standards can also be used in combination with (single or multiple capillary) CE/CGE instruments or with (U)HPLC instruments of other manufacturers. In general, the migration time alignment of DNA fragment sizes (as used in genomics for e.g. short tandem repeat (STR) or restriction fragment length polymorphism (RFLP) analysis), as well as of carbohydrates in CE/CGE and xCGE is currently realized by the use of base pair size standards, as exemplarily shown in
[0263] While the long-term alignment quality of an unknown DNA fragment to a DNA-based base pair size standard is very good, the long-term alignment quality of oligosaccharides to a base pair size standard is not as good. The aligned migration times of carbohydrates to a base pair size standard show some fluctuation over a longer time and for different polymer lots (see
[0264] However, the second (orthogonal) alignment step compensates the most part of these fluctuations in the long-term also for carbohydrates, but not completely. The reason for a less good alignment power in long-term are the different physicochemical properties of the base pair standard and the labeled carbohydrates. While for instance a 360 base pair long fragment (peak 10 in
[0265] The here presented invention enables the use of a carbohydrate-based standard-mix for the migration time alignment of a carbohydrate. A complete set of new fluorescent dyes was developed to label the oligosaccharide sample and/or these carbohydrate standards/-mix. The new developed fluorescent dyes have different spectral properties than the fluorescent dye used for the labeling of the unknown sample. This enables a co-injection of the fluorescently labeled sample together with the fluorescently labeled carbohydrate alignment standard and a simultaneous detection of both analytes in different dye/wavelength traces as shown in
[0266] For the here presented example human citrate plasma N-glycans were analyzed by xCGE-LIF as described in Hennig et al. 2016 using the dyes as described herein. Briefly, citrate plasma proteins were denaturized and linearized. N-glycans were enzymatically released by PNGase F and labeled with 8-aminopyrene-1,3,6-trisulfonic acid (APTS). After HILIC-SPE purification APTS-labeled N-glycans were analyzed by multiplexed capillary gel electrophoresis with laser-induced fluorescent detection (xCGE-LIF) using an Applied Biosystems® 3130 Genetic Analyzer. For internal migration time alignment APTS-labeled samples were co-injected with a 6-Me-labeled carbohydrate-based alignment standard (6-Me.sup.b), see
[0267] A spectral calibration of the instrument to 15.sup.a, 19, 20, 6-Me.sup.a and APTS.sup.a was performed as described in Example 3. APTS samples were recorded at 522 nm, 6-Me.sup.b at the 575 nm and LIZ500 at the 655 nm dye trace. For migration time alignment to LIZ500 13 standard peaks were picked as shown in
[0268] By performing an orthogonal adjustment of the LIZ500 alignment as described in U.S. Pat. No. 8,293,084 an improved migration time alignment could be archived (see
TABLE-US-00004 TABLE 4 Comparison of alignment precision for N-glycans aligned to a base pair ladder LIZ500, to a LIZ500 base pair ladder improved by an additional bracketing carbohydrate re-alignment and to an acridone dye-labeled carbohydrate standard (6-Me.sup.b) only. Root-mean-squared-error (RMSD) of citrate plasma N-glycans was calculated for samples shown in FIG. 10. The 15 picked peaks are depicted in FIG. 10 B. N-glycan groups contain peaks: 10-15 for neutral, 9-7 for single charged, 2-6 for double charged and peak 1 for triple charged (for a detailed annotation of glycan peaks see Hennig et al. 2016). The absolute RMSD is given in base pairs for LIZ500 alignment, in migration time units for LIZ500 + bracketing carbohydrate (oligosaccharide) re-alignment and in carbohydrate (oligosaccha- ride) units for 6-Me.sup.b only alignment. Alignment to LIZ500 + bracketing Alignment to