CN106554305B - Marker, preparation method thereof, marking method and application - Google Patents
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- CN106554305B CN106554305B CN201510639645.4A CN201510639645A CN106554305B CN 106554305 B CN106554305 B CN 106554305B CN 201510639645 A CN201510639645 A CN 201510639645A CN 106554305 B CN106554305 B CN 106554305B
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07D—HETEROCYCLIC COMPOUNDS
- C07D213/00—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members
- C07D213/02—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members
- C07D213/04—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom
- C07D213/24—Heterocyclic compounds containing six-membered rings, not condensed with other rings, with one nitrogen atom as the only ring hetero atom and three or more double bonds between ring members or between ring members and non-ring members having three double bonds between ring members or between ring members and non-ring members having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen or carbon atoms directly attached to the ring nitrogen atom with substituted hydrocarbon radicals attached to ring carbon atoms
- C07D213/36—Radicals substituted by singly-bound nitrogen atoms
- C07D213/38—Radicals substituted by singly-bound nitrogen atoms having only hydrogen or hydrocarbon radicals attached to the substituent nitrogen atom
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H1/00—Processes for the preparation of sugar derivatives
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- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H5/00—Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium
- C07H5/04—Compounds containing saccharide radicals in which the hetero bonds to oxygen have been replaced by the same number of hetero bonds to halogen, nitrogen, sulfur, selenium, or tellurium to nitrogen
- C07H5/06—Aminosugars
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- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N30/00—Investigating or analysing materials by separation into components using adsorption, absorption or similar phenomena or using ion-exchange, e.g. chromatography or field flow fractionation
- G01N30/02—Column chromatography
- G01N30/62—Detectors specially adapted therefor
- G01N30/72—Mass spectrometers
- G01N30/7233—Mass spectrometers interfaced to liquid or supercritical fluid chromatograph
Abstract
The invention provides a marker, a preparation method, a marking method and application thereof, wherein the marker is 1-alkyl-4 aminomethyl pyridine. The invention also provides a preparation method and application of the marker. The marker is particularly suitable for marking saccharides, and is favorable for enhancing mass spectrum signals and improving the sensitivity of mass spectrum detection.
Description
Technical Field
The invention relates to a marker, in particular to a saccharide marker, a preparation method and application thereof.
Background
It is well known that monosaccharides and polysaccharides are the most basic and important molecules that make up a living body. In the field of life sciences such as energy metabolism, nutrition, glycobiology, biopharmaceuticals, and emerging glycomics, with the progress of experiments, the need for sugar discovery and structural characterization is becoming more and more apparent. However, since the complexity of the characteristics and structure of sugar molecules far exceeds that of proteins and amino acids, even today the analytical techniques are so advanced, the analysis of sugar molecules is not a simple process.
Generally, most current analytical procedures rely on chemical labeling. The following objectives can be achieved using chemical labeling: most sugars are hydrophilic neutral molecules and the use of suitable labels can help to distinguish between different sugar molecules by changing their properties, e.g. the addition of a charge to a sugar molecule can help to carry out electrophoretic separation; imparting hydrophobicity to them may improve their resolution in reverse phase HPLC; addition of a chiral label allows discrimination of diastereomers.
Since most sugar molecules have neither chromophores nor fluorophores, intact sugars are typically detected by Ultraviolet (UV) at 195nm, or by amperometric or refractive detection. Unfortunately, the detection sensitivity of these techniques is low. Generally, saccharides are labeled by adding a luminescent group or fluorophore to the molecule to increase their sensitivity for ultraviolet or fluorescence detection. A number of reagents have been found to achieve this labelling, as described by Karamanosost, the glycosaminating [ D.T.Li, J.F.Screen, and G.R.Her.J.Am Soc Mass Spectrum 2000, 11, 292-.
Although UV and fluorescence detection are simple and feasible, both methods show problems of lack of selectivity and specificity when the sample is very complex. Nowadays, the latest sophisticated technology Mass Spectrometry (MS) is the most efficient and commonly used analytical tool. However, intact carbohydrates are not suitable for direct MS detection. Due to their weak polarity, thermal instability, relatively low volatility, they cannot be efficiently ionized in soft ionization or Fast Atom Bombardment (FAB) or electrospray ionization (ESI) or Matrix Assisted Laser Desorption Ionization (MALDI) plasma sources. It is expected that chemical labeling can help obtain simpler spectra and can improve the responsivity of mass spectrometric detection.
