A method for dissociating adducted metal ions and enriching target ions and a device therefor

Abstract

The application discloses a method for dissociating added metal ions and enriching target ions, which comprises the following steps: first, preparing a to-be-tested target product into a solution, injecting the solution into a capillary in a continuous sampling mode and making the solution flow in the capillary, wherein the capillary is provided with an electrode 1 and an electrode 2, and no voltage is applied in the sampling stage; then, controlling the voltage of the electrode 1 to be 0 to -50 kv, controlling the voltage of the electrode 2 to be 0 to +50 kv, and continuously making the to-be-tested target product solution flow in the capillary, so that the added metal ions in the to-be-tested target product solution are dissociated, and the to-be-tested target product from which the metal ions are removed is enriched at the position of the electrode 2; finally, adjusting the voltage of the electrode 1 and the electrode 2 to 0, collecting the to-be-tested target product and performing subsequent detection. The method can quickly promote the separation of metal ions, realize the instantaneous capture of target ions, and improve the sensitivity and resolution of mass spectrometric detection of target ions.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of chemical analysis detection, in particular to a method and device for on-line dissociation of adduct ions and enrichment of target sample ions in a liquid phase environment. BACKGROUND

[0002] High-resolution mass spectrometry is an indispensable sample analysis technology in the field of analytical chemistry and the field of life science research. Biological mass spectrometry analysis has the characteristics of high throughput, high sensitivity, high accuracy and automation, which is required for nucleic acid and protein analysis at the level of omics. However, due to the polarity and electronegativity of nucleic acid molecules, a large number of alkali metal ions such as Na + , K + , etc. are easily absorbed to form various basic ion adducts of different molecular weights, which poses a great challenge to mass spectrometry in nucleic acid analysis.

[0003] Therefore, it is particularly important to study a detection method for analyzing and detecting nucleic acid molecules, protein molecules or small molecule substances that are easily adducted with cations under liquid phase conditions, while removing cations (such as some alkali metal ions). SUMMARY

[0004] In order to overcome the above technical problems, the purpose of the present application is to provide a method for dissociating adducted metal ions and enriching target ions, which can quickly promote the separation of metal ions and realize the instantaneous capture of target ions, thereby improving the sensitivity and resolution of mass spectrometry detection of target ions.

[0005] Another purpose of the present application is to provide a device for dissociating adducted metal ions and enriching target ions, which can greatly improve the sensitivity and resolution of mass spectrometry nucleic acid detection in negative mode, and has good compatibility with mass spectrometry.

[0006] In order to achieve the above purpose, the present application provides a method for dissociating adducted metal ions and enriching target ions, which comprises the following steps:

[0007] 1) Sample injection stage: first prepare the target product to be tested into a solution, inject it into a capillary in a continuous injection mode and make it flow in the capillary, wherein the capillary is provided with an electrode 1 and an electrode 2, and the electrode 1 is not powered during the sample injection stage;

[0008] 2) Dissociation stage: then control the voltage of the electrode 1 to be 0 to -50kv, and control the voltage of the electrode 2 to be 0 to +50kv, and the target product solution still continuously flows in the capillary, and the adducted metal ions in the target product solution are dissociated, and the target product from which the metal ions are removed is enriched at the position of the electrode 2;

[0009] 3) release stage: finally, the electrode 1 and electrode 2 voltage is adjusted to 0, the target product to be tested will be collected for subsequent detection.

[0010] Wherein, the target product to be tested is a nucleic acid molecule, a protein molecule or a small molecule substance of cationic addition reaction, and the cation is Na + , K + , etc.

[0011] The target product to be tested is prepared into a solution, and a buffer solution is added to the target product to be tested to configure a solution, the buffer solution is a mass spectrometry compatible buffer solution, such as a mixed solution of one or more of acetonitrile, methanol, ammonium acetate, ammonium formate, formic acid or acetic acid, the concentration is 10 -10 μmol / L-10 10 mol / L, preferably 10 -10 μmol / L-1mol / L.

[0012] The flow rate of the target product solution in the capillary is controlled to be 0.1nL-10mL / min, preferably 0.1nL-1mL / min.

[0013] In step 1), the target product solution is injected into the capillary by a syringe, and a peristaltic pump is used as the driving power.

[0014] The sampling stage time period is controlled to be 0.01-60min, preferably 5-20min.

[0015] In step 2), the electrode 1 control voltage is 0 to-20kv, and the electrode 2 control voltage is 0 to+20kv.

