CN109243965B

CN109243965B - Protein high-charge ion generation method based on polarity-reversal nano-spray ion source

Info

Publication number: CN109243965B
Application number: CN201810872089.9A
Authority: CN
Inventors: 龚晓云; 方向; 李畅; 戴新华; 江游
Original assignee: National Institute of Metrology
Current assignee: National Institute of Metrology
Priority date: 2018-08-02
Filing date: 2018-08-02
Publication date: 2020-08-11
Anticipated expiration: 2038-08-02
Also published as: CN109243965A

Abstract

The invention discloses a method for generating protein high-charge ions based on a polarity-reversal nano-spray ion source, which comprises the following steps: s1, adding 1-10 mM high-charge reagent into the sample solution; s2, injecting a sample solution from the tail end of the nano-spray needle; s3, inserting the metal electrode into the nano-spray needle from the tail end of the nano-spray needle until the metal electrode contacts with the sample solution; s4, sealing the tail end of the spray needle with an insulating end cover and connecting a high-voltage power supply and a metal electrode; s5, turning on a high-voltage power supply, outputting a voltage of-2.5 kV to-5.0 kV to the metal electrode, and continuously outputting for 3S to 12S; then, the metal electrode is output with a voltage of +1.5 kV- +2.0kV for generating nano-spray. The method comprises the steps of firstly adding a high-charge reagent with a specific concentration into a sample solution, then pretreating the sample solution by using a reverse-phase negative high voltage, and then generating nano-spray by using a normal-phase high voltage. Compared with the protein high-charge ion generation method based on common nano-spray, the method has higher practicability and more convenient use.

Description

Protein high-charge ion generation method based on polarity-reversal nano-spray ion source

Technical Field

The invention relates to a method for generating high-charge protein ions, in particular to a method for generating high-charge protein ions based on a polar reversal nano-spray ion source.

Background

Biomacromolecules such as proteins form ions with multiple charges after electrospray. The number of charges carried by the molecule will vary depending on the electrospray conditions. When the number of charges carried by the molecule is small, it is called a low-charge ion; when the number of charges carried by the molecule is large, the molecule is converted into a highly charged ion. The formation of highly charged ions provides a great deal of convenience for the detection of biological macromolecules such as proteins:

1. the formation of highly charged ions greatly expands the range of protein molecular weights that can be detected by mass spectrometry. This is because the mass-to-charge ratio (m/z) of the ions is detected by the mass spectrometer detector, and after the protein molecules are charged more, the m/z of the protein molecules is reduced significantly, so that the m/z of the protein molecules falls within the mass-to-charge ratio range which can be detected by the ordinary mass spectrometer. At present, except Time of flight mass spectrometry (TOF-MS), most commercial mass spectrometry can detect mass-to-charge ratio in the range of about 50 to 3000. Whereas the molecular weight of a protein molecule is usually several tens kDa, even several hundreds kDa. When the protein molecules have more charges, the m/z of most common proteins falls within the range of 1000-3000, and can be easily detected by mass spectrometry.

2. The high-charge protein ions can obtain higher sensitivity and resolution when being detected by mass spectrometry. The sensitivity and the resolution of the mass spectrum are obviously reduced along with the improvement of m/z, and obviously better detection effect can be obtained when the detection is carried out at the low-mass end of m/z. This phenomenon is more pronounced with some mass spectrometry mass analyzers that are sensitive to ion charge, such as the Orbitrap mass analyzer (Orbitrap) and the Fourier transform ion cyclotron resonance mass analyzer (FTICR).

3. Highly charged protein ions do not readily form metal ion adducts. Protein molecules easily form metal ion adducts with trace metal cations in a solution, so that a spectrum peak is widened, the sensitivity is reduced, and the identification of the spectrum peak is seriously influenced. In contrast, the highly charged ions of the protein are less prone to form metal ion adducts than the less charged ions, which is more advantageous for the detection of the protein.

4. When multi-stage fragmentation is carried out on the high-charge protein ions, the fragmentation efficiency is higher, more fragment ions are generated, and the method is more favorable for structural identification of protein molecules. This phenomenon is more pronounced when Electron capture fragmentation (ECD) and Electron transfer fragmentation (ETD) are performed.