Among all labeled methods, the complete methylation method has been used for several years of experience [ Pilsoo Kang, Yehia Mechref, Iveta Klouckova, et al. rapid Commun Mass spectra.2005; 19, 3421-3428] [ Hakomori SI. J biochem (Tokyo) 1964; 55: 205] [ Ciucanu I, Kerek F. Carbohydrares 1984; 131: 209], the greatest benefit of this process is the greatly increased efficiency of ionization of the methylated saccharide. Under the combined action of electrospray ion source (ESI) and Collision Induced Dissociation (CID), a very detailed structural information of the fully methylated sugar can be obtained. Recently, Wottish introduced a set of N-glycan mass spectrometry detection kits. This kit can accelerate deglycosylation, simplify labeling procedures, and highlight the ionization capacity to maximize the amount of mass spectrometric information [ Matthewa. Lauber, Ying-Qing Yu, Darryl W, et al. anal. chem.2015, 87, 5401-. The disadvantage of this method is that the labeling can only be applied to polysaccharides from glycoproteins, which still carry amino groups, rather than the usual free sugars.
In 2006, professor Yang and his group discovered a preassigned charge-labeling technique that could increase the MS minimum detection limit for polar molecules [ Wen-Chu Yang, Hamid Mirzaei, Xiuping Liu, Fred E.Regnier, anal.chem.2006, 78, 4702-. The marking method skillfully introduces a quaternary amine with a hydrophobic carbon chain into a polar molecule to improve the ESI ionization efficiency, and meanwhile, the method can also be used for obtaining a corresponding stable isotope marker. This in turn provides a way to achieve relative and absolute quantitation. This technique has significantly improved the sensitivity of MS detection of amino acids [ Wen-Chu Yang, Hamid Mirzaei, Xiuping Liu, Fred E.Regnier, anal. chem.2006, 78, 4702-.
Therefore, there is a need in the art to develop new labels that are suitable for this labeling method and that improve the sensitivity of mass spectrometry and facilitate the enhancement of mass spectrometry signals.
Disclosure of Invention
The invention aims to provide a marker which is particularly suitable for marking saccharides, is beneficial to enhancing mass spectrum signals and improves the sensitivity of mass spectrum detection.
The invention also aims to provide a preparation method and application of the marker.
In a first aspect of the invention, there is provided a marker which is a 1-alkyl-4 aminomethyl pyridine of the formula:
wherein R is CnH2n +1 or CnD2n +1, wherein n is an integer of 1-3.
The above n is 1.
The marker is 1-methyl-4-aminomethyl pyridine or 1-trideuteromethyl-4-aminomethyl pyridine.
The above n is 2.
The marker is 1-ethyl-4-aminomethyl pyridine or 1-pentadeutero-ethyl-4-aminomethyl pyridine.
The above n is 3.
The marker is 1-propyl-4-aminomethyl pyridine or 1-heptadeuteropropyl-4-aminomethyl pyridine.
In a second aspect of the invention, there is provided the use of the above-mentioned marker for the marking of carbohydrates.
In a third aspect of the invention, there is provided the use of a label as described above in mass spectrometric detection.
In a fourth aspect of the present invention, there is provided a method for producing the above-mentioned marker, comprising the steps of:
(1) heating 4-aminomethyl pyridine and phthalic anhydride for reaction, and recrystallizing with methanol to obtain compound A, wherein the molecular formula of the compound A is as follows:
(2) dissolving the compound A in a mixed solution of alkyl iodide and methanol, and crystallizing to prepare a compound B;
(3) and reacting the compound B with HBr, and extracting to obtain the 1-alkyl-4-aminomethyl pyridine.
In a fifth aspect of the present invention, there is provided the labeling method for a labeling substance as described above, wherein the chemical reaction formula of the labeling method is:
in a sixth aspect of the present invention, there is provided the labeling method for a labeling substance, wherein the chemical reaction formula of the labeling method is:
the marker of the invention has the following advantages:
the marking is completed by the reaction of amino and aldehyde in reducing sugar, and the reaction condition is relatively simple;
its permanent positive charge promotes gas phase ionization and enhances mass spectral signal;
alkyl chain (C) with adjustable lengthnH2n+1) Enables us to vary the retention time of the reactants on the liquid chromatography column and the sensitivity of the mass spectrometric detection;
the stable isotope label makes it possible to use it as an Internal Standard (IS) for MS quantification.