[0016] The dissociation stage time period is controlled to be 0.01-60min, preferably 5-20min.

[0017] In step 3), the release stage time period is controlled to be 0.01-10min, preferably 5-10min.

[0018] The device for the method of dissociating the added metal ions and enriching the target ions of the application comprises: a peristaltic pump, a syringe, a fluid channel in which two electrodes and a capillary are connected in series, and a power supply for controlling the electrodes, the peristaltic pump drives the syringe, and the syringe is connected to the capillary through a filter head.

[0019] Wherein, the device is also provided with a sampling valve, which is arranged between the syringe and the capillary. A quantitative ring is arranged in the sampling valve.

[0020] The collected target product to be detected needs to be detected by mass spectrometry, and a capillary can also be connected with a spray needle to directly enter a mass spectrometer for detection.

[0021] The researchers of the present application find that some nucleic acid molecules, protein molecules or small molecule substances are easy to adduct with some cations under liquid phase conditions to form various molecular weight basic ion adducts (or chelates), so that cation adduct peaks are easy to form when these substances are analyzed and detected, causing problems such as low detection sensitivity and poor specificity. Therefore, electrode control is determined in multiple experimental studies, and only two electrodes are set, one is a negative electrode and the other is a positive electrode (such as a ground electrode or a positive electrode), and the negative electrode or the positive electrode is in a dissociation enrichment mode when there is voltage, and is in a non-dissociation enrichment mode when there is no voltage.

[0022] As shown in Figure 1 When the negative electrode has voltage, an electric field is formed between the negative electrode and the positive electrode, and under the action of the electric field, the chelate of the sample to be detected adducted with metal ions weakly interacts to generate positively charged metal salt ions and negatively charged sample ions to be detected, and the metal ions are small in charge and small in molecule, and are weakly affected by the electric field, and will not be enriched at the negative electrode, but will flow out of the capillary with constant liquid flow. The sample ions to be detected (such as nucleic acid ions) have many negative groups, and are enriched near the positive electrode due to adsorption.

[0023] Although the electric field applied in the same direction as the carrier flow direction, the ions can accelerate to the lowest potential under the joint action of the electric field force and the viscous resistance, and flow out of the electric field with the carrier flow, but in the dissociation process, the sample ions to be detected will not flow out of the electric field with the carrier flow, but will be enriched near the positive electrode under the action of the adsorption principle. Ultimately, the metal chelate is dissociated in the electric field region, the dissociated sample ions to be detected are negatively charged and are adsorbed on the positive electrode, effectively removing the adducted alkali metal ions of the sample to be detected.

[0024] The technical scheme provided by the present application removes the alkali metal ions combined with the sample to be detected by the potential difference generated by the external direct current voltage in the liquid passage, and simultaneously realizes the enrichment and release of the electronegative sample to be detected at the electrode, which can greatly improve the detection sensitivity and resolution of the sample to be detected in the negative mode. The method of the present application can improve the phenomena such as low sensitivity in the negative ion mode, mass spectrometry signal suppression and multiple cation adduct peaks of the sample to be detected, is simple and flexible to operate, has a wide application range, has good compatibility with mass spectrometry, and can realize automatic control. BRIEF DESCRIPTION OF DRAWINGS

[0025] Figure 1 The method principle diagram for dissociating the adducted metal ions and enriching the target ions is shown in the present application;

[0026] Figure 2-1 、 2-2 Electrode voltage time sequence diagram and extracted ion chromatogram of 8nt single-stranded DNA (ssDNA-1) analyzed by the continuous sampling device of the present application;

[0027] Figure 3-1 、 3-2 Mass spectrum diagram of non-enrichment mode and enrichment mode of 8nt single-stranded DNA (ssDNA-1) analyzed by the continuous sampling device of the present application;

[0028] Figure 4-1 、 4-2 , 4-3 Mass spectrum diagram and abundance percentage of 53nt single-stranded DNA (ssDNA-2) in non-enrichment mode and enrichment mode analyzed by the continuous sampling device of the present application after being placed at room temperature for different time;

[0029] Figure 5-1 、 5-2 , 5-3 Effect diagram of the continuous sampling device of the present application in analyzing single-stranded DNA of different lengths (15nt, 29nt, 115nt);

[0030] Figure 6-1 、 6-2 , 6-3 Effect diagram of the valve sampling device of the present application in analyzing 8nt single-stranded DNA (ssDNA-1) of different concentrations. DETAILED DESCRIPTION

[0031] Embodiments of the present application will be described below with reference to the accompanying drawings. The elements and features described in one drawing or one embodiment of the present application can be combined with the elements and features shown in one or more other drawings or embodiments. It should be noted that the representation and description of components or processes unrelated to the present application, which are known to those of ordinary skill in the art, are omitted from the drawings and the description for the purpose of clarity.