carbohydrate LIZ500 as re-alignment Alignment described in EP according to US to 6-Me.sup.b N-glycan group 2112506 A1 2009/028895 A1 only root-mean- 15 picked peaks 8.388 1.782 0.029 squared error Neutral N-glycans 11.226 2.168 0.037 Single charged N-glycans 8.028 1.606 0.019 Double charged N-glycans 5.881 1.433 0.024 Triple charged N-glycans 4.978 1.745 0.032 root-mean- 15 picked peaks 3.151 0.727 0.359 squared Neutral N-glycans 3.326 0.660 0.357 error in % (of Single charged N-glycans 3.158 0.658 0.236 mean) Double charged N-gly cans 3.008 0.782 0.391 Triple charged N-glycans 2.801 1.059 0.570
Example 5—Utilizing Pyrene Fluorescent Dye Derivates According to the Present Invention for the Internal Migration Time Alignment
[0269] The migration time alignment of DNA fragment sizes as well as of carbohydrates in CE/CGE and xCGE is currently realized by the use of base pair size standards (EP 2112506 A1), as exemplarily shown in
[0270] While the long-term alignment quality of an unknown DNA fragment to a DNA based base pair size standard is very good, the long-term alignment quality of carbohydrates to base pair size standards is not as good. The aligned migration times of oligosaccharides to a base pair size standard show some variation over several days and different polymers lots (see
[0271] After alignment to the carbohydrate-based size standard 15.sup.b an improved long-term reproducibility could be achieved as shown in
[0272] This improved alignment procedure can also be performed by the use of other oligosaccharide ladders, like chitin, cellulose, maltose, pullulan, glycosaminoglycans, as well as by the use of complex carbohydrates like the glycomoiety of glycolipids, O-glycans, N-glycans and milk oligosaccharides (e.g. lactose, lacto-N-tetraose, lacto-N-hexaose and their fucose and/or lactose elongations).
TABLE-US-00005 TABLE 5 Comparison of alignment precision for N-glycans aligned to a base pair ladder LIZ500 (align- ment to LIZ500), to a base pair ladder improved by an additional carbohydrate re-alignment (alignm. to LIZ500 + 15.sup.b) and to a pyrene dye (15) labeled carbohydrate standard (15.sup.b) only. Root-mean- squared-error (RMSD) of citrate plasma N-glycans was calculated for samples shown in FIG. 12. The 15 picked peaks are depicted in FIG. 12 C. N-glycan groups contain peaks: 10-15 for neutral, 9-7 for single charged, 2-6 for double charged and peak 1 for triple charged (for a detailed annota- tion of glycan peaks see Hennig et al. 2016). The absolute RMSD is given in base pairs for LIZ500 alignment, or in carbohydrate (oligosaccharide) units for LIZ500 + 15.sup.b and for 15.sup.b only alignment. Alignment to LIZ500 As described in Alignment to N-glycan group EP 2112506 A1 LIZ500 + 15.sup.b Alignment 15.sup.b only root-mean- 15 picked peaks 8.388 0.121 0.078 squared error Neutral N-glycans 11.226 0.213 0.127 Single charged N- 8.028 0.114 0.071 glycans Double charged N- 5.881 0.036 0.036 glycans Triple charged N- 4.978 0.017 0.017 glycans root-mean- 15 picked peaks 3.151 0.929 0.627 squared error Neutral N-glycans 3.326 1.398 0.837 in % (of Single charged N- 3.158 1.031 0.640 mean) glycans Double charged N- 3.008 0.442 0.445 glycans Triple charged N- 2.801 0.241 0.236 glycans
For the presented example human citrate plasma N-glycans were analyzed by xCGE-LIF as described in Hennig et al. 2016 using the dyes as described herein. Briefly, citrate plasma proteins were denaturized and linearized by incubation with SDS at 60° C. N-glycans were enzymatically released by PNGase F and labeled with 8-aminopyrene-1,3,6-trisulfonic acid (APTS). After HILIC-SPE purification APTS labeled N-glycans were analyzed by multiplexed capillary gel electrophoresis with laser induced fluorescent detection (xCGE-LIF) using an Applied Biosystems® 3130 Genetic Analyzer. A spectral calibration of the instrument to 15.sup.a, 19, 20, 6-Me.sup.a and APTS.sup.a was performed as described in Example 3.