There will be sufficient experimental data and experimental procedures to support the above conclusions.
Drawings
FIG. 1 shows the general formula of the marker NA-AMP of the present invention (1-alkyl-4-aminomethyl pyridine).
FIG. 2 shows a molecular formula of a specific compound of NA-AMP, which is a marker of the present invention.
FIG. 3 shows the chemical reaction formulae (method a, method b) of two different methods for labeling saccharides with the labeling substance of the present invention.
FIG. 4 shows secondary mass spectra of DP5 labeled by method a with (A) C1-AMP or (B) CD3-AMP, respectively. The mass spectrum has a series of major fragment ions, and the specific structural information of the major fragment ions corresponds to the fragments in the inset.
FIG. 5 shows secondary mass spectra of DP5 labeled by method B with (A) C1-AMP or (B) CD3-AMP, respectively. The mass spectrum has a series of major fragment ions, and the specific structural information of the major fragment ions corresponds to the fragments in the inset.
FIG. 6 is a comparison of the spectra labeled by method a (Panel A) and method B (Panel B) for DP3 with AMP, C1-AMP, C2-AMP and C3-AMP, respectively.
The above spectra show that the spectrum signal in the MRM acquisition mode is gradually enhanced with the increase of alkyl chain in the label.
FIG. 7. secondary mass spectrum of DP 5. There are 3 major fragments in the figure: a, B, C, their structure details see the figure above the chemical formula insert.
FIG. 8 the signal intensity in MRM was increased 20-150 fold for oligosaccharide (B) after labeling with C3-AMP relative to unlabeled primary oligosaccharide (A).
8A: primary oligosaccharides;
8B: oligosaccharide after labeling of C3-AMP by method b, peak 1: DP 3; peak 2: DP 4; peak 3: DP 5.
FIG. 9: by the method a, the separation effect graph after marking oligosaccharides DP3, DP4 and DP5 by C3-AMP respectively;
FIG. 10: and b, a separation effect graph after marking the oligosaccharides DP3, DP4 and DP5 by using C3-AMP respectively.
FIG. 11 is a map of an equivalent amount of DP4 labeled with C1-AMP and CD3-AMP, respectively, by method a.
FIG. 12 is a map of an equivalent DP 5-labeled antibody using C1-AMP and CD3-AMP, respectively, by method b.
Detailed Description
The following description of the embodiments of the present invention will be made with reference to the accompanying drawings.
Under the existing innovative technology, a novel marker for detecting saccharides by mass spectrometry, namely 1-alkyl-4 aminomethyl pyridine (NA-AMP), is designed, and the molecular structure of the marker is shown in figure 1.
Wherein R ═ CnH2n+1When n is 1-3, it is a common marker, R is CnD2n+1When n is 1-3, the compound is an isotope label and can be used as an isotope internal standard in the quantification; in the figure, the 1-position is a substitution position of an alkyl group, and the 4-position is a substitution position of an aminomethyl group.
Chemical reagent
Reliable oligosaccharide standard: isomaltotriose (DP3), maltotetraose (DP4), maltopentaose (DP5) and chemicals used for synthesis were all from Sigma-Aldrich, the remaining organic reagents: acetonitrile (ACN), methanol, dimethyl sulfoxide (DMSO) and formic acid were purchased from Fisher Scientific.
Technical solution
EXAMPLE 1 Synthesis of the markers of the invention
The marker of the invention is synthesized by the following method:
equimolar amounts of free 4-aminomethylpyridine (4-aminomethylpyridine, abbreviated to AMP) and phthalic anhydride were mixed, then heated to 140 ℃ and 160 ℃ until no water was produced (about 30 minutes), the residue was cooled, and recrystallized from methanol to obtain Compound A.
0.01mol of Compound A was dissolved in a mixture of 20mL of methanol and 5mL of alkyl iodide, and the mixture was placed in a reflux condenser to continue the reaction until completion (4 to 72 hours). The solution was cooled, the solid was filtered off and recrystallized from methanol to yield compound B.
Compound B was placed in 20mL of 48% HBr and the mixture was placed in a reflux condenser to react until substantial phthalic acid precipitation occurred. The solution was cooled to room temperature, 20mL of water was added, and phthalic acid was filtered off. The filtrate was extracted with 10mL of diethyl ether, and the extraction was repeated once, evaporated to dryness with suction filtration, and recrystallized from aqueous ethanol to obtain the product N-alkyl-4-aminomethylpyridine (NA-AMP) (1-alkyl-4-aminomethylpyridine).