[0032] Example 1 Electric field dissociation of nucleic acid adduct ions and enrichment of nucleic acid ions

[0033] In this embodiment, in the continuous sampling embodiment, the sampling system selects a peristaltic pump and a 500μl syringe, the sample separation channel selects a quartz capillary with an inner diameter of 150μm and an outer diameter of 360μm, wherein the liquid channel from the filter head to the two-way titanium plus electrode adopts a quartz capillary with a length of 50cm, the liquid channel from the two-way titanium plus electrode to the two-way alloy ground electrode adopts a quartz capillary with a length of 5cm, and the liquid channel from the two-way alloy ground electrode to the spray needle adopts a quartz capillary with a length of 5cm.

[0034] The nucleic acid ssDNA-1 (5'att ccg aa) concentration is 1 μmol / L (the buffer is acetonitrile:water=1:1), the sample is injected into the channel by a peristaltic pump in a continuous injection mode, and the peristaltic pump flow rate is 120 μL / h. The injection stage is 0-5 min without electricity, and the sample flows uniformly in the pipeline under the action of liquid flow driving; the dissociation and enrichment stage is 5-10 min, the metal joint electrode 1 is adjusted to-9.0 kV; the release stage is 10-15 min, and the HV of the metal joint electrode 1 is adjusted back to 0.

[0035] As can be seen from FIG. 2, when a strong electric field is formed between the electrode 1 and the electrode 2, the small-molecule cation added to the nucleic acid is dissociated under the action of the electric field. At the anode of the electric field, the nucleic acid with the added cation is negatively charged and is enriched at the anode position of the electrode 2; the release stage is 10 min to 15 min, at which time the pipeline is not electrified, and there is no potential difference in the pipeline, so that the sample cannot be enriched. Under the driving of the liquid flow, the enriched nucleic acid is directly eluted from the liquid phase ion trap and released into the mass spectrometry detection. Figure 2-1 、2、2)。

[0036] As can be seen from the mass spectrum of the nucleic acid ( Figure 3-1 、3、2), in the non-enrichment mode, a large number of metal-added ion peaks of the nucleic acid ions are measured by the mass spectrometry, while in the enrichment mode, the metal-added ion peaks are weakened, and the nucleic acid detection sensitivity is improved by 167 times.

[0037] As can be seen, the peak intensity of the nucleic acid obtained by the method of the present application is greater than that of the conventional mode, which can be enhanced by tens of times, and the peak width is narrower. In an alternative quantitative ring injection implementation mode, the peak intensity of the nucleic acid can be improved by hundreds of times, which indicates that the present application can effectively improve the sensitivity and resolution of the mass spectrometry detection in the negative mode.

[0038] The present embodiment can improve the phenomena of low sensitivity of nucleic acid in negative ion mode, mass spectrum signal suppression, and multiple cation addition peaks, and simultaneously realize the separation of ions combined with the nucleic acid through weak interaction and the transient capture and enrichment of the nucleic acid without destroying the primary structure of the nucleic acid.

[0039] Example 2 Dissociation of nucleic acid adducts placed at room temperature for different times

[0040] In this embodiment, the sample injection system uses a peristaltic pump and a 500 μL syringe, and the sample separation channel uses a quartz capillary with an inner diameter of 150 μm and an outer diameter of 360 μm. The liquid channel from the filter head to the two-way titanium electrode is a quartz capillary with a length of 50 cm, the liquid channel from the two-way titanium electrode to the two-way alloy ground electrode is a quartz capillary with a length of 5 cm, and the liquid channel from the two-way alloy ground electrode to the spray needle is a quartz capillary with a length of 5 cm.

[0041] The nucleic acid ssDNA-2 (53 nt, 5' cct ctc tat ggg cag tcg gtg atc gca gta cga tct atc aga gct gca) has a concentration of 1 μmol / L (the buffer is acetonitrile: water = 1:1 (the buffer used at the time of the experiment)), and the sample is injected into the channel by a peristaltic pump in a continuous injection mode, and the flow rate of the peristaltic pump is 120 μL / h. The injection stage is 0-5 min without electricity, and the sample flows uniformly in the pipeline under the action of the liquid flow; the dissociation and enrichment stage is 5-10 min, and the metal linker electrode is adjusted to -9.0 kV; the release stage is 10 min to 15 min, and the HV of the metal linker electrode 1 is adjusted back to 0.