Example 6—Pyrene and/or Acridone Labeled Carbohydrates as a Universal Alignment Standard
[0273] The current example includes the use of modified commercial DNA Genetic Analyzer 310, 3100, 3130(xl), 3730(xl) and 3500 (all manufactured by Applied Biosystems, now Thermo Scientific). Nevertheless, the here presented carbohydrate-based alignment standards can also be used in combination with CE/CGE and with (U)HPLC instruments (single or multiple capillary) of other manufacturers.
[0274] In general, the migration time alignment of DNA fragment and of carbohydrates in (x)CE/(x)CGE is currently realized by the use of base pair size standards (EP 2112506 A1). For this purpose, the migration times of an unknown sample is aligned to a co-injected base pair size standard. While a base pair size standard based alignment shows good results for DNA, the aligned of a carbohydrates sample shows big variations as shown in Example 2 and 3. This variation is more apparent when using different: [0275] Instruments (
During this stress test these parameters were modified and the alignment procedure (base pairs vs. carbohydrate standard) was compared. For all examples the carbohydrate alignment procedure showed a superior performance. For the most variations a stable migration time could be archived, as shown for example for the different capillary lengths. This means by using the carbohydrate alignment procedure a comprehensive carbohydrate database can be used, also if experimental settings, instrument parameters or instruments are alternated. This is impossible with a base pair-based alignment standard.
TABLE-US-00006 TABLE 6 Comparison of alignment precision for N-glycans aligned to a base pair ladder LIZ500 (alignm. to LIZ500), to a LIZ500 base pair ladder improved by an additional bracketing (b) carbohydrate (oligosaccharide (OS)) re-alignment (alignm. to LIZ500 + bOS, = bracketing OligoSaccharide), to a LIZ500 base pair ladder improved by an additional pyrene dye (23) labeled carbohydrate standard (23.sup.c) (alignm. to LIZ500 + 23.sup.c) and to a pyrene dye (23) labeled carbohydrate standard (23.sup.c) only (alignm. to 23.sup.c only). Root-mean-squared-error (RMSD) of citrate plasma N-glycans was calculated for 15 picked peaks as shown in FIGURE 12 C. N-glycan groups contain peaks: 10-15 for neutral, 9-7 for single charged, 2-6 for double charged and peak 1 for triple charged (for a detailed annotation of glycan peaks see Hennig et al. 2016). The absolute RMSD is given in base pairs for LIZ500 alignment, in migration time units for LIZ500 + bracketing carbohydrate re-alignment and in carbohydrate units for LIZ500 + 23.sup.c and 23.sup.c only alignment. For instrument comparison, data of FIGURE 15 was used (6 different instruments). For polymer lot comparison, citrate plasma N-glycans were measured inside 3130xl1 using four different POP7 polymer lots (lot: 1612560, 1701565, 1703117 and 1705571). For polymer age comparison citrate plasma N-glycans were measured inside 3130xl_1 with fresh polymer (lot: 1708574), fresh opened one year old polymer (lot: 1411512), opened one year old polymer (lot: 1411512) and opened five years old polymer (lot: 1208456). For all comparison cases a reduction of RMSD by a factor of five (10.697 to 2.172) up to seven (2.246 to 0.334) could be archived. Instrument Comparison Polymer Lot Polymer Age (see Figure 17 A, B, C & D) Comparison Comparison Alignm. Alignm. Alignm. Alignm. Alignm. Alignm. To To To Alignm. To Alignm. to N-glycan to LIZ500 + LIZ500 + 23.sup.c To 23.sup.c To 23.sup.c group LIZ500 bOS 23.sup.c only LIZ500 only LIZ500 only root- 15 peaks 4.446 1.133 0.018 0.013 5.905 0.015 31.838 0.100 mean- Neutral 5.365 1.060 0.010 0.007 7.722 0.010 45.485 0.053 squared Single 4.240 1.225 0.015 0.017 5.687 0.013 29.895 0.109 error charged Double 3.646 1.125 0.027 0.017 4.283 0.020 19.606 0.144 charged Triple 3.547 1.334 0.035 0.024 3.764 0.027 16.942 0.129 charged root- 15 peaks 1.715 0.487 0.417 0.298 2.246 0.334 10.697 2.172 mean- Neutral 1.572 0.318 0.137 0.089 2.296 0.126 12.111 0.689 squared Single 1.665 0.505 0.284 0.325 2.251 0.240 10.785 2.036 error in charged % (of Double 1.860 0.614 0.707 0.445 2.204 0.540 9.292 3.711 mean) charged Triple 1.995 0.816 1.050 0.739 2.136 0.829 8.973 3.783 charged
Example 7—Recalibration of a DNA Sequencer Using New Sets of Fluorescent Acridone and Pyrene Dyes According to the Invention
[0278] Commercial CE-systems may have a multi-wavelength detector and therefore several color channels.