FIG. 2 shows some different NA-AMPs, followed from left to right by N-methyl-4-aminomethyl-pyridine (abbreviated as C1-AMP, Chinese name: 1-methyl-4-aminomethylpyridine), N-methyl-d 3-4-aminomethylpyridine (abbreviated as CD3-AMP, Chinese name: 1-trideuteromethyl-4-aminomethylpyridine), N-ethyl-4-aminomethylpyridine (abbreviated as C2-AMP, Chinese name: 1-ethyl-4-aminomethylpyridine), and N-propyl-4-aminomethylpyridine (abbreviated as C3-AMP, Chinese name: 1-propyl-4-aminomethylpyridine). See in particular the following formulae:
EXAMPLE 2 labeling procedure
There are two different labelling methods for saccharides, as shown in the following reaction scheme.
The method a comprises the following steps: the working solution is a mixed standard solution of 1 mu g/mL DP3, DP4 and DP5 in DMSO as a solvent. mu.L of the working solution was taken and 100. mu.L of 10mg/mL of the labeled reactant in water was added. mu.L triethanolamine was added to the mixture to bring the pH of the solution to 7.5. The mixture was shaken at room temperature for 2 hours. From this 50. mu.L of the mixture was taken, 150. mu.L of Acetonitrile (ACN) was added, and then the mixture was subjected to detection by machine LC-MS.
The method b: the working solution is a mixed standard solution of 1 mu g/mL DP3, DP4 and DP5 in DMSO as a solvent. Adding 50 μ L of DMSO as solvent into 100 μ L of working solution containing 1M sodium borocyanide (NaCNBH)3) To the reaction mixture, 50. mu.L of glacial acetic acid was added, and the reaction mixture was labeled at 10 mg/mL. The mixture was shaken for two hours at 50 ℃. 50 μ L of the mixture was removed, 150 μ L of ACN was added, and the mixture was then placed in an LC-MS apparatus for detection.
Conditions of the apparatus and method
Liquid phase system and liquid phase conditions
The liquid phase system of Shimadzu, Japan, included an LC20AD HPLC pump and an SIL-20AC XR autosampler. Liquid chromatography column: waters ACQUTY UPLC BEH Amide 1.7um, 2.1X100mm (part # 186004801). Elution method of labeled oligosaccharides: flow rate: 0.4 ml/min; mobile phase: (A) 0.05% formic acid acetonitrile solution/(B) 0.05% formic acid aqueous solution; gradient: 70% mobile phase (A), 5min down to 0%. Elution of unlabeled oligosaccharide, constant flow rate, mobile phase: (A) 0.1% ammonium hydroxide acetonitrile solution/(B) 0.1% ammonium hydroxide aqueous solution; gradient: 70% mobile phase (A), 5min down to 0%.
Mass Spectrometry System and Mass Spectrometry parameters
The mass spectrometry system is 5500Qtrap from AB Sciex, USA, and is used with liquid phase system from Shimadzu, Japan. Both the labelled and unlabelled oligosaccharides were used in mass spectrometry using electrospray ionisation (ESI) mode, which differs: the former uses positive ion mode (ESI +), the latter uses negative ion mode (ESI-). The mass spectrum related parameters are shown in table 1.
TABLE 1 comparison of the relevant Mass Spectrometry parameters for labeled and unlabeled oligosaccharides
Quantification was obtained by multiple reaction monitoring mode (MRM) scanning. The MRM parameters after labeling oligosaccharides with method a are shown in Table 2, the MRM parameters after labeling oligosaccharides with method b are shown in Table 3, and the MRM parameters without labeling oligosaccharides are shown in Table 4.
TABLE 2 Mass Spectrometry parameter List after labeling oligosaccharides Using method a
TABLE 3 Mass Spectrometry parameter List after labeling oligosaccharides Using method b
TABLE 4 Mass Spectrometry parameter List for unlabeled oligosaccharides
Data support for the performance of the designed reactants:
as mentioned above, the reactants have the following advantages: 1) reacting with reducing saccharides; 2) enhancing MS signal by the charge of quaternary amines; 3) the retention time is adjusted by adjusting the length of the alkyl chain; 4) quantification was by isotopic labeling.
