CN107449650B - Rapid quantitative analysis method and application of N-glycans based on MALDI-MS and stable isotope labeling - Google Patents

Abstract

The invention discloses a rapid quantitative analysis method and application of N-glycan based on MA L DI-MS and stable isotope labeling, belonging to the field of glycoproteomics analysis, wherein the method comprises the steps of (1) PNGase F releases N-glycan in the form of glycosaminoglycan under the assistance of microwave, (2) d0/d 5-benzoyl chloride respectively performs nucleophilic reaction with glycosaminoglycan, and then is purified, (3) d 0-benzoyl chloride and glycan derived from d 5-benzoyl chloride are mixed according to the molar ratio of 1:1, (4) N-glycan derived from d0/d 5-benzoyl chloride after mixing is performed with a methylamine reaction, (5) MA L DI-MS detection, and (6) data analysis.

Description

A rapid quantitative analysis method based on MALDI-MS and stable isotope-labeled N-glycans and application

technical field

The invention belongs to the field of glycoproteomics analysis, and in particular relates to a rapid quantitative analysis method of N-glycans based on MALDI-MS and stable isotope labeling.

Background technique

Matrix-assisted laser desorption ionization mass spectrometry (MALDI-MS) technology was born in the late 1980s. It is a "soft ion" mass spectrometry technology. It has the characteristics of difficult sample cracking, strong molecular ion peaks and high sensitivity. Thermally unstable and non-volatile biological samples provide a new approach and gradually become the preferred method for the analysis of protein samples, peptides, and nucleic acids.

Stable isotope labeling technology is mainly developed by using the characteristics of stable isotopes and their compounds. In nature, stable isotopes and their compounds have the same chemical and biological properties as the corresponding common elements and compounds, but have different Therefore, stable isotopes can be used as tracer atoms to make labeled compounds (such as labeled inorganic salts, labeled amino acids, labeled drugs, labeled proteins, etc.) instead of corresponding non-labeled compounds.

Glycosylation is an important post-translational modification of proteins, and abnormal glycosylation of proteins is often associated with various diseases. Therefore, specific detection methods that can analyze the trace changes of oligosaccharides are urgently needed. Today, many analytical detection techniques are used to monitor changes in glycans, such as capillary electrophoresis, liquid chromatography, and mass spectrometry. However, these techniques have more or less deficiencies, such as sample-to-sample variability caused by different ionization efficiencies, instrumental Differences between response values and lack of internal or external standards, etc. In addition, although some isotopic quantification techniques have also been applied to the quantitative analysis of glycans, there are still many disadvantages, such as the lengthy reaction time (10h-18h), the destruction of acidic groups caused by extreme reaction conditions (acidity, high temperature), Post-source and in-source resolution of acid sugars in mass spectrometry detection severely limit the quantitative analysis of glycans.

SUMMARY OF THE INVENTION

In view of the deficiencies in the existing technology, we have established a fast and effective method for quantitative glycan based on stable isotope. Simultaneous quantitative analysis of sexual sugars and acidic sugars, reducing experimental costs, improving experimental efficiency, and shortening reaction time, have medical development and application value.

In order to realize the purpose of the present invention, the inventor finally obtained the following technical solutions through a large number of experimental studies and unremitting exploration:

A method for rapid quantitative analysis of N-glycans based on MALDI-MS and stable isotope labeling, the method comprises the following steps:

(1) Microwave-assisted rapid digestion: standard glycoprotein (10ug) or serum (5ul) was dissolved in 20ul reaction solution (containing 10mM sodium phosphate, 0.13% sodium dodecyl sulfate, 10mM dithiothreitol), Then boil in a 100°C water bath for 10min, add 2.4ul of 10% NP-40, add 0.5ul of PNGase F after mixing, and digest the enzyme for 20min under microwave-assisted conditions (the maximum output power of the microwave oven used in the experiment is 700W. lowest gear), which releases N-glycans in the form of glycosamines.

(2) Rapid derivatization reaction between sugar amine obtained in step (1) and d0/d5-benzoyl chloride (d0 represents non-isotopic labeling; d5 represents isotopic labeling), reaction conditions: prepare benzoyl chloride solution (because benzoyl chloride is volatile And it is prone to hydrolysis, so this reagent should be used and prepared immediately), add benzoyl chloride to the enzyme cleavage reaction solution in step (1), mix quickly, and leave at room temperature for 30min.

(3) SPE purification: Purify the reaction solution in step (2), the steps are: ① 3ml deionized water to wash the MCC purification column; ② 3ml balance solution (n-butanol:ethanol:water=4:1:1) to The MCC is balanced; 3. 800ul of the equilibrated solution is added to the glycan reaction system of step (2), fully mixed, and sampled; 4. 3 ml of the equilibrated solution (n-butanol:ethanol:water=4:1:1) is applied to the MCC Rinse to remove all impurities; ⑤1ml eluent (ethanol:water=1:1) to elute the sample, evaporate to dryness for later use.

