Quantitative determination method for UDP-HexNAc in biological sample

Abstract

The invention discloses a quantitative determination method for UDP-HexNAc in a biological sample, and belongs to the technical field of biological analysis. The method comprises the following steps: firstly, derivatizing a pretreated biological sample, carrying out separation and quantitative analysis on UDP-HexNAc in a biological sample to be detected through high performance liquid chromatography-tandem mass spectrometry, separating the derivatized sample through high performance liquid chromatography, entering tandem mass spectrometry, converting the sample into electric phase ions in an ESI ion source, and carrying out quantitative analysis on UDP-HexNAc in the biological sample to be detected through high performance liquid chromatography-tandem mass spectrometry. Performing analysis and detection through triple quadrupole-tandem mass spectrometry; the UDP-HexNAc comprises uridine diphosphate N-acetyl glucosamine (UDP-GlcNAc) and uridine diphosphate N-acetyl galactosamine (UDP-GalNAc), and the UDP-HexNAc comprises uridine diphosphate N-acetyl glucosamine (UDP-GlcNAc). The method disclosed by the invention is good in reproducibility and high in accuracy, is suitable for quantitative analysis and detection of UDP-GlcNAc and UDP-GalNAc in a complex biological sample, and has a relatively good application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of biological analysis, and particularly relates to a method for quantitatively determining UDP-HexNAc in a biological sample. Background Art

[0002] UDP-HexNAc is the end product of the hexose synthesis pathway HBP in mammalian cells, and its quantitative determination is an effective means to study HBP flux. UDP-HexNAc in cells has two chiral forms, one is UDP-GlcNAc, and the other is UDP-GalNAc, which can be converted into each other under the catalysis of allosteric enzymes. UDP-GlcNAc and UDP-GalNAc are glycosyl donors for glycosylation of various proteins, and participate in a variety of protein glycosylation modifications including N-glycan modification, mucin-type O-glycan modification and O-GlcNAc modification. In view of the important regulatory role of protein glycosylation modification in gene expression regulation, cell signal transduction, metabolism and other physiological and pathological processes, the quantitative analysis of UDP-HexNAc, an important sugar donor in cells, is of great significance in cell biology research.

[0003] In recent years, the rapid development of liquid chromatography-tandem mass spectrometry (LC-MS / MS) technology has provided a possible solution for the quantitative analysis of UDP-HexNAc. Compared with indirect enzymatic determination, liquid chromatography-tandem mass spectrometry shows great advantages in accuracy, precision, selectivity and sensitivity. However, there is no report on a liquid chromatography-tandem mass spectrometry (LC-MS / MS) method with high sensitivity and good selectivity for the determination of UDP-HexNAc in biological samples in existing studies. The reasons are as follows: first, UDP-HexNAc has strong polarity and is easy to ionize, so it is not well retained on most reversed-phase chromatographic columns. Secondly, ionized UDP-HexNAc can react with a variety of metal ions to form adducts, or exist in a multi-charged form, so it is difficult to use liquid chromatography-tandem mass spectrometry (LC-MS / MS) to stably quantify. Thirdly, since the two isomers of UDP-HexNAc, UDP-GlcNAc and UDP-GalNAc, serve as sugar donors for protein glycosylation modification, they are responsible for adding acetylglucosamine GlcNAc and acetylgalactosamine GalNAc to naked protein peptides or glycopeptides, respectively. The incorporation of different acetylated glycosamine residues determines the different types of glycosylation modification, which means different regulatory outcomes for the modified proteins. Therefore, the differentiation and quantification of isomers UDP-GlcNAc and UDP-GalNAc are of great significance for the study of protein glycosylation modification in biological samples. However, since UDP-GlcNAc and UDP-GalNAc only have stereoisomers in the hexosamine structure, it is difficult to distinguish them directly using chromatography. Finally, the high matrix effect interference of the biological sample itself also poses a considerable challenge to the mass spectrometry detection of UDP-HexNAc.