Various methods have been developed to generate highly charged ions of proteins. Most commonly, denaturing agents such as acids and bases are added to the solution to change the pH of the solution, thereby denaturing the protein molecules and destroying the structure, thereby generating highly charged ions. The addition of an organic solvent to the solution can be used as an auxiliary means to further disrupt the structure of the protein molecules and improve the efficiency of the generation of highly charged ions of the protein. Reagents such as acids and bases may be added directly to the solution or may be added in the gas phase. Acid and alkali are added into a dry gas of an electrospray ion source, and in the solvent removing process of electrospray, acid and alkali steam and charged liquid drops formed by electrospray interact with each other, so that the pH value of the liquid drops is changed, the molecular structure of protein is damaged, and high-charge protein ions are generated. Most of the common acids, alkalis and organic solvents have strong volatility and toxicity, so that the use of the common acids, alkalis and organic solvents inevitably brings some personal safety concerns and operational inconvenience, and the common acids, alkalis and organic solvents need to be used under the condition of a fume hood and related protective measures.

In recent years, some reagents with high charge besides acid and base have been discovered, and these reagents are organic solvents, such as m-NBA, sulfolane, Dimethyl sulfoxide (DMSO), etc. To the solution is added a proportion of these organic solvents, generally 1% to 15% (v/or-_v) When the sample is ionized, highly charged ions of the protein can be generated. The use of these organic solvents also poses certain safety concerns.

In addition, an electrothermal high-charge ion generation method has been proposed. The method entails adding about 100mM ammonium bicarbonate (NH) to the solution₄HCO₃). When electrospray is performed using a lower voltage, low charge protein ions are produced, and when electrospray is performed using a higher voltage, high charge protein ions are produced. This approach avoids the use of many volatile toxic organic reagents. But high concentration of NH₄HCO₃The use of (2) has a large influence on the signal of the target protein, and the detection sensitivity is significantly reduced.

Disclosure of Invention

In view of the above-mentioned shortcomings and drawbacks of the prior art, the present invention provides a method for generating high-charge protein ions based on a polar reversed sodium salt spray ion source, which is convenient, practical, low-toxic and low-harmful, and an operation method thereof.

The purpose of the invention is realized by the following technical scheme:

a protein high-charge ion generation method based on a polarity-reversal nanospray ion source comprises the following steps:

s1, adding 1-10 mM high-charge reagent into the sample solution;

s2, injecting a sample solution from the tail end of the nano-spray needle;

s3, inserting the metal electrode into the nano-spray needle from the tail end of the nano-spray needle until the metal electrode contacts with the sample solution;

s4, sealing the tail end of the spray needle with an insulating end cover and connecting a high-voltage power supply and a metal electrode;

s5, turning on a high-voltage power supply, outputting a voltage of-2.5 kV to-5.0 kV to the metal electrode, and continuously outputting for 3S to 12S; then, the metal electrode is output with a voltage of +1.5 kV- +2.0kV for generating nano-spray.

Preferably, in step S5, after the high voltage power supply is turned on, a voltage of-2.5 kV to-5.0 kV is output to the metal electrode for 3S to 12S; then, the metal electrode is output with a voltage of +1.5 kV- +2.0 kV.

Preferably, the 1-10 mM high-charge reagent is a strong acid salt or a medium-strong acid salt.

Preferably, the strong acid salt is bromide, chloride, iodide or nitrate, and the medium strong acid salt is phosphate.

Compared with the prior art, the embodiment of the invention at least has the following advantages:

the method comprises the steps of firstly adding a high-charge reagent with a specific concentration into a sample solution, then pretreating the sample solution by using a reverse-phase negative high voltage, and then generating nano-spray by using a normal-phase high voltage. Compared with a protein high-charge ion generation method based on common Nano-spray (Nano-ESI), the method is more practical and more convenient to use, and specifically comprises the following steps:

firstly, the invention uses common inorganic salt or organic salt (such as sodium chloride (NaCl), potassium chloride (KCl) and the like) as high-charge reagents which are very common and easy to obtain, have extremely low toxicity, even are nontoxic and are more convenient to use. In the method based on the common nano-spray, reagents such as strong acid and strong base or special organic reagents such as m-Nitrobenzyl alcohol (m-NBA) and sulfolane are generally used as high-charge reagents, which are not common in common laboratories, have high toxicity and volatility, require special facilities such as a fume hood during operation, and are harmful to human bodies.

