Electro-spray ionization mass spectrometry ion source based on low-frequency alternating voltage and use method thereof

Abstract

The invention discloses an electrospray ionization mass spectrometry ion source based on a low-frequency alternating-current voltage strategy and a use method of the electrospray ionization mass spectrometry ion source. The ion source comprises a nanoliter electrospray needle used for loading a sample solution; the metal electrode is inserted into the nanoliter electrospray needle and is in direct contact with the sample solution; the signal generator has a plurality of signal output functions; and the high-voltage amplifier has a voltage amplifying function, a signal input end is connected with the signal generator, and an output end is connected with the metal electrode. Firstly, a signal generator is used for generating an alternating current signal with specific waveform, frequency, peak-to-peak value and duty ratio, and the signal is amplified by a high-voltage amplifier and then directly acts on a nanoliter electrospray needle to generate electrospray; meanwhile, the amplified signal is monitored in real time by an oscilloscope. The low-frequency alternating-current nano-liter electrospray ion source involved in the invention is named as LFAC-nESI. Compared with a conventional nanoliter electrospray (nESI) ionization method, the method provided by the invention has an online desalting function, can significantly improve the signal-to-noise ratio and analysis sensitivity of a to-be-detected object, and is more practical.

Description

Technical Field

[0001] The present invention relates to an on-line desalting high-sensitivity mass spectrometry ion source device and an analysis method thereof, in particular to an on-line desalting high-sensitivity mass spectrometry analysis method based on a low-frequency alternating current high-voltage nanoliter electrospray ion source, belonging to the technical field of on-line desalting high-sensitivity mass spectrometry analysis methods. Background Art

[0002] The analysis of biological samples always faces challenges such as complex matrices. Nanoliter electrospray evolved from electrospray, using a finer emitter to generate a spray at the nL / min level, which can effectively improve the ionization efficiency and ion transmission efficiency. In addition, another significant feature of nanoliter electrospray is that it can also effectively alleviate the influence of ion suppression and matrix effects compared with electrospray, with longer analysis time and less sample consumption, being simple and durable.

[0003] A simple method is to reduce the size of the nanospray needle to the sub-micron level to achieve a good desalting effect. The separation of biomolecules and salt components can also be achieved by crystallization at the tip of the nanospray needle. This method has been successfully used for the detection of various proteins and peptides. Flexible high-voltage application also has a good desalting effect. Applying a stepped step voltage or non-contact induced voltage to the nanoliter electrospray separates the analyte and salt components due to the difference in electrophoretic migration rates. Conventional nanoliter electrospray combined with an additional dual-electrode auxiliary electric field can also create electrophoretic conditions to achieve the analysis effect of complex sample analysis or desalting.

[0004] Although nESI has been continuously improved and widely used in the analysis of biological samples, in view of the high-sensitivity detection requirements of actual sample analysis for nanoliter electrospray, its analysis performance still has deficiencies. Therefore, an on-line desalting high-sensitivity mass spectrometry analysis method based on a low-frequency alternating current high-voltage nanoliter electrospray ion source (LFAC-nESI) is designed to solve the above problems. Summary of the Invention

[0005] The main object of the present invention is to provide an electrospray mass spectrometry ion source based on a low-frequency alternating current voltage and its on-line desalting and high-sensitivity usage method.

[0006] The object of the present invention can be achieved by adopting the following technical solutions:

[0007] A usage method of an electrospray mass spectrometry ion source based on a low-frequency alternating current voltage strategy includes the following steps:

[0008] Step 1: Load the sample solution by using a nanoliter electrospray needle;

[0009] Step 2: Insert a metal electrode into the nanoliter electrospray needle to directly contact the sample solution;

[0010] Step 3: Use a signal generator to generate an alternating current signal with a specific waveform, frequency, peak-to-peak value, and duty cycle. After being amplified by a high-voltage amplifier, the signal directly acts on the nanoliter electrospray needle to generate an electrospray.

[0011] Step 4: The amplified signal is monitored in real time by an oscilloscope.

[0012] Preferably, in Step 3, the signal input terminal is connected to the signal generator, and the output terminal is connected to the metal electrode.

[0013] Preferably, in Step 3, the application of high voltage is changed from direct current high voltage to low-frequency alternating current high voltage.