[0004] In summary, establishing an accurate and sensitive UDP-HexNAc typing and quantitative analysis method is an urgent problem to be solved in the field of cell metabolism research and protein glycosylation modification research. Summary of the invention

[0005] In order to solve the technical problems in the background technology, the purpose of the present invention is to provide a UDP-HexNAc typing and quantitative analysis method suitable for biological samples.

[0006] In order to achieve the above object, the present invention provides the following technical solutions:

[0007] The present invention provides a method for detecting UDP-HexNAc in a biological sample. The pretreated biological sample is derivatized, and UDP-HexNAc in the biological sample to be tested is separated and quantitatively analyzed by high performance liquid chromatography-tandem mass spectrometry. The derivatized sample is separated by high performance liquid chromatography and then enters the tandem mass spectrometry, where it is converted into charged phase ions in an ESI ion source, and analyzed and detected by a triple quadrupole-tandem mass spectrometry.

[0008] Based on the above technical solution, further, the UDP-HexNAc includes UDP-GlcNAc and UDP-GalNAc.

[0009] Based on the above technical scheme, further, the high performance liquid chromatography separation conditions are as follows: chromatographic column: Cosmosil5NH2-MS amino chromatographic column, 4.6mm×250mm ID, 5μm particle size; mobile phase: mobile phase A is an aqueous solution containing 1mM ammonium acetate, and mobile phase B is an acetonitrile solution containing 1mM ammonium acetate; forward chromatographic separation, 100% mobile phase A balance; 40% mobile phase A and 60% mobile phase B isocratic elution, flow rate 1mL / min; column temperature: 30°C.

[0010] Based on the above technical scheme, further, the conditions of tandem mass spectrometry are: Q-trap 6500 tandem mass spectrometer, equipped with ESI ionization source and Analyst data processing software; ion source: ESI ionization source; ion spray voltage 4500v, source temperature 500°C; negative ion detection mode; source gas 1: nitrogen pressure 40psi; source gas 2: nitrogen pressure 60psi; curtain gas: nitrogen pressure 35psi; multiple reaction monitoring (MRM) scanning mode is adopted.

[0011] Based on the above technical scheme, further, a stable high-response ion pair m / z 634.0→282.1 was selected for the quantitative determination of UDP-GlcNAc and UDP-GalNAc; detection conditions: parent ion m / z 634.0, daughter ion m / z 282.1; declustering voltage: 60 V; collision energy: 40 eV; detection conditions for the internal standard carbenicillin: parent ion m / z 283.9, daughter ion m / z 239.9; declustering voltage: 50 V; collision energy: 20 eV.

[0012] Based on the above technical solution, further, when the biological sample is a cell, the pretreatment process is: resuspend the cells in a methanol-water solution with a volume ratio of 10:1 to 15:1 to a concentration of 1 to 9×10 6 cells / mL, disrupt the cells with ultrasound on ice, centrifuge at 10000-15000 rpm for 2-10 min at 1-4°C, and collect the supernatant.

[0013] Based on the above technical scheme, the specific process of derivatization is as follows: 100 μL of pretreated biological sample, 10 μL of internal standard and 100 μL of trimethylsilyldiazomethane solution are derivatized at a temperature of 20 to 35°C in the dark for 20 to 60 minutes, dried under nitrogen, re-dissolved in 500 μL of acetonitrile, mixed, centrifuged at 10,000 to 15,000 rpm at 1 to 4°C for 2 to 10 minutes, and the supernatant is collected.

[0014] Based on the above technical scheme, further, the trimethylsilyldiazomethane solution is a 400-600 mM trimethylsilyldiazomethane solution prepared with a methanol-water solution in a volume ratio of 10:1 to 15:1, and the internal standard is a 50-150 ng / mL carbenicillin solution prepared with a methanol-water solution in a volume ratio of 10:1 to 15:1.