Second, the present invention requires low concentrations of highly charged reagents. The salt concentration in the solution is 1-10 mM (10)^-3mol/L) grade. Whereas the concentration of highly charged reagents required for the conventional nanospray-based process is significantly higher. When m-nitrobenzyl alcohol or sulfolane are used, concentrations of the two reagents of 1% to 5% (v/v) are required, almost reaching their solubility limits.

Again, the present invention has better adaptability to biological samples. Many biological samples dissolve in phosphate and chloride buffers during pretreatment. The presence of these salts can have a profound effect on the signal of a conventional nanospray. Therefore, the conventional nano-spray based method requires a desalting step before detection. These additional operations can result in sample loss and contamination. The invention is built on a polarity-reversal nano-spray ion source and has extremely strong tolerance to a salt solution. Furthermore, the buffer salts commonly used in biological samples are just highly charged reagents useful in the present invention. Thus, the present invention allows for the direct analysis of biological samples.

Drawings

FIG. 1a is a graph showing the results of an ionization effect test on cytochrome c (Cytc, from horse heart, purchased from Sigma Aldrich) using a common nanospray with 1% (v/v) acetic acid (HAc) as a highly charged reagent;

FIG. 1b is a graph showing the results of cytochrome c detection using a polar reversed nanospray method with 5mM NaCl as the high charge reagent;

FIG. 1c is a graph showing the results of myoglobin (Mb, equine heart origin, Sigma Aldrich) detection using a conventional nanospray with 1% (v/v) acetic acid (HAc) as the high charge reagent;

FIG. 1d is a graph showing the results of a detection using a polar reversed nanospray method with 5mM NaCl as the high charge reagent;

FIG. 1e is a graph showing the results of the detection of calmodulin (CaM, from bovine heart, purchased from Sigma Aldrich) using a conventional nanospray, with 1% (v/v) acetic acid (HAc) as the highly charged reagent;

FIG. 1f is a graph showing the results of a detection using a polar reversed nanospray method with 5mM NaCl as the high charge reagent;

FIG. 1g is a graph showing the results of lysozyme (Lys, derived from ovalbumin, purchased from Sigma Aldrich) using a conventional nanospray with 1% (v/v) acetic acid (HAc) as a high charge reagent;

FIG. 1h is a graph showing the results of a polarity-reversed nanospray assay with 5mM NaCl as the high-charge reagent;

FIG. 2a shows a method of spraying with polar reversed sodium at 5mM NH₄The detection result of the Cl as the high-charge reagent on the cytochrome c is shown in the figure;

FIG. 2b is a graph showing the results of cytochrome c detection using polar-reversed nanospray with 5mM KCl as the high charge reagent;

FIG. 3 is a graph showing the results of cytochrome c detection using a polar reversed nanospray method with 5mM LiCl as the high charge reagent;

FIG. 4a is a process of spraying with polar reversed sodium, with 5mM NaH₂PO₄A schematic diagram of the result of detection of cytochrome c as a highly charged reagent;

FIG. 4b shows a method of spraying with polar reversed sodium, 5mM Na₂HPO₄A schematic diagram of the result of detection of cytochrome c as a highly charged reagent;

FIG. 4c is a graph showing the results of cytochrome c detection using polar-reversed nanospray with 2mM NaI as the highly charged reagent;

FIG. 4d shows a method of spraying with polar reversed nanospray with 5mM NaNO₃A schematic diagram of the result of detection of cytochrome c as a highly charged reagent;

FIG. 5a is a process of spraying with polar reversed sodium NaHCO at 5mM₃A schematic diagram of the detection result of cytochrome c as an additive reagent;

FIG. 5b shows a method of spraying with polar reversed sodium at 5mM NH₄HCO₃As additivesThe detection result of the added reagent on the cytochrome c is shown in a schematic diagram;