[0014] Preferably, perform LFAC-nESI to detect biological samples.

[0015] A method for using an electrospray ionization mass spectrometry ion source based on low-frequency alternating current voltage, including a signal generator, which is coupled to a high-voltage amplifier;

[0016] The high-voltage amplifier is coupled to an oscilloscope and forms a mass spectrometry by injecting an electrospray through high voltage into a sample tube.

[0017] The beneficial technical effects of the present invention:

[0018] The present invention provides an electrospray ionization mass spectrometry ion source based on low-frequency alternating current voltage and its online desalting and highly sensitive use method, including a nanoliter electrospray needle for loading a sample solution; a metal electrode inserted into the nanoliter electrospray needle and in direct contact with the sample solution; a signal generator with various signal output functions; a high-voltage amplifier with the function of amplifying voltage, the signal input terminal is connected to the signal generator, and the output terminal is connected to the metal electrode. First, the present invention uses a signal generator to generate an alternating current signal with a specific waveform, frequency, peak-to-peak value, and duty cycle. After being amplified by a high-voltage amplifier, the signal directly acts on the nanoliter electrospray needle to generate an electrospray; at the same time, the amplified signal is monitored in real time by an oscilloscope. Compared with the ionization method of ordinary nanoliter electrospray (nESI), the present invention has the function of online desalting, can significantly improve the signal-to-noise ratio of the analyte and the analysis sensitivity, and is more practical. The technical solution provided by the present invention is applicable to the direct analysis of biological samples. Description of the Drawings

[0019] Figure 1 It is a comparative diagram of the nESI and LFAC-nESI analysis spectra of insulin in a preferred embodiment of an online desalting and highly sensitive mass spectrometry analysis method based on a low-frequency alternating current high-voltage nanoliter electrospray ionization source according to the present invention;

[0020] Figure 2Comparison chart of nESI and LFAC-nESI analysis spectra of cytochrome c (a, b) and myoglobin (c, d) for a preferred embodiment of an online desalting high-sensitivity mass spectrometry analysis method based on a low-frequency alternating current high-voltage nanoliter electrospray ionization source according to the present invention;

[0021] Figure 3 Ion source condition optimization for a preferred embodiment of an online desalting high-sensitivity mass spectrometry analysis method based on a low-frequency alternating current high-voltage nanoliter electrospray ionization source according to the present invention, including waveform (a), frequency (b), negative voltage (c), and duty cycle (d) diagram;

[0022] Figure 4 Soft ionization analysis diagram of LFAC-nESI for insulin (a) and cytochrome c (b) for a preferred embodiment of an online desalting high-sensitivity mass spectrometry analysis method based on a low-frequency alternating current high-voltage nanoliter electrospray ionization source according to the present invention;

[0023] Figure 5 Insulin analysis sensitivity evaluation diagram for a preferred embodiment of an online desalting high-sensitivity mass spectrometry analysis method based on a low-frequency alternating current high-voltage nanoliter electrospray ionization source according to the present invention;

[0024] Figure 6 Insulin salt addition analysis diagram for a preferred embodiment of an online desalting high-sensitivity mass spectrometry analysis method based on a low-frequency alternating current high-voltage nanoliter electrospray ionization source according to the present invention;

[0025] Figure 7 Online analysis of LFAC-nESI for nano-LC for a preferred embodiment of an online desalting high-sensitivity mass spectrometry analysis method based on a low-frequency alternating current high-voltage nanoliter electrospray ionization source according to the present invention. (a, b) Schematic diagram and physical diagram of nano-LC; (c, d) Ion current diagram and mass spectrometry diagram of cytochrome c analysis;

[0026] Figure 8 Analysis effects of nESI and LFAC-nESI on small molecule metabolites for a preferred embodiment of an online desalting high-sensitivity mass spectrometry analysis method based on a low-frequency alternating current high-voltage nanoliter electrospray ionization source according to the present invention. (a-c) Mass spectrometry diagrams of typical deoxynucleoside 2-deoxycytidine, ribonucleoside cytidine, and nucleoside analogue ATP; (d-f) Mass spectrometry diagrams of typical basic amino acid glutamate, neutral amino acid serine, and acidic amino acid histidine; (g-i) Mass spectrometry diagrams of typical lipids phosphatidylcholine PC, phosphatidylethanolamine PE, and cholesterone. N.D. = Not Detected diagram;