(4) methylamination reaction: before the methylamination treatment, d0-benzoyl chloride and d5-benzoyl chloride-derived N-glycans were mixed at a 1:1 molar ratio, and then 25ul of methylamine hydrochloride solution (with 100ul DMSO to dissolve 7mg methylamine hydrochloride, then add 5.5ul methylmarine solution) and 25ul PyAOP solution (dissolve 2.6mg PyAOP in 100ul DMSO) and add to the dry mixed sample. It was left standing for 30 min at room temperature and in the dark, and purified by SPE, and the steps were the same as above. (Note: As shown in Figure 3, to verify the stability of the quantitative capability of this method, d0-benzoyl chloride and d5-benzoyl chloride-derived standard glycoproteoglycans were mixed in a molar ratio of 1:1, followed by methylamination treatment; as shown in Figure 4, when verifying the application of this method in the quantitative linear range, the above-mentioned d0-benzoyl chloride and d5-benzoyl chloride-derived N-glycans were mixed in 7 molar ratios, followed by methylamination treatment ; it can be seen from Figure 5 that the method has a 10-fold linear quantitative range).

(5) Detection of glycan samples by MALDI-MS: Dissolve the double-derivatized N-glycans in step (4) in 10 μL of solution (acetonitrile: water = 1:1), take 1.0 μL of the sample solution and 1.0 μL of the solution. μL of DHB solution was mixed and spotted onto a 384-well plate, with about 1.0 μL of liquid per spot. The plate was left to stand horizontally for 15 min to evaporate the solvent, and then N-glycan detection was performed by MALDI-MS 5800 (SCIEX).

The relevant parameters of the matrix-assisted laser desorption ionization mass spectrometer are set as follows: detection mode, ReflectorPositive; laser intensity (laser intensity), 5200; voltage multiplier (detector voltagemultiplier), 0.60; ion mass range (TOF masses), 1000-5000m/z.

(6) Data analysis: MALDI-MS data was analyzed by the software Data Explorer 4.0; the structural schematic diagram of N-glycans was specified by the software GlycoWorkbench 2.1. (Note: This method has potential application value in tumor marker mining, as shown in Figure 6).

An application of a rapid quantitative analysis method based on MALDI-MS and stable isotope-labeled N-glycan, which can be used for serum glycan analysis.

Compared with the prior art, the advantages of the present invention are:

1. Improve experimental efficiency: The use of microwave-assisted rapid enzyme digestion greatly shortens the reaction time, so that the overall reaction can be completed within 2 hours, which greatly improves the experimental efficiency.

2. Eliminate the interference of system differences: The use of stable isotopes (d0/d5-benzoyl chloride) to analyze N-glycans can minimize the differences in sample ionization efficiency and instrument response values.

3. Low cost: The isotopes used in this method have been commercialized and have low cost. At the same time, due to the use of isotope quantification technology, there is no need to introduce internal or external standards, which greatly reduces the experimental cost and experimental complexity.

4. Mild reaction conditions: The experiment can react under weak alkaline and normal temperature conditions, and the derivatization efficiency exceeds 99%, which will not cause damage to acidic groups.

5. Simultaneous quantitative analysis of neutral sugars and acidic sugars: In this method, since the methylamination method is used to neutralize the acidic sugars, the acidic groups (sialic acid) are effectively protected to prevent them from Interference caused by in-source and post-source cleavage in later mass spectrometry detection (Note: If there is no neutralization treatment such as methylamination, sialic acid will undergo in-source and post-source cleavage during mass spectrometry detection, causing permanent damage to the acidic sugar structure. Destruction, not only makes the quantification of acidic sugars into bubbles, but also produces many impurity peaks, which greatly interferes with data analysis).

6. Improve quantitative ability: This method exhibits a 10-fold dynamic linear range in glycan quantification, and can accurately analyze glycans at picomolar level.

7. Medical application value: In order to reflect the value in the medical field, this method is also used for serum glycan analysis. By comparing the changes of serum glycans in different pathological stages, specific markers that can be used as rapid pathological diagnosis can be screened out.

Description of drawings

Figure 1 is a flow chart of the rapid quantitative analysis of MALDI-MS and stable isotope-labeled N-glycans.

Figure 2 is a diagram showing the optimization of the derivatization conditions of sugar amine and benzoyl chloride.

Figures 3-A to 3-C illustrate the application of this method in the quantitative analysis of standard glycoprotein N glycans.

Figure 4 quantifies d0/d5-benzoyl chloride derivatized RNase B glycans at seven different molar ratios.

Figure 5 shows the 10-fold linear quantitative range of this method.