FIG. 5c is a graph showing the results of cytochrome c detection using a polar reversed nano-spray method with 5mM NaHS as the reagent added;

FIG. 5d shows a method of spraying with polar reversed sodium at 5mM (NH)₄)₂S is used as a detection result schematic diagram of the added reagent on the cytochrome c;

FIG. 5e is a graph showing the results of cytochrome c detection using a polar reversed nanospray method with 5mM NaAc as the additive reagent;

FIG. 5f shows a polar reversed nanospray method with 5mM NH₄A schematic diagram of the detection result of the cytochrome c by using Ac as an added reagent;

FIG. 6 shows a method of spraying with polar reversed nano-particles at 5mM (NH)₄)₂CO₃A schematic diagram of the detection result of cytochrome c as an additive reagent;

FIG. 7 shows a method of spraying with reversed polarity nanospray, with 5mM ammonium formate (HCOONH)₄) A schematic diagram of the detection result of cytochrome c as an additive reagent;

FIG. 8a is a schematic diagram of the negative high voltage step in the formation of highly charged ions of basic protein molecules;

FIG. 8b is a schematic diagram of the positive high pressure step in the formation of highly charged ions of basic protein molecules;

FIG. 9a is a schematic diagram of the negative high pressure step in the formation of highly charged ions of acidic protein molecules;

FIG. 9b is a schematic diagram of the positive high pressure step in the formation of highly charged ions of acidic protein molecules;

FIG. 10a is a spectrum obtained with KCl;

FIG. 10b is NH₄A spectrum obtained by Cl;

FIG. 10c is NaH₂PO₄Obtaining a spectrogram;

FIG. 10d is Na₂HPO₄Obtaining a spectrogram;

FIG. 10e is a NaI spectrum;

FIG. 10f is a spectrum obtained with LiI;

FIG. 11 is a schematic diagram of the structure of an ion source for the protein high-charge ion generation method based on a polar-reversed nanospray ion source according to the present invention;

FIG. 12a is a schematic view of the nano-spray needle and its internal sample solution and the distribution of various ions in FIG. 8 a;

FIG. 12b is a schematic view of the nanospray nozzle needle and its internal sample solution and the distribution of various ions in FIG. 8 b;

FIG. 13a is a schematic view of the nano-spray needle of FIG. 9a and its internal sample solution and the distribution of various ions;

FIG. 13b is a schematic view of the nano-spray needle and its internal sample solution and the distribution of various ions in FIG. 9 b.

Detailed Description

The detection effect of the nanospray ion source based on the polarity inversion voltage strategy is verified by the specific embodiment.

A nanospray ion source based on a polarity inversion voltage strategy comprises

A nano-spray needle for loading a sample solution; the material of the nano-spray needle is not limited, and the nano-spray needle is generally glass or quartz; the inner diameter of the tip is consistent with that required by common nano-spray and is 1-10 mu m. The size of the Nano-tip tail is not limited.

The metal electrode is inserted into the nano-spray nozzle needle and is directly contacted with the sample solution; the electrode material is not limited, but should be chemically inert and not prone to corrosion and dissolution caused by chemical reaction with the sample solution. Typically platinum wire.

The insulating end cover is plugged at the tail end of the spray needle to prevent electric leakage;

the high-voltage power supply has a positive and negative bidirectional output function and is connected with the metal electrode, and the wire connected with the metal electrode penetrates through the insulating end cover.

The positive output voltage range of the high-voltage power supply is 0 to +3kV, and the reverse output voltage range is 0 to-5 kV.

The method for generating the protein high-charge ions comprises the following steps:

firstly, 1-10 mM of high-charge reagent, namely salt, is added into a sample solution. The kind of salt is required to be certainSalts of strong acids, such as chloride, bromide, iodide, nitrate, and the like, or salts of strong acids, such as phosphate. Salts of weak acids are not capable of becoming highly charged agents such as sulfides, carbonates, formates and acetates. The type of cation of the salt has no significant effect on the effectiveness of the highly charged agent. Li⁺、Na⁺、K⁺And NH₄ ⁺And the like, can be used as the high charge agent.

