CN110879247A

CN110879247A - Method for improving electrospray charge number of protein molecules based on strong acid salt

Info

Publication number: CN110879247A
Application number: CN201910993155.2A
Authority: CN
Inventors: 龚晓云; 戴新华; 冯璐璐; 方向; 谢洁; 江游
Original assignee: National Institute of Metrology
Current assignee: National Institute of Metrology
Priority date: 2019-10-18
Filing date: 2019-10-18
Publication date: 2020-03-13

Abstract

The invention provides a method for improving the electrospray charge number of a protein molecule based on a strong acid salt, which relates to the technical field of mass spectrum, can obviously improve the charge number carried by the protein molecule during electrospray ionization, and has the advantages of simple operation, low toxicity and low harm; the method adopts a common nano-spray ion source to carry out ionization operation on a protein solution, and a strong acid salt and a weak acid additive are added into the protein solution; the concentration of the strong acid salt in the protein solution is 1-10 mM; the weak acid additive accounts for 0.001-1% of the protein solution by volume; the strong acid salt is one or more of chloride, bromide, iodide, nitrate trifluoroacetate and trichloroacetate; the weak acid additive is one or more of formic acid, acetic acid, oxalic acid, citric acid and lactic acid. The technical scheme provided by the invention is suitable for the process of carrying out electrospray on the protein solution.

Description

Method for improving electrospray charge number of protein molecules based on strong acid salt

[ technical field ] A method for producing a semiconductor device

The invention relates to the technical field of mass spectrometry, in particular to a method for improving the electrospray charge number of protein molecules based on a strong acid salt.

[ background of the invention ]

Electrospray ionization is a soft ionization method that can generate ions with multiple charges, which is beneficial for analyzing biological macromolecules such as intact proteins. The extent to which a protein is charged in electrospray ionization is related to its conformation. In general, a low charge state is associated with a folded protein structure, while a high charge state is associated with an unfolded protein structure. Increasing the charge state of protein molecules is advantageous for Mass Spectrometry (MS) based analysis.

First, it reduces the mass-to-charge ratio (m/z) of the protein, thereby extending the effective mass range. The molecular weight of a protein molecule can usually reach tens or even hundreds of kDa, and if the charge is small, the molecular weight exceeds the m/z range which can be detected by a common mass spectrometer. The method for increasing the number of charges can obviously reduce m/z and greatly improve the upper limit of the mass range detectable by the instrument. Second, in top-down proteomics, protein molecules with higher charge states dissociate more readily and have higher sequence coverage. This phenomenon is more pronounced when Electron-based tandem mass spectrometry is used, such as Electron capture fragmentation (ECD) and Electron transfer fragmentation (ETD). In addition, the resolution of most MSs increases as m/z decreases. The sensitivity of MS is also higher at lower m/z, especially for those charge sensitive detectors, including fourier transform ion cyclotron resonance (FT-ICR) and orbitrap mass spectrometers.

Early experiments produced highly charged protein ions by several methods of denaturation. The structure of the molecule is destroyed if the protein in solution is exposed to extreme pH or high temperatures. The expanded structure, where unfolding occurs, can retain more charge during electrospray. Some can also achieve unfolding of proteins at the ESI interface by introducing acidic or basic vapors in the blowback. The acids or bases used in this process are generally volatile and toxic.

In recent years, methods of highly charged reagents based on organic solvents have also been extensively studied. The most commonly used additives include m-NBA, sulfolane and DMSO. These organic solvents have higher boiling points, and the solvents are enriched during the desolvation process of ESI, and the high concentration of organic solvents finally produced has an influence on the surface tension of the droplets and the conformation of the protein. The additive used in this method has high concentration and certain toxicity.

In addition, an electrothermal high-charge ion generation method (ETS) and a high-charge ion generation method of PR-noesi have also been developed. ETS ammonium bicarbonate (NH) was added prior to analysis₄HCO₃) Was added to the sample solution at a concentration of 100 mM. The charge state of the protein can be increased by applying a higher spray voltage. However, high concentrations of ammonium bicarbonate can affect the signal response of the target protein as well as instrument sensitivity. The PR-nESI method needs a high-voltage power supply with positive and negative bidirectional output functions, a high-charge reagent with a certain concentration and then certain voltage switching operation, and the duration for generating a high-charge ion signal is short.

Accordingly, there is a need to develop a method for increasing electrospray charge number of protein molecules based on a strong acid salt to address the deficiencies of the prior art and to solve or mitigate one or more of the problems set forth above.