[0015] Based on the above technical solution, further, the quantitative determination of UDP-HexNAc is carried out by an external standard method.

[0016] Based on the above technical scheme, further, the external standard method includes the following steps: UDP-GlcNAc and UDP-GalNAc are respectively prepared into a series of UDP-GlcNAc solutions and UDP-GalNAc solutions with a volume ratio of 10:1 to 15:1, and the UDP-GlcNAc solution and UDP-GalNAc solution of the same concentration are mixed at a volume ratio of 1:1 to obtain a series of standard solutions of concentrations, and the obtained series of standard solutions of concentrations are respectively derivatized, and detected by the quantitative determination method, with the concentration as the abscissa and the ratio of the peak area to be measured to the peak area of ​​the internal standard as the ordinate, to prepare a standard curve, and the detection result of the sample solution to be measured is substituted into the standard curve to obtain the content of UDP-HexNAc in the sample solution to be measured.

[0017] Based on the above technical solution, further, the concentration range of UDP-HexNAc in the standard curve is 1 to 50 μM.

[0018] Compared with the prior art, the present invention has the following beneficial effects:

[0019] 1) The present invention proposes for the first time a UDP-HexNAc typing and quantitative detection based on a TMSCHN2 derivatization strategy, using a pre-column derivatization method to replace the active hydrogen of the hydroxyl group on the chiral structure of UDP-HexNAc with a larger methyl group, thereby stabilizing the compound and making the chromatographic separation of UDP-GlcNAc and UDP-GalNAc possible.

[0020] 2) The present invention utilizes the high resolution and high mass accuracy of liquid chromatography-tandem mass spectrometry (LC-MS / MS) to establish a multiple reaction monitoring (MRM) analysis method to achieve quantitative analysis of UDP-GlcNAc and UDP-GalNAc. This method has good reproducibility and high accuracy and is suitable for the quantitative analysis and detection of UDP-GlcNAc and UDP-GalNAc in complex biological samples. BRIEF DESCRIPTION OF THE DRAWINGS

[0021] Figure 1 Schematic diagram of the derivatization of the UDP-HexNAc standard obtained in Example 1 with TMSCHN2.

[0022] Figure 2 This is the scanning mass spectrum of the UDP-HexNAc standard obtained in Example 1 after derivatization with TMSCHN2.

[0023] Figure 3 The secondary mass spectrum of the UDP-HexNAc standard derivative fragment obtained in Example 1 and the chromatogram corresponding to the characteristic ion pair m / z 648.0→296.0.

[0024] Figure 4 The secondary mass spectrum of the UDP-HexNAc standard derivative fragment obtained in Example 1 and the chromatogram corresponding to the characteristic ion pair m / z 634.0→282.1.

[0025] Figure 5 This is the standard curve diagram after UDP-HexNAc derivatization obtained in Example 2.

[0026] Figure 6 This is the chromatogram corresponding to the characteristic ion pair m / z 634.0→282.1 after UDP-HexNAc derivatization in the biological sample obtained in Example 3. DETAILED DESCRIPTION

[0027] The present invention is further described below in conjunction with specific embodiments, but the present invention is not limited to the following specific embodiments.

[0028] Example 1: Scanning confirmation of characteristic ion pairs of UDP-HexNAc derivatives

[0029] Weigh 1 mg of UDP-GlcNAc disodium salt and UDP-GalNAc disodium salt respectively and dissolve them in 1.535 mL ddH2O to obtain 1 mM stock solution. Dilute the two stock solutions to 1 μM concentration with methanol-water (v / v=12 / 1) solution, and mix them in a volume ratio of 1:1 to obtain a mixed solution with a concentration of 1 μM, which is the UDP-HexNAc standard.