The polar reversed nanospray method was then performed to perform the detection of the protein sample. The mass spectrogram obtained at this time is the spectrogram of protein with high charge.

(1) NaCl for the production of highly charged ions of different proteins

The high-charge ion generation method established by the invention has universality on all protein samples. Here, we selected 4 most common protein samples as the subjects to be tested for method feasibility verification. The most common salt, namely NaCl, is used as a high-charge reagent, and the protein is detected by using the method and compared with the detection result of common nano-spray.

FIG. 1a shows the ionization effect on cytochrome c (Cytc, from horse heart, purchased from Sigma Aldrich) using a normal nanospray with 1% (v/v) acetic acid (HAc) as the highly charged reagent. Two clusters of peaks can be seen in the figure, namely a cluster of low charge peaks and a cluster of high charge peaks. Wherein, the low charge peak cluster takes a spectrum peak 9+ peak with 9 charges as a main peak and has obviously higher signal intensity. And the high-charge peak cluster takes a spectrum peak 17+ peak with 17 charges as a main peak and has obviously lower signal intensity. Weighted average charge number q_avThe method is used for calculating the average charge number of the target protein molecule and can be used for comparison among different methods. q. q.s_avIs defined as shown in formula 1. Wherein q is_iIs the number of charges corresponding to the ith peak, W_iIs the signal intensity of the ith peak and N is the total number of peaks. Under the detection conditions of the nano-spray, the average charge number q of cytochrome c_av＝10.6+。

FIG. 1b shows the results of cytochrome c detection using a polar reversed nanospray method with 5mM NaCl as the high charge reagent. The significant difference from the detection result of the common nano-spray is that in the method claimed by the invention, only the high-charge peak cluster exists, no low-charge peak cluster exists, and the main peak of the high-charge peak cluster is an 18+ peak. From the point of view of the kind of the spectrum peak and the signal intensity, the method using polarity-reversed nano-spraying produces cytochrome c ions with an overall charge number significantly higher than that of the ordinary nano-spraying method. Average charge number q of cytochrome c_av16.7+ is a significant improvement over the results of conventional nanospray.

FIG. 1c shows the results of myoglobin (Mb, from horse heart, purchased from Sigma Aldrich) detection using a common nanospray with 1% (v/v) acetic acid (HAc) as the high charge reagent. From the figure, a high charge peak cluster can be seen, with the 18+ peak as the main peak. Average number of charges q of calmodulin_av18.5 +. FIG. 1d shows the results of the measurement using a polar reversed nanospray with 5mM NaCl as the highly charged reagent. From the figure, a cluster of high charge peaks is seen, with the 23+ peak as the main peak. The average charge number of the myoglobin is obviously improved, q_av＝21.8+。

FIG. 1e shows the results of the detection of calmodulin (CaM, from bovine heart, purchased from Sigma Aldrich) using a conventional nanospray with 1% (v/v) acetic acid (HAc) as the highly charged reagent. From the figure, a high charge peak cluster can be seen, with the 18+ peak as the main peak. Average number of charges q of myoglobin_av18.6 +. FIG. 1f shows the results of a measurement using a polar reversed nanospray with 5mM NaCl as the highly charged reagent. From the figure, a high charge peak cluster is seen, with the 20+ peak as the main peak. The average charge number of calmodulin is obviously increased, q_av＝20.2+。

FIG. 1g shows the results of lysozyme (Lys, derived from egg albumen, purchased from Sigma Aldrich) using 1% (v/v) acetic acid (HAc) as a high charge reagent using a common nanospray. From the figure, a cluster of high charge peaks is seen, with the 10+ peak as the main peak. Average charge number q of lysozyme_av＝9.8+。FIG. 1h shows the results of the measurement with a polar reversed nanospray using 5mM NaCl as the highly charged reagent. From the figure, a cluster of high charge peaks is seen, with the 11+ peak as the main peak. The average charge number of the lysozyme is obviously improved, q_av＝11.2+。

From the above results, it can be seen that the method can obtain highly charged ions of different kinds of proteins by using NaCl as a highly charged reagent, and the average charge number of the protein ions is significantly higher than that of the common nano-spray method.