Figure 6 shows the application of this method in tumor marker screening.

Detailed ways

The following are specific embodiments of the present invention to further describe the technical solutions of the present invention, but the protection scope of the present invention is not limited to these embodiments. All changes or equivalent substitutions that do not depart from the concept of the present invention are included in the protection scope of the present invention.

Example 1

A method for rapid quantitative analysis of N-glycans based on MALDI-MS and stable isotope labeling, the method comprises the following steps (see Figure 1):

(1) Microwave-assisted rapid digestion: standard glycoprotein (10ug) or serum (5ul) was dissolved in 20ul reaction solution (containing 10mM sodium phosphate, 0.13% sodium dodecyl sulfate, 10mM dithiothreitol), Then boil in a 100°C water bath for 10min, add 2.4ul of 10% NP-40, add 0.5ul of PNGase F after mixing, and digest the enzyme for 20min under microwave-assisted conditions (the maximum output power of the microwave oven used in the experiment is 700W. lowest gear), which releases N-glycans in the form of glycosamines.

(2) Rapid derivatization reaction between sugar amine obtained in step (1) and d0/d5-benzoyl chloride (d0 represents non-isotopic labeling; d5 represents isotopic labeling), reaction conditions: prepare benzoyl chloride solution (because benzoyl chloride is volatile And it is prone to hydrolysis, so the reagent should be used and prepared immediately), add benzoyl chloride to the enzyme cleavage reaction solution in step (1), mix quickly, and leave at room temperature for 30min.

(3) SPE purification: Purify the reaction solution in step (2), the steps are: ① 3ml deionized water to wash the MCC purification column; ② 3ml balance solution (n-butanol:ethanol:water=4:1:1) to The MCC is balanced; 3. 800ul of the equilibrated solution is added to the glycan reaction system of step (2), fully mixed, and sampled; 4. 3 ml of the equilibrated solution (n-butanol:ethanol:water=4:1:1) is applied to the MCC Rinse to remove all impurities; ⑤1ml eluent (ethanol:water=1:1) to elute the sample, evaporate to dryness for later use.

(4) Methylamination reaction: before the methylamination treatment, d0-benzoyl chloride and d5-benzoyl chloride-derived N-glycans were mixed at a molar ratio of 1:1 to obtain a mixed sample, and then 25 ul of methylamine hydrochloric acid was added. Salt solution (dissolve 7mg methylamine hydrochloride in 100ul DMSO, then add 5.5ul methylmarine solution) and 25ul PyAOP solution (dissolve 2.6mg PyAOP in 100ul DMSO) were added to the above mixed sample , stand for 30 min at room temperature and in the dark, and purify by SPE, the steps are the same as above.

(5) Detection of glycan samples by MALDI-MS: Dissolve the double-derivatized N-glycans in step (4) in 10 μL of solution (acetonitrile: water = 1:1), take 1.0 μL of the sample solution and 1.0 μL of the solution. μL of DHB solution was mixed and spotted onto a 384-well plate, with about 1.0 μL of liquid per spot. The plate was left to stand horizontally for 15 min to evaporate the solvent, and then N-glycan detection was performed by MALDI-MS 5800 (SCIEX).

The relevant parameters of the matrix-assisted laser desorption ionization mass spectrometer are set as follows: detection mode, ReflectorPositive; laser intensity (laser intensity), 5200; voltage multiplier (detector voltagemultiplier), 0.60; ion mass range (TOF masses), 1000-5000m/z.

(6) Data analysis: MALDI-MS data was analyzed by the software Data Explorer 4.0; the structural schematic diagram of N-glycans was specified by the software GlycoWorkbench 2.1.

Example 2

In order to verify the derivatization effect of benzoyl chloride and sugar amine, the pH, temperature and reaction time that will affect the derivatization efficiency were optimized and explored (the results are shown in Figure 2). In the experiment, benzoyl chloride and Man5GlcNAc2 (a standard glycoprotein The derivatization efficiency of one of the five glycans of ribonuclease B) was discussed as a benchmark. Figure 2-A shows the effect of pH of the reaction environment on the derivatization efficiency. It can be seen that when the pH reaches 9.0, it can reach The best derivatization effect; Figure 2-B: the effect of reaction temperature on the derivatization efficiency, it can be seen that when the temperature is 20 °C, the best derivatization effect can be achieved; Figure 2-C: The effect of reaction time on the derivatization efficiency can be It can be seen that the best derivatization effect can be achieved when the reaction time reaches 40min.