[0030] Pre-column derivatization reaction: UDP-HexNAc is derivatized with trimethylsilyldiazomethane TMSCHN2. The methyl group CH3 can replace the active hydrogen of the hydroxyl group OH on the chiral structure of UDP-GlcNAc and UDP-GalNAc, making the difference in the chiral structure of the two easy to be resolved by chromatography while increasing their stability. The derivatization reagent is a trimethylsilyldiazomethane solution (500 mM) prepared with a methanol-water (v / v=12 / 1) solution, and the internal standard is a probenecid solution (100 ng / mL) prepared with a methanol-water (v / v=12 / 1) solution.

[0031] Take 100 μL of UDP-HexNAc standard solution, 10 μL of internal standard solution and 100 μL of derivatization reagent and derivatize for 30 min at 25°C in the dark; dry under nitrogen. Then, re-dissolve in 500 μL of acetonitrile, mix by pipetting, centrifuge at 4°C and 12000 rpm for 5 min to remove insoluble matter and collect the supernatant.

[0032] Take 10 μL for LC-MS / MS analysis, and the chromatographic separation conditions are as follows: high performance liquid chromatography; chromatographic column: Cosmosil5NH2-MS amino chromatographic column, 4.6 mm×250 mm ID, 5 μm particle size; mobile phase: mobile phase A is an aqueous solution containing 1 mM ammonium acetate, and mobile phase B is an acetonitrile solution containing 1 mM ammonium acetate; forward chromatographic separation, 100% mobile phase A for balance; isocratic elution with 40% mobile phase A and 60% mobile phase B, flow rate 1 mL / min; column temperature: 30°C.

[0033] The mass spectrometry conditions were as follows: Q-trap 6500 tandem mass spectrometer, equipped with ESI ionization source and Analyst data processing software; ion source: ESI ionization source; ion spray voltage 4500v, source temperature 500℃; negative ion detection mode; source gas 1: nitrogen pressure 40psi; source gas 2: nitrogen pressure 60psi; curtain gas: nitrogen pressure 35psi; multiple reaction monitoring (MRM) scanning mode was used, and the characteristic ion pairs of UDP-HexNAc derivatives were found to be m / z 648.0→296.0 and m / z 634.0→282.1 ( Figure 2). Detection condition 1: parent ion m / z 648.0, daughter ion m / z 296.0; declustering voltage: 36V; collision energy: 39eV. Detection condition 2: parent ion m / z 634.0, daughter ion m / z 282.1; declustering voltage: 60V; collision energy: 40eV. Detection conditions for internal standard (carbosulfan): parent ion m / z 283.9, daughter ion m / z 239.9; declustering voltage: 50V; collision energy: 20eV, record chromatogram.

[0034] Through experiments and data processing, it was found that compared with the m / z 648.0→296.0 ion pair, m / z 634.0→282.1 can stably and effectively distinguish UDP-GlcNAc and UDP-GalNAc derivatives ( Figure 3 and Figure 4 ), and confirmed m / z 634.0→282.1 as the characteristic ion pair for typing and quantitative detection of UDP-HexNAc.

[0035] Example 2: Drawing of standard curve

[0036] Weigh 1 mg of UDP-GlcNAc disodium salt and UDP-GalNAc disodium salt, respectively, and dissolve them in 1.535 mL ddH2O to obtain 1 mM stock solution. Dilute the stock solutions of the two substances to 50 μM, 20 μM, 10 μM, 5 μM, 2 μM, and 1 μM concentrations, respectively, with methanol-water (v / v=12 / 1) solution, and mix them at a volume ratio of 1:1 to obtain a mixed solution, and obtain UDP-HexNAc solutions with concentrations of 50 μM, 20 μM, 10 μM, 5 μM, 2 μM, and 1 μM, respectively; prepare internal standard solution (carbenicillin, 100 ng / mL) and derivatization reagent (trimethylsilyldiazomethane, 500 mM) with methanol-water (v / v=12 / 1) solution.