More options for labelling methods
According to the literature, the aldehyde groups in the reducing sugars can be labeled with primary amines by methods a and b (see fig. 3). All of the data in this report can prove that both methods are fully feasible when using the designed reagents of the present invention. Comparing the results obtained using these two methods separately, we can conclude that: 1) the oligosaccharides labeled using method a differ by 2 mass units compared to method b (see fig. 4 and 5); 2) the retention time of the labeled oligosaccharides on the amide column using method a was short (see fig. 6); 3) the MS sensitivity of the oligosaccharide labeled using method a was 50 times lower than that of the oligosaccharide labeled using method b (see fig. 6); 4) the process of labeling using method a is relatively simple and mild. It can be done in aqueous phase at neutral pH, room temperature. This method is therefore simpler and particularly suitable for readily labelled saccharides such as: sialic acid-containing oligosaccharides, and the like. In summary, we can choose a more suitable method according to practical situations under different application conditions.
Spectrogram interpretation and structural description
As shown in FIGS. 4 and 5, the labeled oligosaccharide C3-AMP-DP5 only formed a series of fragments of poor mass 162 when linked to the hemiacetal. In fig. 7 it can be seen that the unlabeled DP5 forms three series of fragments. The contrast between the two results shows that the complexity of the mass spectrum of the marked reactant is greatly reduced, which is more beneficial to the reading of the spectrogram and the explanation of the structure.
Lifting of mass spectrum signal
The sensitivity of MS detection can be improved by adding a charge to the analyte through the alkalization reaction, and in addition, as shown in fig. 6, increasing the length of the alkyl chain of the label can further improve the efficiency of MS detection (see the summary of table 5). FIG. 6 is a chromatogram of DP3 labeled with AMP, C1-AMP, C2-AMP and C3-AMP by method a and method b. Figure 6 clearly shows that the mass spectral signal intensity increases significantly as the alkyl chain grows from 0 to 3. Table 5 summarizes the details. Using C3-AMP-DP3 as an example, a 4-fold and 176-fold increase in sensitivity was obtained using method a and method b, respectively, compared to unlabeled DP 3.
Table 5: quantitative characterization alignment after labeling with unlabeled saccharide.
Calculation based on fig. 6 and 8A
Calculation based on fig. 8A and 9
This phenomenon was first discovered by Baker and his groups in the amino acid labeling [ Wen-Chu Yang, Hamid Mirzaei, Xiuping Liu, Fred E.Regnier, anal.chem.2006, 78, 4702-: during ionization in electrospray ionization mass spectrometry (ESI-MS), substances that are both hydrophobic and cationic migrate to the surface of the droplets, and when the concentration is high enough, they push the relatively hydrophilic molecules of weak cations off the surface of the droplets, even inhibiting ionization of those molecules. In ESI-MS, ionization occurs at the surface of the droplet, and the above process can be considered as an ion suppression effect. Based on the ESI-MS model, increasing the alkyl chain length of the labeled saccharide is likely to give the analyte surfactant-like properties, which in turn increases the concentration of the analyte at the droplet surface under ESI-MS, thereby increasing ionization efficiency. In addition, increasing the alkyl chain length also increases the hydrophobicity of the labeled product, thereby allowing the labeled analyte to elute more rapidly in normal phase liquid chromatography (amide column) under relatively high proportion of organic phase conditions. This in turn can further improve ESI ionization efficiency and test sensitivity.
This labeling method also improves the thermal stability of the analyte, which explains the high mass spectral response of the labeled assay product, while the low mass spectral response of the unlabeled oligosaccharide. In fig. 8A, the unlabeled DP3 and DP4 chromatographic peaks are not unique, and the peak-off time of their second peak is the same as the retention time of DP4 and DP5, respectively. These interference peaks are actually endogenous ion fragments of DP4 and DP5, which decompose into DP3 and DP4 when DP4 and DP5 enter the ion source, respectively, due to their thermal instability. These endogenous fragments of ions interfere with the concentration of the corresponding analyte, thereby reducing its own mass spectral intensity. Conversely, by labeling, the stability of the analyte can be improved without interference from fragmentation peaks, and the sensitivity of the assay can be increased. As shown in fig. 8B, after labeling, there were no unwanted interference peaks in the spectra.