Example 3

Three standard glycoproteins - ribonuclease B, fetal bovine protein Fetuin and immunoglobulin IgG were analyzed respectively (the results are shown in Figure 3). Figure 3-A is: quantitative analysis of ribonuclease B glycans, when d0- After the N-glycans derived from benzoyl chloride and d5-benzoyl chloride were mixed in a molar ratio of 1:1, it could be seen that almost all of the five N-glycans in the standard glycoprotein existed in the form of equal height ion pairs (equal height). The state presented by the ion pair is consistent with the molar ratio of 1:1); Figure 3-B: Quantitative analysis of Fetuin glycans in fetal bovine protein, when d0-benzoyl chloride and d5-benzoyl chloride-derived N glycans are in a molar ratio After mixing at 1:1, it can be seen that the five N glycans in the standard glycoprotein are almost all in the form of equal ion pairs; Figure 3-C: Quantitative analysis of immunoglobulin IgG glycans, when d0-benzene After the N-glycans derived from formyl chloride and d5-benzoyl chloride were mixed in a molar ratio of 1:1, it could be seen that almost all of the N-glycans in the standard glycoprotein existed in the form of isomeric glycan ion pairs. The above results fully demonstrate the stability of this method in glycan quantification.

Example 4

To verify the linear dynamic range of this quantitative method, d0-benzoyl chloride and d5-benzoyl chloride derivatized RNase B glycans were mixed in seven different molar ratios (results are shown in Figure 4), (A) 10: 1. (B)5:1, (C)2:1, (D)1:1, (E)1:2, (F)1:5, (G)1:10, it can be seen from the above results The glycan peaks derived from d0-benzoyl chloride and d5-benzoyl chloride are displayed in the form of the above-mentioned seven different proportional ion pairs, indicating the linear feasibility of this method in terms of quantification.

The linear statistical analysis of the results in Figure 4 shows that since the above five glycans are mixed in seven proportions, each line presents seven separation points. Statistical analysis shows that the experimental ratio is very close to the theoretical ratio, and the five poly The R values of the fitting coefficients of sugars were all higher than 0.999, indicating that the method has a very good linear quantitative range. The specific results are shown in Figure 5.

Example 5

Normal human serum glycans and serum glycans from patients with early myeloma were labeled with d0-benzoyl chloride and d5-benzoyl chloride, respectively (the results are shown in Figure 6). It can be seen from Figure 6-A that most of the glycan ion pairs All of them are of the same height, indicating that most of the glycans in normal human serum and serum of myeloma patients have not changed much in content, and several glycan ion pairs were randomly selected from these The enlarged graph better illustrates that most of the glycans do not change much at the peak; however, there are still some glycans that show some change in the two sera (Fig. 6-E-G), where Fig. These two glycans in 6-E-F were up-regulated in the serum of patients with early myeloma compared with normal human serum, but this glycan in Fig. 6-G was down-regulated. Application value of quantitative methods in tumor marker mining.

Claims (1)

1. a fast quantitative analysis method of N-glycan based on MALDI-MS and stable isotope labeling, it is characterized in that: the method applies MALDI-MS and stable isotope labeling technology, to the fast quantitative analysis of N-glycan, specifically comprises the steps: (1) Dissolve the standard glycoprotein or serum in the reaction solution, then boil it in a water bath at 100°C for 10 minutes, add 10% NP-40, mix well, then add PNGase F, and digest with microwave for 20 minutes to make N Glycans are released in the form of glycosamines; The reaction solution contains 10 mM sodium phosphate, 0.13% sodium dodecyl sulfate, 10 mM dithiothreitol; (2) Mix the sugar amine obtained in step (1) with d0/d5-benzoyl chloride solution, place at room temperature for 30min, and the reaction environment pH=9.0; (3) SPE purification treatment is carried out on the reaction solution in step (2), the steps are: (1) washing the MCC purification column with deionized water; (2) equilibrating the MCC with a balance solution; (3) adding the balance solution to the glycan reaction in step (2) In the system, mix thoroughly and load the sample; ④ The balance solution is used to rinse the MCC to remove all impurities; (4) Mix the N-glycans derived from d0-benzoyl chloride and d5-benzoyl chloride at a molar ratio of 1:1, add methylamine hydrochloride solution and PyAOP solution to the above mixed sample, and store at room temperature in the dark. Place stand for 30min, SPE purification, steps are the same as above; (5) Dissolve the double-derivatized N-glycan in step (4) in 50% acetonitrile solution, take the sample solution and mix it with DHB solution, and place it on the sample well plate, and let the sample well plate stand horizontally for 15 minutes to make the solvent volatilization followed by N-glycan detection by MALDI-MS 5800; The 50% acetonitrile solution is acetonitrile: water=1:1; The relevant parameters of the matrix-assisted laser desorption ionization mass spectrometer are set as follows: detection mode, Reflector Positive; laser intensity 5200; voltage multiple 0.60; ion mass range 1000-5000m/z; (6) The MALDI-MS data was analyzed by the software Data Explorer 4.0; the structural schematic diagram of N-glycans was specified by the software GlycoWorkbench 2.1.

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