(2) Use of other chloride salts for the production of highly charged ions of cytochrome c

As mentioned above, the polar reversal nano-spraying method established by the invention has certain requirements on the type of salt, needs strong acid or medium strong acid salt, and the cation of the salt has little influence on the detection effect. Here, K is treated by changing the kind of cation in addition to NaCl⁺、NH₄ ⁺And Li⁺For examination, the method was used for the generation of highly charged cytochrome c ions.

FIG. 2a shows the result of cytochrome c detection using a polar reversed nano-spray method with 5mM NH4Cl as the highly charged reagent. From the figure, a high charge peak cluster can be seen, with the 18+ peak as the main peak. Average charge number q of cytochrome c_av＝16.6+。

FIG. 2b shows the result of cytochrome c detection using polar reversed nanospray with 5mM KCl as the high charge reagent. From the figure, a cluster of high charge peaks is seen, with the 17+ peak as the main peak. Average charge number q of cytochrome c_av＝16.2+。

FIG. 3 shows the result of cytochrome c detection using a polar reversed nano-spray method using 5mM LiCl as a high-charge reagent. From the figure, a cluster of high charge peaks is seen, with the 17+ peak as the main peak. Average charge number q of cytochrome c_av＝16.2+。

From the above results, it can be seen that the method using polar reversed nanospray with different chloride salts as the highly charged agent can produce the practical effect of highly charged cytochrome c ions. Plus NaCl as previously investigated, the chloride salts we investigated include: LiCl, NaCl, KCl and NH₄Cl, these chloride salts all produce highly charged ions of cytochrome c and are not very different in effect. It can be seen that the change in the cation of the salt has little effect on the generation of highly charged ions.

(3) Other strong or medium-strong acid sodium salts for the generation of highly charged ions of cytochrome c

Salts of strong or medium strong acids can be used to generate highly charged ions of proteins. Here, on the basis of NaCl, the anions were changed, respectively for I^-、NO₃ ^-、H₂PO₄ ^-And HPO₄ ^2-A study was made.

FIG. 4a is a process of spraying with polar reversed sodium, with 5mM NaH₂PO₄As a result of detection of cytochrome c by the highly charged reagent. From the figure, a cluster of high charge peaks is seen, with the 17+ peak as the main peak. Average charge number q of cytochrome c_av＝16.9+。

FIG. 4b shows a method of spraying with polar reversed sodium, 5mM Na₂HPO₄As a result of detection of cytochrome c by the highly charged reagent. From the figure, a cluster of high charge peaks is seen, with the 17+ peak as the main peak. Average charge number q of cytochrome c_av＝16.7+。

FIG. 4c shows the result of cytochrome c detection using polar reversed nanospray with 2mM NaI as the highly charged reagent. From the figure, a high charge peak cluster can be seen, with the 18+ peak as the main peak. Average charge number q of cytochrome c_av＝17.2+。

FIG. 4d shows a method of spraying with polar reversed nanospray with 5mM NaNO₃As a result of detection of cytochrome c by the highly charged reagent. From the figure, a high charge peak cluster can be seen, with the 18+ peak as the main peak. Average charge number q of cytochrome c_av＝15.3+。

From the above results, it can be seen that the method using polar reversed nanospray with different strong or medium strong acid sodium salts as the high charge agent can produce the practical effect of cytochrome c high charge ions. Plus NaCl as previously investigated, the sodium salts we investigated include: NaCl, NaI, NaNO₃、NaH₂PO₄And Na₂HPO₄These sodium salts all produce highly charged ions of cytochrome c and are not very different. It can be seen that when the anion of the salt is a strong or medium acid radical, a highly charged ion of cytochrome c is produced.

(4) Salts incapable of producing highly charged ions of cytochrome c

Salts of weak acids do not produce highly charged ions of proteins. Here, sulfide, carbonate, acetate, and formate were examined and examined.