[0042] The mass spectrum of the nucleic acid with a high intensity of -24 charges is observed, and from Figure 4-1 It can be seen that, under the non-enrichment time, with the extension of the placement time, the nucleic acid is combined with a large amount of alkali metal ions to form an adduct; from Figure 4-2 It can be seen that, after using the electric field dissociation and enrichment function, the nucleic acid ions removed from the adduct are mainly detected; by comparing the two methods, from Figure 4-3 It can be seen that, under the non-enrichment mode, with the extension of the nucleic acid placement time at room temperature, the adduct ion peak will be more and more, and the non-adduct ion peak will be weaker and weaker, which will greatly affect the mass spectrometric detection result of the nucleic acid; and in the enrichment mode, since the adduct ion peak is removed, it will not be affected by the adduct ion peak, and the nucleic acid ion peak changes little in each time period, and a relatively stable nucleic acid ion peak (with an abundance ratio of about 80%) can be detected.

[0043] It can be seen that, by using the method of this embodiment, the length of the placement time has no obvious effect on the mass spectrometric detection of the target sample.

[0044] Example 3 Dissociation and enrichment of single-stranded DNA (15 nt, 29 nt, 115 nt) of different lengths

[0045] In this embodiment, the sample injection system selects a peristaltic pump and a 500 μL syringe in the continuous injection mode. The sample separation channel selects a quartz capillary with an inner diameter of 150 μm and an outer diameter of 360 μm. The liquid channel from the filter head to the two-way titanium electrode is a quartz capillary with a length of 50 cm. The liquid channel from the two-way titanium electrode to the two-way alloy ground electrode is a quartz capillary with a length of 5 cm. The liquid channel from the two-way alloy ground electrode to the spray needle is a quartz capillary with a length of 5 cm.

[0046] The nucleic acid ssDNA (15 nt, 20 nt, 115 nt) has a concentration of 1 μmol / L (the buffer is acetonitrile: water = 1:1). The sample is injected into the channel by the peristaltic pump in the continuous injection mode. The flow rate of the peristaltic pump is 120 μL / h. The injection stage is 0-5 min without electricity. The sample flows uniformly in the pipeline under the action of the liquid flow. The dissociation and enrichment stage is 5-10 min. The metal linker electrode 1 is adjusted to -9.0 kV. The release stage is 10-15 min. The HV at the metal linker electrode 1 is adjusted back to 0.

[0047] As can be seen from FIG. 5, in the non-enrichment mode, as the molecular weight of the nucleic acid increases, the adduct ion peaks increase more and more, and the non-adduct ion peaks become weaker and weaker. When the 115 nt nucleic acid is analyzed, the nucleic acid molecule ion peak cannot be detected. Figure 5-1 、 Figure 5-2 、 Figure 5-3 As can be seen from FIG. 5, in the non-enrichment mode, as the molecular weight of the nucleic acid increases, the adduct ion peaks increase more and more, and the non-adduct ion peaks become weaker and weaker. When the 115 nt nucleic acid is analyzed, the nucleic acid molecule ion peak cannot be detected.

[0048] Therefore, the method is suitable for different molecular weight target samples, and can be determined with high sensitivity and high resolution.

[0049] Example 4 Analysis of dissociation and enrichment of single-stranded DNA with different concentrations by valve injection

[0050] In this embodiment, the sample injection valve with a quantitative ring is selected to control the sample injection. The sample injection part is composed of a peristaltic pump, a syringe, a six-way injection valve, and a quantitative ring. The quantitative ring selects a capillary with an inner diameter of 150 μm, an outer diameter of 360 μm, and a length of 25 cm. One injection peristaltic pump with a flow rate of 100 μL / h carries single-stranded DNA-1 (5'att ccg aa) with a concentration of 0.1 μmol / L and 1 μmol / L to the six-way valve quantitative ring within 2-4 min. After injection, the valve is switched to the analysis flow path. The microliter pump carries the buffer to push the sample in the quantitative ring into the analysis liquid channel at a flow rate of 1 μL / min. The injection stage is 2-4 min. The enrichment stage is 4-14 min. The release stage is 14-20 min.