[0027] Figure 9Tandem mass spectrometry analysis of cytidine by nESI and LFAC-nESI for a preferred embodiment of an online desalting high-sensitivity mass spectrometry analysis method based on a low-frequency alternating current high-voltage nanoliter electrospray ionization source according to the present invention. (a) Tandem mass spectrometry diagram of cytidine; (b) Schematic diagram of fragmentation of high-abundance fragment ions; (c) Schematic diagram of fragmentation of high-abundance fragment ions of cytidine in tandem mass spectrometry analysis;

[0028] Figure 10 Application analysis diagrams of BSA solution reference material (a) and single onion cell (b) for a preferred embodiment of an online desalting high-sensitivity mass spectrometry analysis method based on a low-frequency alternating current high-voltage nanoliter electrospray ionization source according to the present invention;

[0029] Figure 11 Mechanism exploration diagram for a preferred embodiment of an online desalting high-sensitivity mass spectrometry analysis method based on a low-frequency alternating current high-voltage nanoliter electrospray ionization source according to the present invention;

[0030] Figure 12 Schematic diagram of the device for a preferred embodiment of an online desalting high-sensitivity mass spectrometry analysis method based on a low-frequency alternating current high-voltage nanoliter electrospray ionization source according to the present invention. Detailed implementation manners

[0031] To make the technical solutions of the present invention clearer and more definite to those skilled in the art, the present invention will be further described in detail below in conjunction with embodiments and the accompanying drawings, but the implementation manners of the present invention are not limited thereto.

[0032] The detection effect of the nanospray ionization source based on the low-frequency alternating current high-voltage strategy is verified below through specific embodiments.

[0033] (1) LFAC-nESI can enhance the protein analysis effect

[0034] In this example, insulin was first selected as the research object to demonstrate the promoting effect of LFAC-nESI on protein mass spectrometry analysis. Insulin is a protein hormone secreted by pancreatic islet β cells after stimulation and has the effect of lowering blood sugar. Insulin is similar to most human proteins and has an isoelectric point of 5.6. Therefore, under neutral conditions, insulin molecules are more likely to lose protons and carry negative charges, so the analysis effect is not ideal in the positive mode. A 5 μM aqueous solution of insulin was prepared for analysis. For nESI analysis, a 1.75 kV DC high voltage was applied. For LFAC-nESI analysis, a low-frequency electrical signal of 1.75 kV positive high voltage and -3.5 kV negative high voltage was alternately applied. Mass spectrometry signals were collected to compare the analysis effects of nESI and LFAC-nESI ( Figure 1) In nESI analysis, the unimodal charge state distribution ranges from 3+ to 5+ and is concentrated at 4+. When using the LFAC-nESI experiment, the signal is well improved. The unimodal charge state distribution is from 3+ to 6+ and is concentrated at 4+. This indicates that the protein structure does not unfold during LFAC-nESI analysis and remains in its native state. Magnifying the mass spectrum of 4+ ions shows that in the analysis of nESI, insulin mainly has Na+ adduct peaks, and its content is much higher than that of H+ adduct peaks. The magnified 4+ ion spectrum shows that in the analysis of LFAC-nESI, the protonated signal dominates, the Na+ adduct peak disappears, and the spectrum is greatly simplified, which is more conducive to the identification of compounds. To quantify the improvement in the analysis effect, the signal-to-noise ratio of insulin ions was calculated. The signal-to-noise ratio of the main peak 4+ ions of insulin after ionization by nESI is about 6, and after ionization by LFAC-nESI, the signal-to-noise ratio is about 163, showing a 27-fold increase. For the protonated signal, the signal-to-noise ratio is increased by about 60 times.