[0279] There are so-called “virtual light filters” in those systems, where the software defines certain wavelength-areas for the collection of the fluorescent emissions from different dyes.
[0280] These areas are called virtual filters. Each of them is associated with a relatively narrow range of the visible light emitted only by one dye (
[0281] Importantly, the emission of APTS dye and its conjugates with glycans always appears in the channel with shortest wavelength, and the absence of cross-talk with the reference channel is crucial. After labeling with APTS, the electropherograms of the complex glycan mixtures contain peaks with intensities varying in the orders of magnitude. Thus, the fluorescence signal in APTS channel has to be completely free from the emission “leaking” from the reference channel. The reference sample contains a mixture labeled with another fluorescent dye and injected simultaneously with the analyzed sample. This requirement of a “complete” absence of the cross-talk between the observation channel (APTS dye or its substitute) and the reference channel seems to be easy to fulfill, but is not the case, because both dyes have to be excited with the same light source and their emission spectra overlap. Up to now, a LIZ dye (attached to a “DNA ladder” used as an internal alignment standard in glycan analysis) was used as an additional color in a 655 nm observation channel. For the detection of a LIZ dye, a virtual filter set G5 (including 6-Fam™, VIC®, NED™, PET® and LIZ®) is used in ABI 3100 DNA sequencer (ABI user manual). This dye consists of a FRET pair—a donor dye, and an acceptor dye. This combination (similar to a dye with very large Stokes shift) provides an absence of cross-talk, because a donor dye is efficiently excited with green light, transfers energy to an acceptor, and the latter emits only red light. However, FRET pairs with complete energy transfer, multiple negative charges, and an aromatic amino group are too complex and therefore hardly synthetically available. Therefore, the present invention provides fluorescent dyes with enlarged Stokes shifts. As substitutes for an internal alignment standard, these dyes give no emission in the APTS (observation) channel.
[0282] In order to eliminate cross-talk with an APTS channel, it was necessary to re-calibrate the commercial DNA sequencer (manufactured by Applied Biosystems) using other sets of fluorescent dyes. According to the manufacturer, there can be any number of (various) virtual filters (observation windows). Therefore, the new detection channels may be designated. For example, the emission maxima of 5 arbitrary fluorescent dyes define 5 (new) detection windows (filters). To minimize cross-talk, the absorption maxima of the new reference dyes have to be spread more or less uniformly in the range from 500 nm to 655 nm. The “crosstalk” (overlap) between emission colors on the CCD array is corrected by a matrix file in the software. This procedure is well-known and called “linear unmixing” (T. Zimmermann, et al., Methods Mol. Biol. 2014, 1075, 129-148).
[0283] The matrix file is generated from a separate, “matrix” run in which the reference dyes or their derivatives are subjected to capillary electrophoresis, separated into individual peaks and their emission spectra are registered in the whole spectral range. The matrix file contains information about the inputs of the individual dyes into the emitted light falling onto a certain filter (detected within a certain observation window). For each filter (detection window), the input of one dye is maximal, but there are also contributions from the other dyes “contaminating” the overall signal passing through the certain filter.