[0037] Take 100 μL of UDP-HexNAc solution, 10 μL of internal standard solution and 100 μL of derivatization reagent and derivatize for 30 minutes at 25°C in the dark; dry under nitrogen. Then, re-dissolve in 500 μL of acetonitrile, mix by blowing with a pipette tip, centrifuge at 12000 rpm at 4°C for 5 minutes, remove insoluble substances, and collect the supernatant. Take 10 μL for LC-MS / MS analysis, and record the chromatogram corresponding to the characteristic ion pairs; use the solution concentration as the horizontal axis, the ratio of the peak area of ​​the analyte to the internal standard solution as the vertical axis, and use the weighting W=1 / x 2 The least squares method is used for regression calculation to obtain the linear regression equation, which is the standard curve ( Figure 5 ).

[0038] Example 3: UDP-HexNAc typing and quantitative detection of HepG2 cell samples

[0039] Human hepatocellular carcinoma cells Huh7 and HepG2 were cultured in 10 cm dishes. Culture conditions: MEM complete medium (containing 10% fetal bovine serum, 10% penicillin-streptomycin) at 37°C, 5% CO2. When the confluence reached about 90%, trypsin was used for digestion, and the cells were washed three times with pre-cooled PBS buffer. The cells were resuspended in PBS buffer and counted on a hemocytometer. 5×10 6 cells, centrifuge, and carefully aspirate all the liquid.

[0040] The protein precipitation method was used to remove some matrix interference in the biological samples. Methanol-water solution was selected as the precipitant. The cells were resuspended in 1000 μL methanol-water (v / v=12 / 1) solution, and then sonicated on ice (120 W), with 1 s impact and 2 s interval, for a total working time of 2 min (note that the centrifuge tube cap should be covered immediately after the end of sonication to avoid volume error caused by evaporation). The supernatant was collected after centrifugation at 4°C and 12000 rpm for 15 min.

[0041] Take 100 μL of the supernatant obtained in the above step, 10 μL of the internal standard solution (benzylbenzenesulfonamide, 100 ng / mL) and 100 μL of the derivatization reagent (trimethylsilyldiazomethane, 500 mM) and derivatize for 30 minutes at 25°C in the dark; dry under nitrogen. Then, re-dissolve in 500 μL of acetonitrile, mix by blowing with the pipette tip, centrifuge at 4°C and 12000 rpm for 5 minutes, remove insoluble matter, and collect the supernatant. Take 10 μL for LC-MS / MS analysis, and record the chromatogram corresponding to the characteristic ion pair m / z 634.0→282.1 ( Figure 6 ).

[0042] Through experiments and data processing, it was found that the concentration of UDP-HexNAc in Huh7 cells was 1.55×10 -6 nmol / cell, the concentration of UDP-HexNAc in HepG2 cells was 2.71×10 -5 The UDP-HexNAc in both cell samples was successfully typed, and the peak area ratio was UDP-GlcNAc:UDP-GalNAc=10:3.

[0043] The description presented in the above exemplary embodiments is only used to illustrate the technical solution of the present invention, and is not intended to be exhaustive, nor is it intended to limit the present invention to the precise form described. Obviously, it is possible for a person of ordinary skill in the art to make many changes and variations based on the above teachings. The exemplary embodiments are selected and described to explain the specific principles of the present invention and its practical application, so that other technicians in the field can easily understand, implement and use the various exemplary embodiments of the present invention and its various selected forms and modified forms. The scope of protection of the present invention is intended to be defined by the attached claims and their equivalent forms.

Claims

1. A method for detecting UDP-HexNAc in a biological sample, characterized in that: The pretreated biological sample is derivatized, and UDP-HexNAc in the biological sample to be tested is separated and quantitatively analyzed by high performance liquid chromatography-tandem mass spectrometry. The derivatized sample is separated by high performance liquid chromatography and then enters the tandem mass spectrometry, converted into charged phase ions in an ESI ion source, and analyzed and detected by triple quadrupole-tandem mass spectrometry; the UDP-HexNAc includes uridine diphosphate nitrogen acetyl glucosamine UDP-GlcNAc and uridine diphosphate nitrogen acetyl galactosamine UDP-GalNAc.