[0035] In addition to insulin, LFAC-nESI also has the effect of improving the signal-to-noise ratio and desalting for other proteins such as cytochrome c and myoglobin ( Figure 2 ). Figure 2 a, b), its ionization efficiency is very high under neutral conditions, and the spectrum of nESI is very clean; and the magnified spectrum shows that the H+ addition peak is the main one, but at the same time, there are also serious alkali metal adduct ions. In contrast, the spectrum of LFAC-nESI is also very clean. Looking at the highest magnified 8+ ions, it can be seen that there are almost no alkali metal adduct peaks during the ionization of cytochrome c, and all are protonated signals, and its signal-to-noise ratio is significantly improved. For myoglobin ( Figure 2 c, d), its signal-to-noise ratio is also improved. In the analysis of nESI, the CSD of myoglobin ranges from 7+ to 10+ and is concentrated at 8+. The magnified spectrum of 8+ ions is similar to that of insulin and cytochrome c, and also mainly has sodium ion adduct signals. In the analysis of LFAC-nESI, the CSD of myoglobin ranges from 8+ to 10+ and is concentrated at 9+. The slight shift of the base peak indicates that the structure of myoglobin may unfold during LFAC-nESI analysis, resulting in an increase in the number of charges, but there is no obvious apo-Mb with the loss of the prosthetic group, indicating that the heme is still retained in a non-covalent form.

[0036] (2) Exploration of influencing factors

[0037] The application of low-frequency alternating high voltage in this invention is crucial for the results of mass spectrometry analysis. Therefore, we studied the influence of different electrical parameters on the improvement effect of signal-to-noise ratio. Insulin was still used as the analysis object, and we optimized the waveform, frequency, negative voltage and duty cycle of the ion source in turn. First was the optimization of the waveform, and low-frequency alternating sine wave and square wave were applied respectively. The results showed that for both insulin 4+ ions and 5+ ions, the improvement effect of the signal-to-noise ratio of the low-frequency alternating square wave was better than that of the sine wave( Figure 3 a). Secondly, the frequency of the low-frequency alternating current was optimized, and the results showed that the optimal application frequency was 5 Hz( Figure 3 b). When the frequency increased further, the signal improvement effect gradually decreased. When the frequency was 500 Hz, perhaps because the frequency changed too fast to generate spray, no signal was detected at this time. In the optimization of the negative high voltage, experiments were carried out every 0.5 kV from -1 kV to -5 kV. The results showed that as the negative high voltage gradually increased, the improvement effect of LFAC-nESI was gradually enhanced, and the peak appeared at -4 kV. However, since the higher the negative voltage, the easier it was to have violent discharge, making the spray unstable, so -3.5 kV was selected as the optimal voltage, and subsequent experiments were carried out based on this( Figure 3 c). Finally, the duty cycle of the positive and negative high voltages was optimized, and the optimal duty cycle was 70%, that is, 1.75 kV was applied for 140 ms in the positive mode, and -3.5 kV was applied for 60 ms in the negative mode, and they were cycled in turn( Figure 3 d).

[0038] (3) Soft ionization and high-sensitivity analysis characteristics of LFAC-nESI

[0039] In addition to improving the signal-to-noise ratio and online desalting, we also studied other characteristics of this ion source, including soft ionization characteristics and high-sensitivity analysis characteristics. First, the ionization voltage was reduced to explore the ionization situation of proteins. The positive high voltage gradually increased from 0 V, increasing by 100 V each time, and the starting voltage when protein signals began to be generated was recorded. For insulin, its starting voltage was 1.3 kV in the nESI mode, while LFAC-nESI generated signals at a lower 0.6 kV( Figure 4 a). Under the starting voltage condition, insulin 3+ ions obviously had higher abundances in the LFAC analysis. And the obvious improvement of the signal-to-noise ratio could still be observed. For cytochrome c, its ionization effect was better than that of insulin, and the starting voltage decreased to 0.8 kV in the nESI mode( Figure 4 b). The nESI-LFAC analysis of cytochrome c still had a lower starting voltage and generated signals at 0.6 kV. Similarly, under the starting voltage condition, cytochrome c 7+ ions obviously had higher abundances in the LFAC analysis, which also illustrated the soft ionization ability of LFAC.

[0040] Analysis of samples at low concentrations can effectively evaluate the sensitivity of the method. For an insulin sample with a concentration of 1 μM, the signal-to-noise ratio is only 3 after nESI ionization ( Figure 5 ), and the concentration cannot be further reduced for detection; while after LFAC-nESI ionization, the signal-to-noise ratio is about 95, which is about 32 times higher. Compared with the previous results, LFAC-nESI shows a more significant improvement in performance under low-concentration conditions. When the insulin concentration is reduced by 10 times to 100 nM, the signal-to-noise ratio of LFAC-nESI analysis is about 9; when the concentration is further reduced to 50 nM, the signal-to-noise ratio is about 5; the concentration cannot be further reduced for detection. Therefore, under the LFAC-nESI analysis mode, the detection limit of insulin is about 50 nM.