[0284] In
[0285]
[0286] For that, the negatively charged fluorescent dyes 19, 20, 6-R and 15 (see below) were chosen and used together with APTS in a new set for the spectral calibration of the electrophoresis unit integrated into a DNA sequencing device. With these dyes, a new matrix file was generated and used in correcting the spectral overlap.
##STR00031##
[0287] Table 7 indicates the properties of fluorescent dyes, including rhodamines 19 and 20 (see K. Kolmakov, et al., Chem. Eur. J. 2012, 18, 12986-12998 and K. Kolmakov, et al., Chem. Eur. Journal, 2013, 20, 146-157.), 6-R and 15 and their conjugates with oligosaccharides consisting of maltose units. Remarkably, the conjugate of dye 8-H with maltohexaose has a much shorter retention time (13.1 min) that the APTS derivative obtained from maltotetraose (16.5 min). Though the hydrodynamic ratios of dyes 8-H and 15 are larger than that of APTS, the presence of six negative charges in these dyes (versus three in APTS) strongly increases their electrophoretic mobilities in the electric field.
TABLE-US-00007 TABLE 7 Properties of fluorescent dyes 6-R, 15, 19, 20 and 23 used in a new set together with APTS for the spectral calibration of the fluorescence detection unit integrated into a DNA sequencing device. Migration time,.sup.b Free dye absorption Free dye emission (see also FIGS. in Dye λ.sub.max, nm (ε, M.sup.−1 cm.sup.−1) λ.sub.max, nm (ϕ.sub.fl) Conjugate with attachment) 6-H.sup.a 217 (13500), 260 (26000) 586 (0.05) maltotriose 15.5 min, 575 nm 295 (28000), 420 (3700) 2 × OP(O)(OH).sub.2 6-Me.sup.a 219 (10300), 263 (18600) 585 (0.05) maltotriose 15.0 min, 575 nm 299 (18500), 430 (2900) 2 × OP(O)(OH).sub.2 8-H.sup.a 465 (3 × OP(O)(OH).sub.2) 530 (0.94) free dye 7.3 min, 522/544 nm.sup.c maltohexaose 13.1 min, 554 nm 15.sup.a 477 (3 × OP(O)(OH).sub.2) 542 (0.94) free dye 6.8 min, 554 nm maltotriose 9.5 min, 554 nm APTS.sup.a 425 (3 × SO.sub.3H) 457 maltotetraose 16.5 min, 522 nm 19 635 (75000) 655 (0.55).sup.b free dye 11.2 min 20 581 (60000) 607 (0.95) free dye 11.7 min 23.sup.a 486 (23000) 3 × SO.sub.3H 542 (0.83) free dye 9.9 min, 554 nm maltotriose 16.9 min, 554nm .sup.aConjugation to carbohydrates and/or N-alkylation of amino-substituted dyes shifts the absorption and emission bands to the red spectral region by ca. 20 nm (see Table 1). .sup.bRetention (migration) time in the additional color channel where the dye has the largest emission, as measured in a gel at pH = 8. .sup.cConjugates of dye 8-H have a large cross-talk between 522 and 544 nm channels.
[0288] In fact, if one compares the emission maxima for the color channels in
[0289] For obtaining the color traces depicted in
[0290]
[0291]
[0292]
[0293] For dye 6-Me (and 6-H), the time difference between peaks is ca. 1.5 min, which corresponds to four negative charges on the dye residue. The right side of
LITERATURE
[0294] Feng H T, et al., Electrophoresis (2017) 38, 1788-1799. doi: 10.1002/elps.201600404. Epub 2017 May 11. [0295] Hennig R, et al., Biochimica et Biophysica Acta—General Subjects 2016, 1860, 1728-1738. [0296] Hennig R, et al., Methods Molecular Biology 2015, 1331, 123-143. [0297] Meininger M, et al., Journal of Chromatography B 2016, 1012, 193-203. [0298] Reusch D, rt al., MAbs. 2015, 7, 167-179. doi: 10.4161/19420862.2014.986000. [0299] Ruhaak L R, et al., Journal of Proteome Research 2010, 9, 6655-6664.