[ summary of the invention ]

In view of the above, the present invention provides a method for increasing electrospray charge number of protein molecules based on a strong acid salt, which can significantly increase the charge number carried by the protein molecules during electrospray ionization, and has the advantages of simple operation, low toxicity and low harm.

The invention provides a method for improving the electrospray charge number of protein molecules based on a strong acid salt.

The above aspects and any possible implementations further provide an implementation in which the strong acid salt is one or more of chloride, bromide, iodide, nitrate trifluoroacetate and trichloroacetate.

The above-described aspects and any possible implementations further provide an implementation in which the weak acid additive is one or more of formic acid, acetic acid, oxalic acid, citric acid, and lactic acid.

The aspect as defined above and any one of the possible implementations, further providing an implementation in which the concentration of the added strong acid salt in the protein solution is 1-10 mM.

The above aspects and any possible implementations are further provided with an implementation where the weak acid additive is added at a volume fraction of 0.001% to 1% in the protein solution.

The above aspects and any possible implementations further provide an implementation in which the cation species of the strong acid salt is not limited.

The above aspects and any possible implementations further provide an implementation where the average charge number of the protein cytochrome c is 14.4+ -19.7 +.

The above aspects and any possible implementations further provide an implementation where the protein is myoglobin containing a heme prosthetic group, and the ionized protein cytochrome c has an average charge of 18.5+ to 19.7 +.

The aspect and any possible implementation manner as described above, further providing an implementation manner that the strong acid salt is added at a concentration of 5 mM.

The above aspect and any possible implementation further provides an implementation where the weak acid additive is 1% by volume.

Compared with the prior art, the invention can obtain the following technical effects: by adding a strong acid salt as a high-charge reagent into a protein sample solution, the number of charges carried by protein molecules during electrospray ionization can be obviously increased; compared with the toxicity of the additive used in the prior art, the strong acid salt has the advantages of low toxicity, low harm and difficult volatilization; the method can be realized on a common Nano-ESI ion source without improvement on the device or change of the ion source condition, and is simple to operate and easy to realize.

Of course, it is not necessary for any one product to achieve all of the above-described technical effects simultaneously in practicing the invention.

[ description of the drawings ]

In order to more clearly illustrate the technical solutions of the embodiments of the present invention, the drawings used in the embodiments will be briefly described below, and it is obvious that the drawings in the following description are only some embodiments of the present invention, and it is obvious for those skilled in the art that other drawings can be obtained according to these drawings without creative efforts.

FIG. 1 is a block diagram of the enhancement of electrospray charge number for protein molecules based on a strong acid salt according to one embodiment of the present invention;

FIG. 2 is a mass spectrum of the result of cytochrome c detection with different amounts of NaCl as a high-charge reagent according to an embodiment of the present invention;

FIG. 3 is a mass spectrum of the result of cytochrome c detection using different sodium salts as high charge reagents according to one embodiment of the present invention;

FIG. 4 is a mass spectrum of the result of cytochrome c detection using different weak acid anions as high-charge reagents according to an embodiment of the present invention;

FIG. 5 is a mass spectrum of the cytochrome c detection result of the high charge reagent with different cations according to one embodiment of the invention;

FIG. 6 is a summary of the charge states of cytochrome c under the high charge reagents of different cations provided by one embodiment of the invention;

FIG. 7 is a graph showing the effect of NaCl as a high charge reagent and HAc as a weak acid additive on cytochrome c assay results according to one embodiment of the present invention;

FIG. 8 is a graph showing the effect of NaCl as a high charge reagent and HAc as a weak acid additive on the results of a hemoglobin assay containing a heme prosthetic group according to one embodiment of the present invention;

FIG. 9 is a mass spectrum of cytochrome c obtained using chloroacetic acid and HCl according to an embodiment of the present invention.

Wherein, in the figure:

1. a high voltage power supply; 2. an insulating end cap; 3. an electrode; 4. a spray needle is contained; 5. a mass spectrum sample inlet; 6. a protein solution.

[ detailed description ] embodiments

For better understanding of the technical solutions of the present invention, the following detailed descriptions of the embodiments of the present invention are provided with reference to the accompanying drawings.

It should be understood that the described embodiments are only some embodiments of the invention, and not all embodiments. All other embodiments, which can be derived by a person skilled in the art from the embodiments given herein without making any creative effort, shall fall within the protection scope of the present invention.