[0041] Detection of samples in complex matrices can also be used to evaluate analysis sensitivity. We added 1 mM NaCl to an aqueous solution of 5 μM insulin. According to the results of nESI, whether it is different ion adduct states or different charge states of insulin ions, the signals are completely covered by noise and cannot be identified. After applying LFAC-nESI, very obvious insulin signals can be obtained, and the charge states are between 3+ and 5+, and all are protonated signals for the detection object, without Na+ adduct signals, indicating that LFAC-nESI analysis has a very significant desalting effect and can effectively reduce the interference caused by high-concentration metal ions to detection ( Figure 7 ).

[0042] (4) Online continuous analysis characteristics of LFAC-nESI

[0043] Our research group previously reported a polarity-reversed nano-spray ion source. First, turn on the high-voltage power supply to output a voltage of -2.5 kV to -5.0 kV to the metal electrode for 6 s; then output a voltage of +1.5 kV to +2.0 kV to the metal electrode to generate nano-spray. The application of such a polarity-reversed voltage can also improve the signal-to-noise ratio of protonated signals, but the too low voltage frequency and long switching period greatly limit the online analysis of substances, especially not suitable for chromatographic coupling analysis. Here, we coupled LFAC-nESI with nano-LC to achieve online, continuous and highly sensitive analysis of cytochrome c ( Figure 8 ).

[0044] (5) Expansion of the analysis object of LFAC-nESI

[0045] In addition to large molecular protein samples, the technology described in this patent also has the same analytical improvement effect on small analytical metabolites, including desalting and improving sensitivity, which is another feature different from the polarity-reversed nanoelectrospray ionization source. First, nucleosides and their analogs were selected as the analysis objects. 2'-Deoxycytidine, cytidine, and ATP with a concentration of 5 μM were respectively prepared. The results showed that compared with nESI, nucleosides and their analogs could achieve better analytical effects in LFAC-nESI analysis. The signal-to-noise ratios of the proton adduct peaks of 2'-deoxycytidine and cytidine were both increased by about 6 times ( Figure 8 a, b). It is worth noting that ATP could not be detected in nESI analysis, but the signal-to-noise ratio reached 38 in LFAC-nESI analysis, and the improvement effect was very significant ( Figure 8 c). For 2'-deoxycytidine and cytidine, [M + H] + and [M + Na] + peaks would be simultaneously formed in nESI analysis, and [M + Na] + would dominate, with an intensity about 2 times that of [M + H] + ; however, in LFAC-nESI analysis, there was no [M + Na] + peak, and the [M + H] + peak was greatly enhanced, making the spectrum cleaner. Further, we compared and analyzed the performance of nESI and LFAC-nESI in tandem mass spectrometry analysis. The spectrum obtained by cytidine in the LFAC-nESI analysis mode was cleaner, without interference from miscellaneous peaks, and the fragments were easy to identify, which was helpful for the identification and characterization of metabolites ( Figure 9 ).

[0046] Amino acids are important amphoteric metabolites, containing both acidic carboxyl functional groups and basic amino functional groups. The tendency of each amino acid to dissociate into cations and anions is different, and it has different isoelectric points. Typical basic amino acids, neutral amino acids, and acidic amino acids were respectively selected for experiments. The isoelectric point of glutamic acid is 3.22, which is a typical acidic amino acid. Its solution is acidic in pure water, so its analytical effect in nESI is the worst, and a concentration of 5 μM cannot be detected. In LFAC-nESI analysis, glutamic acid easily forms proton adducts, and the detection effect is good, with a signal-to-noise ratio of 20 ( Figure 8 d). In addition, methionine (isoelectric point 5.74), a neutral amino acid, and histidine (isoelectric point 7.59), a basic amino acid, with a concentration of 5 μM were also prepared. The signal-to-noise ratios were increased by 5 times and 4 times respectively ( Figure 8 e, f), and the signals of metal adduct ions were significantly reduced.