FIG. 5a is a process of spraying with polar reversed sodium NaHCO at 5mM₃As a result of detection of cytochrome c by the added reagent. As can be seen from the figure, no high charge peak cluster is generated, only one low charge peak cluster is generated, and the 9+ peak is taken as the main peak. Average charge number q of cytochrome c_av＝9.6+。

FIG. 5b shows a method of spraying with polar reversed sodium at 5mM NH₄HCO₃As a result of detection of cytochrome c by the added reagent. As can be seen from the figure, no high charge peak cluster is generated, only one low charge peak cluster is generated, and the 10+ peak is taken as the main peak. Average charge number q of cytochrome c_av＝10.1+。

FIG. 5c shows the result of cytochrome c detection using a polar reversed nano-spray method using 5mM NaHS as an additive reagent. As can be seen from the figure, no high charge peak cluster is generated, only one low charge peak cluster is generated, and the 9+ peak is taken as the main peak. Average charge number q of cytochrome c_av＝9.0+。

FIG. 5d shows a method of spraying with polar reversed sodium at 5mM (NH)₄)₂S is used as the result of detection of cytochrome c by the added reagent. As can be seen from the figure, no high charge peak cluster is generated, only one low charge peak cluster is generated, and the 8+ peak is taken as the main peak. Average charge number q of cytochrome c_av＝8.0+。

FIG. 5e shows the result of cytochrome c detection using a polar reversed nano-spray method using 5mM NaAc as an additive reagent. As can be seen from the figure, no high charge peak cluster is generated, only one low charge peak cluster is generated, and the 8+ peak is taken as the main peak. Average charge number q of cytochrome c_av＝8.0+。

FIG. 5f shows a polar reversed nanospray method with 5mM NH₄Ac was used as a detection result of cytochrome c by the reagent added. As can be seen from the figure, no high charge peak cluster is generated, only one low charge peak cluster is generated, and the 8+ peak is taken as the main peak. Average charge number q of cytochrome c_av＝7.8+。

FIG. 6 shows a method of spraying with polar reversed nano-particles at 5mM (NH)₄)₂CO₃As a result of detection of cytochrome c by the added reagent. As can be seen from the figure, no high charge peak cluster is generated, only one low charge peak cluster is generated, and the 9+ peak is taken as the main peak. Average charge number q of cytochrome c_av＝9.6+。

FIG. 7 shows a method of spraying with reversed polarity nanospray, with 5mM ammonium formate (HCOONH)₄) As a result of detection of cytochrome c by the added reagent. As can be seen from the figure, no high charge peak cluster is generated, only one low charge peak cluster is generated, and the 8+ peak is taken as the main peak. Average charge number q of cytochrome c_av＝8.0+。

From the above results, it can be seen that when a salt of a weak acid is used as an additive, highly charged ions of cytochrome c cannot be generated. The weak acid salts investigated included three carbonates (NaHCO)₃、NH₄HCO₃And (NH)₄)₂CO₃) Two sulfides (NaHS and (NH)₄)₂S), two acetates (NaAc and NH)₄Ac), and a formate salt (HCOONH)₄) None of these weak acid salts can generate highly charged ions of cytochrome c. From this, it can be seen that when the anion of the salt is an acid group of a weak acid, a highly charged ion of cytochrome c cannot be generated.

(5) Mechanism discussion

The invention utilizes the interaction of the anions of the salt and the protein molecules, and the interaction of the anions in the solution and the functional groups on the surfaces of the protein molecules changes the structure of the protein molecules, causes the damage of the structure of the protein molecules, and releases the functional groups originally wrapped in the protein molecules. Thus, the protein molecules can be charged more, forming highly charged ions.

Basic protein molecules have a positive charge in neutral solution, while acidic protein molecules have a negative charge in solution. Their mechanisms of formation of highly charged ions are slightly different.