[0051] Figure 6-1、 6-2 As shown in 6-3, the enrichment fold of nucleic acid molecules at low concentration of 0.1 μmol / L is 285, and the enrichment fold of nucleic acid molecules at high concentration of 1 μmol / L is 7.8. This is because at low concentration, the ratio of solvent to solute increases, so that nucleic acid can bind more metal ions. If the low-concentration target sample is directly subjected to mass spectrometry detection, there are many adduct ion peaks, which affect the nucleic acid mass spectrometry detection results. The low-concentration target sample is more easily affected by adduct ions than the high-concentration target sample, and in the enrichment mode, the difference between the two is not great.

[0052] In this embodiment, the negatively charged nucleic acid molecules are analyzed, and in the enrichment mode, the target ion chromatogram has higher peak intensity and narrower peak width, and the mass spectrum has no obvious metal ion adduct peak, which improves the sensitivity and measurement accuracy of mass spectrometry analysis of nucleic acid molecules.

[0053] The alkali metal ions combined with the sample to be detected are removed by the potential difference generated by the applied direct current voltage in the liquid passage, and at the same time, the electronegative sample to be detected is enriched and released at the electrode, which can greatly improve the detection sensitivity and resolution of the sample to be detected in the negative mode. The method of the present application can improve the phenomena of low sensitivity in negative ion mode, mass spectrum signal suppression, and multiple cation adduct peaks of the sample to be detected. The operation is simple and flexible, and has wide application range, good compatibility with mass spectrometry, and can realize automatic control of software. It is suitable for the technical field of analysis and detection of nucleic acid molecules, protein molecules or small molecule drugs which are easy to adduct with small molecules, and has high sensitivity, high resolution, high specificity, simple operation and other characteristics, and has good adaptability and wide application prospect.

[0054] Although the present application and its advantages have been described in detail, it should be understood that various changes, substitutions and alterations can be made without departing from the spirit and scope of the application as defined by the appended claims. Moreover, the scope of the present application is not intended to be limited to the particular embodiments of the process, machine, manufacture, composition of matter, means, methods and steps described in the specification. As one of ordinary skill in the art will readily appreciate from the disclosure herein, processes, machines, manufacture, compositions of matter, means, methods or steps, presently existing or later to be developed that perform substantially the same function or achieve substantially the same result as the corresponding embodiments described herein can be utilized according to the present application. Accordingly, the appended claims are intended to cover all processes, machines, manufacture, compositions of matter, means, methods or steps, substantially as set forth in the disclosure herein, and other processes, machines, manufacture, compositions of matter, means, methods or steps that they have substantially the same functions or achieve substantially the same results as those recited in the claims.

Claims

1. A method of dissociating a complexed metal ion and enriching a target ion, characterized by, It comprises the following steps: 1) Injection stage: first, the target product to be tested is prepared into a solution, which is injected into a capillary in a continuous injection mode and flows in the capillary, the capillary is provided with electrode 1 and electrode 2, and the injection stage is not powered; the target product to be tested is a nucleic acid molecule, a protein molecule or a small molecule substance of cationic addition reaction, and the cation is an alkali metal ion; 2) Dissociation stage: then, the voltage of electrode 1 is controlled to be 0 to -50kv, the voltage of electrode 2 is controlled to be 0 to +50kv, the target product solution still continuously flows in the capillary, the added alkali metal ions in the target product solution are dissociated, and the target product solution with dissociated alkali metal ions flows out of the capillary with constant liquid flow, and the target product without alkali metal ions is enriched at the position of electrode 2; 3) Release stage: finally, the voltage of electrode 1 and electrode 2 is adjusted to 0, the target product without alkali metal ions is collected, and subsequent detection is performed.

2. The method of claim 1, wherein, The alkali metal ion is Na+ or K+.

3. The method of claim 1, wherein, The target product to be tested is prepared into a solution, and the target product to be tested is added into a mixed solvent of one or more of acetonitrile, methanol, ammonium acetate, ammonium formate, formic acid or acetic acid to configure into a solution.

4. The method of claim 1, wherein, The flow rate of the target product solution in the capillary is controlled to be 0.1nL-10mL / min, the target product solution is injected into the capillary by a syringe, and a peristaltic pump is used as a driving power.

5. The method of claim 1, wherein, In the injection stage, the time period is controlled to be 0.01-60min, in the dissociation stage, the time period is controlled to be 0.01-60min, and in the release stage, the time period is controlled to be 0.01-10min.

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