[0047] Finally, we selected lipid compounds that are abundant and diverse in cells for research. Similar to nucleosides and amino acids, lipid substances can also significantly inhibit the [M+Na] peak and enhance the [M+H] peak after LFAC-nESI analysis. Taking phosphatidylcholine PC 30:0 as an example, compared with nESI analysis, the signal-to-noise ratio of the proton adduct peak is increased by 10 times in LFAC-nESI analysis, while the metal ion adduct peak is hardly detected ( Figure 8 g). Another lipid, phosphatidylethanolamine, was detected. Its polarity is slightly weaker than that of ethanolamine, and the signal-to-noise ratio is also increased by 10 times ( Figure 8 h). Cholestanone has a weaker polarity, and the signal-to-noise ratio is doubled during LFAC-nESI analysis ( Figure 8 i). The above content shows that LFAC-nESI can improve the analysis effect of various metabolites and has broad application potential. + peak, and enhance the [M+H] + peak. Taking phosphatidylcholine PC 30:0 as an example, compared with nESI analysis, the signal-to-noise ratio of the proton adduct peak is increased by 10 times in LFAC-nESI analysis, while the metal ion adduct peak is hardly detected ([ Figure 8 Figure 8 g). Another lipid, phosphatidylethanolamine, was detected. Its polarity is slightly weaker than that of ethanolamine, and the signal-to-noise ratio is also increased by 10 times ([ Figure 8 Figure 8 h). Cholestanone has a weaker polarity, and the signal-to-noise ratio is doubled during LFAC-nESI analysis ([ Figure 8 Figure 8 i). The above content shows that LFAC-nESI can improve the analysis effect of various metabolites and has broad application potential.

[0048] (6) Application demonstration

[0049] Protein reference materials are standard reference substances used for protein-related detections. They are important references for protein metrology research and standardization organization research and have wide applications in multiple fields. Bovine serum albumin solution reference material is mainly used for the value traceability, quality control of protein content measurement results in food analysis, drug analysis, and clinical tests, as well as the calibration of analytical instruments such as nucleic acid protein analyzers and the confirmation and evaluation of related methods. We purchased a bovine serum albumin solution reference material stored in an aqueous solution of 0.02% sodium azide and 0.02 mol / L NaCl. To ensure protein stability, conformational integrity, and correct protein multimer assembly and maintain its stability and activity, individual proteins, protein mixtures, or non-covalent protein complexes are usually stored in non-volatile salt buffers and other solubilizers such as serum, PBS buffer, glycerol, etc. Generally speaking, the presence of the matrix will seriously interfere with mass spectrometry analysis, resulting in various negative effects such as ion suppression and space charge effects. Therefore, the steps of desalting and replacing the buffer are very necessary, and liquid chromatography-mass spectrometry is required for further analysis, with many operation steps and a long analysis time. Due to the excellent desalting effect of LFAC-nESI, we directly detected the bovine serum albumin solution reference material using LFAC-nESI and compared it with direct nano-spray analysis ([ Figure 11 Figure 11a). No obvious signal could be obtained in the direct nano-spray mass spectrum of BSA, while a very obvious BSA signal was obtained in the LFAC-nESI mass spectrum, with its CSD ranging from 35+ to 55+, and concentrated at 46+. The direct analysis of the protein solution reference material containing matrix saves a large amount of manpower and material resources and has significant environmental friendliness. The AGREE method was used to evaluate the environmental friendliness of the constructed analytical method, and comprehensive, flexible and direct information was carried out from 12 aspects such as the quantity, toxicity, waste generated, and pretreatment steps of the required reagents. After calculation, the total score was 0.58. The total score is shown in the middle of the pie chart. The closer the value is to 1, the higher the environmental friendliness, and the greener the color. The AGREE score of the LFAC-nESI method was 0.76 after evaluation, showing significant advantages in automation and analysis throughput.