The process of formation of highly charged ions of basic protein molecules is first discussed. The entire process of the polar inversion nanospray process is divided into two steps, namely a negative high pressure step and a positive high pressure step, as shown in fig. 8. First, a negative high pressure is applied to the sample solution (fig. 8a and fig. 12a), and both the positively charged metal cations and the basic protein molecules in the solution move away from the tip of the needle. Since metal ions have a significantly smaller collision volume, they migrate a greater distance, whereas protein molecules have a larger collision volume, they can only migrate a very limited distance. The metal ions and protein molecules are thereby separated. The negatively charged anions move toward the tip of the needle and accumulate in the region of the needle tip, increasing in concentration. The electrospray continues, so that the sample solution is kept moving in the direction of the needle tip. When the voltage is switched to a positive high voltage, the migration direction of each component in the solution starts to reverse (fig. 8b and fig. 12 b). The metal cations and protein cations move in the direction of the needle tip, while the anions move from the needle tip in the direction of the electrode. Since the protein molecules are located closer to the needle tip, they will first encounter anions and will interact strongly, eventually leading to a structural change in the protein molecules. As electrospray continues to occur, the solution still moves toward the tip of the needle and carries the structurally modified protein molecules to the tip of the needle to form an electrospray, generating highly charged ions of the protein. Because the solution moves in a single direction towards the tip of the spray needle under negative high pressure or positive high pressure, when the voltage is switched to the positive high pressure, metal ions cannot catch up with protein molecules, and the protein molecules form electrospray, the finally obtained detection signal is a protonated ion peak of the protein molecules, and no spectrum peak of metal ion adducts appears.

The formation of highly charged ions is slightly different for acidic proteins, as shown in FIG. 9. When a negative high pressure is applied to the sample solution, the positively charged metal cations in the solution move away from the tip of the needle (fig. 9a and 13 a). The negatively charged protein molecules and anions move in the direction of the tip of the needle. Protein molecules and anions accumulate and interact at the tip of the needle, eventually leading to disruption of the protein molecular structure. When the high voltage is converted into positive high voltage, the protein molecules gathered at the tip of the spray needle directly form electrospray under the carrying of the solution, and high-charge ions are generated (fig. 9b and fig. 13 b). Since the metal cations also need to migrate to the tip of the needle for a while, they do not form metal ion adducts with the protein molecules.

The interaction of the protein molecules with the anion of the salt is critical in the formation of highly charged ions. From the enlarged image of the obtained mass spectrum, the peak of the adduct of the protein molecule with the anion can be seen (fig. 10). The appearance of these adduct peaks further confirms the strong interaction between the protein molecules and the anions. And (3) respectively taking chloride salt, phosphate and iodide salt as high-charge reagents to detect cytochrome c, and investigating detection results. In the spectrum obtained with KCl, it can be seen that there is a significant presence of a Cl^-Peak of the adduct spectrum (fig. 10 a). At NH₄In the spectrum obtained with Cl, it can also be seen that there is a distinct presence of one Cl^-Peak of the adduct spectrum (fig. 10 b). The results of phosphate detection are also similar. In NaH₂PO₄The resulting spectra were seen to have one, two and three H's evident₂PO₄ ^-Peak of the adduct spectrum (fig. 10 c). In Na₂HPO₄The resulting spectra were also seen to have one, two and three H's evident₂PO₄ ^-Peak of the adduct spectrum (fig. 10 d). The results are similar for the iodonium salts. In the spectra obtained with NaI, distinct bands with one, two and three I can be seen^-Peak of the adduct spectrum (fig. 10 e). In the spectrum obtained with LiI, it is also possible to see the bands marked with one, two and three I^-Peak of the adduct spectrum (fig. 10 f).

Claims

1. A method for generating protein high-charge ions based on a polarity-reversal nanospray ion source is characterized by comprising the following steps:

s1, adding 1-10 mM strong acid salt or medium strong acid salt into the sample solution;

s2, injecting a sample solution from the tail end of the nano-spray needle;

2. The method for generating protein high-charge ions based on a sodium spray ion source with polarity inversion of claim 1, wherein in step S5, after the high voltage power is turned on, a voltage of-2.5 kV to-5.0 kV is output to the metal electrode for 3S to 12S; then, the metal electrode is output with a voltage of +1.5 kV- +2.0 kV.

3. The method for generating protein high-charge ions based on a polar-reversed sodium spray ion source according to claim 1 or 2, wherein the strong acid salt is bromide, chloride, iodide or nitrate, and the medium-strong acid salt is phosphate.