2. The detection method according to claim 1, characterized in that The HPLC separation conditions were as follows: chromatographic column: Cosmosil 5NH2-MS amino chromatographic column, 4.6 mm × 250 mm ID, 5 μm particle size; mobile phase: mobile phase A was an aqueous solution containing 1 mM ammonium acetate, and mobile phase B was an acetonitrile solution containing 1 mM ammonium acetate; normal chromatographic separation, 100% mobile phase A for balance; isocratic elution with 40% mobile phase A and 60% mobile phase B, flow rate 1 mL / min; column temperature: 30°C.

3. The detection method according to claim 1, characterized in that The conditions for tandem mass spectrometry were: Q-trap 6500 tandem mass spectrometer equipped with ESI ionization source and Analyst data processing software; Ion source: ESI ionization source; ion spray voltage 4500v, source temperature 500℃; negative ion detection mode; source gas 1: nitrogen pressure 40psi; source gas 2: nitrogen pressure 60psi; curtain gas: nitrogen pressure 35psi; multiple reaction monitoring (MRM) scanning mode was used.

4. The detection method according to claim 1, characterized in that A stable and highly responsive ion pair m / z 634.0→282.1 was selected for the quantitative determination of UDP-GlcNAc and UDP-GalNAc; detection conditions: parent ion m / z 634.0, daughter ion m / z 282.1; declustering voltage: 60 V; collision energy: 40 eV; detection conditions for the internal standard carbenesulfonamide: parent ion m / z 283.9, daughter ion m / z 239.9; declustering voltage: 50 V; collision energy: 20 eV.

5. The detection method according to claim 1, characterized in that When the biological sample is a cell, the pretreatment process is as follows: resuspend the cells in a methanol-water solution with a volume ratio of 10:1 to 15:1 to a concentration of 1 to 9 × 10 6 cells / mL, disrupt the cells with ultrasound on ice, centrifuge at 10000-15000 rpm for 2-10 min at 1-4°C, and collect the supernatant.

6. The detection method according to claim 1, characterized in that The specific process of derivatization is as follows: 100 μL of pretreated biological sample, 10 μL of internal standard and 100 μL of trimethylsilyldiazomethane solution are derivatized at 20 to 35°C in the dark for 20 to 60 minutes, dried under nitrogen, re-dissolved in 500 μL of acetonitrile, mixed, centrifuged at 10,000 to 15,000 rpm at 1 to 4°C for 2 to 10 minutes, and the supernatant is collected.

7. The detection method according to claim 6, characterized in that The trimethylsilyldiazomethane solution is a 400-600 mM trimethylsilyldiazomethane solution prepared with a methanol-water solution in a volume ratio of 10:1-15:1, and the internal standard is a 50-150 ng / mL carbenicillin solution prepared with a methanol-water solution in a volume ratio of 10:1-15:

1.

8. The detection method according to claim 1, characterized in that The quantitative determination of UDP-HexNAc is performed by the external standard method.

9. The detection method according to claim 8, characterized in that The external standard method comprises the following steps: using a methanol-water solution with a volume ratio of 10:1 to 15:1 to prepare UDP-GlcNAc and UDP-GalNAc into a series of UDP-GlcNAc solutions and UDP-GalNAc solutions respectively, mixing the UDP-GlcNAc solution and UDP-GalNAc solution with the same concentration at a volume ratio of 1:1 to obtain a series of standard solutions, respectively derivatizing the obtained series of standard solutions, detecting by the quantitative determination method, taking the concentration as the abscissa and the ratio of the peak area to be measured to the peak area of ​​the internal standard as the ordinate, preparing a standard curve, substituting the detection result of the sample solution to be measured into the standard curve to obtain the content of UDP-HexNAc in the sample solution to be measured.

10. The detection method according to claim 9, characterized in that The concentration range of UDP-HexNAc in the standard curve is 1 to 50 μM.

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