Flexibility and adjustability of liquid phase separation
In reality, since the saccharide samples come from various substances and contain various impurities, the saccharide samples from different sources need corresponding liquid phase separation conditions, and the designed marker can meet different liquid phase separation requirements. The pyridine ring on the label can be connected with alkyl chains with different lengths so as to provide different degrees of hydrophobicity, so that the whole separation process is adjustable, and the corresponding separation condition can be selected from a series of labeled reactants to be most suitable for meeting.
FIGS. 9 and 10 are examples of separation on an amide column of DP3, DP4 and DP5 labeled with C3-AMP by method a and method b, respectively. The peak in FIG. 9 is essentially the baseline separation peak, while the peak in FIG. 10 at DP3/DP4 is not the baseline separation peak (Rs < 1.5), but because there are two different mass channels in the mass spectrometric detection, it is not disturbed thereby.
The research shows that: the NA-AMP labeled saccharide can be well separated in common reversed phase chromatography, which cannot be achieved by the non-labeling method. Because the carbohydrate has strong polarity and can only be retained in normal phase chromatography, the labeling method can ensure that the labeled product has stronger hydrophobicity and can be retained in reverse phase chromatography. This provides more flexibility in the choice of the mode of separation of the saccharides.
Convenience of isotopic labels in quantitation
Quantitative methods in mass spectrometric detection typically require internal standards to track and compensate for partial deviations during detection, pre-processing. The best internal standard is an isotopic compound corresponding to the analyte, since both have very similar physical and chemical properties. Unfortunately, optimal internal standards in quantitation are not always readily available or are very expensive. The experimental design allows the laboratory to obtain internal standards as needed.
Isotopic labeling is typically achieved by substituting a deuterium atom for a hydrogen atom on an alkyl chain. The reaction of the deuterated marker with the saccharide molecule herein will result in the corresponding deuterated assay product. As shown in fig. 4 (fig. 4) and 5 (fig. 5), all ions of the CD3-AMP labeled DP5 product were 3 mass numbers more than the C1-AMP labeled DP5 product ion, which also effectively demonstrated the success of the preparation of the deuterated internal standard.
In fig. 11, 12, this isotopic label meets its criteria as an internal standard. Firstly, the isotope labeled analyte and the non-isotope labeled analyte have similar properties and can be extracted together; next, by two methods (FIG. 11: method a and FIG. 12: method b), the peak intensities of the analyzed product ions were similar when equal amounts of DP4 or DP5 were labeled with C1-AMP and CD3-AMP, respectively. In practice, a set of analytes may be labeled with isotopically labeled NA-AMP. The purification allows to obtain isotopically labelled analytical products from NA-AMP (non-isotopically labelled) products, which can be used exactly as internal standard for quantitative analytical procedures.
The present invention has been described in terms of embodiments thereof, which are illustrative and not restrictive, and it is to be understood that variations and/or modifications may be effected by those skilled in the art without departing from the scope of the invention which is defined by the appended claims.
Claims (8)
1. Use of a marker for marking a carbohydrate, wherein the marker has a cationic formula of:
wherein R is CnH2n+1Or CnD2n+1Wherein n is an integer of 1 to 3.
2. The use of a label according to claim 1 for labeling a carbohydrate, wherein the label cation is a 1-methyl-4-aminomethylpyridine cation or a 1-trideuteromethyl-4-aminomethylpyridine cation.
3. Use of a label according to claim 1 for the labeling of carbohydrates wherein n is 2.
4. Use of the label of claim 3 for labeling a saccharide, wherein the label cation is 1-ethyl-4-aminomethylpyridine cation or 1-pentadeuteroylethyl-4-aminomethylpyridine cation.
5. Use of a label according to claim 1 for the labeling of carbohydrates wherein n is 3.
6. Use of the label of claim 5 for labeling a saccharide, wherein the label cation is 1-propyl-4-aminomethylpyridine cation or 1-heptadeuteropropyl-4-aminomethylpyridine cation.
7. Use of a label according to claims 1-6 for the labeling of carbohydrates according to the chemical reaction formula:
8. use of a label according to claims 1-6 for the labeling of carbohydrates according to the chemical reaction formula:
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| Miao Wang et al.,.Fatty Acidomics: Global Analysis of Lipid Species Containing a Carboxyl Group with a Charge-Remote Fragmentation-Assisted Approach.《Analytical Chemistry》.2013,第85卷(第19期),第9312-9320页. * |
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