[0050] Next, we also used this ion source to analyze single-cell complex samples. LFAC-nESI was used for the analysis of single purple onion cells. The outer epidermis of the purple onion was torn off and placed under a microscope, and sampling was carried out with a pulled nano-tip. After sampling, methanol / water solution was used as an auxiliary solvent to generate a spray, and high-resolution mass spectrometry was used to analyze unknown metabolites in single cells. The nESI analysis results of the single onion cell extract showed multiple glycan signals, with the degree of polymerization covering 1-9, mainly all detected as [M+K] + peaks, and most were accompanied by [M+Na] + signals ( Figure 11 b). Multiple glycan signals were also detected in the LFAC-nESI signal of the single onion cell extract, and [M+NH4] + peaks were detected. In addition, the LFAC-nESI analysis also showed several new peaks with high intensities, which were identified as onion flavor compounds (γ-L-glutamyl-S-(2-carboxy-1-propyl)cysteine glycine, m / z 394; cyanamide 3-O-(6-O-malonyl)-β-D-glucoside, m / z 535; and flavonol alkaloid-3O-cyclohexenyl-O-malonyl hexanol, m / z 697) after confirmation by high-resolution mass spectrometry and tandem mass spectrometry

[0051] (7) Mechanism exploration

[0052] During the nESI positive mode analysis process, all positively charged ions are forced to migrate towards the tip of the nano-tip, and the same is true for the negative mode. We speculate that during the LFAC-nESI analysis process, the different migration speeds of the charged ions at the tip of the nano-tip generate microelectrophoresis, enabling the analytes to be separated from impurities such as metal ions Na +Separation. During the negative half-cycle of the alternating current, the positively charged substance is driven away from the tip by the electric field; during the positive half-cycle, the analyte is ionized and analyzed by mass spectrometry, thereby enhancing the signal of the analyte ion proton adduct while weakening the signal of the metal ion adduct. Therefore, after the LFAC-nESI analysis, a large amount of metal ions accumulate at the nano-tip. We collected the composition of the substance at the nano-tip after LFAC-nESI using nESI and found a large number of Na+ adduct peaks, with a greater degree of adduct than in the case of the original nESI, thus proving the existence of tip microelectrophoresis.

[0053] Regarding the tendency of sugar molecules to adduct ammonium ions in single-cell metabolite analysis, we speculate that this may be due to the promotion of the conversion and absorption of ammonia in the air by the high-voltage electric flip. Our group previously reported a polarity-reversed nanoelectrospray (PR-nESI) ion source, which can achieve the in-situ synthesis of NH 3 at the plasma-solution interface using negative high-voltage corona discharge and analyze and identify the products using positive-mode electrospray. Here we also use maltose for verification. The direct nanoelectrospray analysis of maltose shows a peak at m / z 365, representing the sodium adduct ion of maltose. When maltose is detected by LFAC-nESI, the signal at m / z 365 disappears, and the peak at m / z 360 dominates, which is consistent with the literature report ( Figure 11 c), confirming the existence of this mechanism.

[0054] As described above, it is only a further embodiment of the present invention, but the protection scope of the present invention is not limited thereto. Any person skilled in the art within the scope disclosed by the present invention, according to the technical solution and its concept of the present invention, makes equivalent substitutions or changes, all belong to the protection scope of the present invention.

Claims

1. A method for using an electrospray mass spectrometry ion source based on low-frequency alternating voltage, characterized in that: The steps include: Step 1: Load the sample solution by using a nano-electrospray needle; Step 2: Insert the metal electrode into the nano-electrospray needle to make it in direct contact with the sample solution; Step 3: Use a signal generator to generate an AC signal with a specific waveform, frequency, peak-to-peak value and duty cycle. After being amplified by a high-voltage amplifier, the signal directly acts on the nanoliter electrospray needle to generate electrospray; Step 4: The amplified signal is monitored in real time by an oscilloscope.

2. The method for using an electrospray mass spectrometry ion source based on a low-frequency alternating voltage according to claim 1, characterized in that: In step three, the signal input terminal is connected to the signal generator, and the output terminal is connected to the metal electrode.

3. The method for using an electrospray mass spectrometry ion source based on low-frequency alternating voltage according to claim 2, characterized in that: In step three, the application of high voltage is changed from DC high voltage to low frequency AC high voltage.

4. The method for using an electrospray mass spectrometry ion source based on low-frequency alternating voltage according to claim 3, characterized in that: Implement a low frequency AC nanoelectrospray ion source for detection of biological samples.

5. A method for using an electrospray mass spectrometry ion source based on low-frequency alternating voltage, characterized in that: including a signal generator coupled to a high voltage amplifier; The high voltage amplifier is coupled to an oscilloscope and the mass spectrometer is composed of an electrospray spray spray sprayed into the sample tube through a high voltage input.

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