The terminology used in the embodiments of the invention is for the purpose of describing particular embodiments only and is not intended to be limiting of the invention. The singular forms "a", "an", and "the" as used in the embodiments of the present invention and the appended claims are intended to include the plural forms as well, unless the context clearly indicates otherwise.

In view of the above-mentioned drawbacks and deficiencies of the prior art, it is an object of the present invention to provide a method for increasing the electrospray charge number of protein molecules based on strong acid salts, which is convenient, practical, low-toxicity, and capable of generating stable high-charge ion signals over a long period of time.

According to the method for improving the electrospray charge number of the protein molecules based on the strong acid salt, as shown in figure 1, an electrospray device is the same as a common Nano-ESI, and is not changed; adding 1-10mM (10 mM) to the protein sample solution^- ³mol/L) of a highly charged agent, i.e., a strong acid salt. The salt is a salt of a strong acid, such as chloride, bromide, iodide, nitrate trifluoroacetate and/or trichloroacetate. Salts of weak acids are not capable of becoming highly charged agents such as sulfides, carbonates, formates and acetates. Of saltThe type of cation 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.

In addition, a weak acid additive is added into the protein solution in a volume ratio of 0.001-1%. Conventional weak acids such as formic acid, acetic acid, oxalic acid, citric acid, lactic acid and the like can be used.

The detection effect of the method for increasing the electrospray charge number of the protein molecule based on the strong acid salt is verified by the specific examples.

Example 1: NaCl for production of highly charged protein molecules

In this example, NaCl and cytochrome c (cytochrome c from horse heart, purchased from SigmaAldrich) were selected as the subjects to be studied, and the effect of NaCl at different concentrations was compared.

FIG. 2a is a graph showing the ionization effect on cytochrome c using a normal nanospray with 1% (v/v) acetic acid (HAc) as the weak acid additive and without the use of highly charged reagents. 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＝10.9+。

FIG. 2b shows the result of cytochrome c detection using 1mM NaCl as a high-charge reagent under the same conditions as in FIG. 2 a. And haveA significant difference with the detection results using the high charge reagent is that the signal intensity of the high charge peak cluster is significantly increased, while the low charge peak cluster is reduced compared to before. From the spectrum peak type and signal intensity, the use of 1mM NaCl high charge reagent produced cytochrome c ion overall charge number significantly higher than the use of high charge reagent method. Average charge number q of cytochrome c_avThe results of the buna spray gave a significant improvement of 15.1 +. The "+" in the average charge number in this application represents a positive charge.

FIG. 2c shows the result of cytochrome c detection using 5mM NaCl as a high-charge reagent under the same conditions as in FIG. 2 a. The charge distribution is mainly high charge peak cluster, and the low charge peak cluster is obviously reduced. The high charge peak cluster takes 16+ charges as a main peak, and the low charge peak cluster takes 9+ charges as a main peak. Average charge number q of cytochrome c_av15.4+, which is further improved compared to fig. 2 b.

FIG. 2d is the result of measurement of cytochrome c using 9mM NaCl as a high-charge reagent under the same conditions as in FIG. 2 a. The charge distribution is mainly based on a high charge peak cluster, the center of the high charge peak cluster is 16+ charges, and the low charge peak cluster is mainly based on 8 +. Number of average charges q of cytochrome c_av15.2+, with a slight drop compared to fig. 2 c.

From the above results, it can be seen that our method can obtain mass spectra of highly charged ions with NaCl as the highly charged reagent, and significantly increase the charge number of protein in the lower concentration range (1-9mM), with 5mM NaCl being the best effect.

Example 2: use of other sodium salts for the production of highly charged ions of cytochrome c

The method established by the invention has certain requirements on the type of salt, and needs strong acid salt. In this embodiment, the species of the anion is changed to Br separately based on NaCl^-、I^-、NO₃ ^-And TFA^-Examined for the generation of highly charged ions of cytochrome c.

FIG. 3a shows a high charge reagent pair with 5mM NaBrResults of cytochrome c detection. 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 9+ peak as a main peak, and the high charge peak cluster takes a 17+ peak as a main peak. The abundance of the high charge peak clusters is much higher than that of the low charge peak clusters. Average charge number q of cytochrome c_av16.7+ slightly higher than NaCl.

FIG. 3b shows the result of cytochrome c detection using 5mM NaI as a high charge reagent. It can be seen from the figure that the abundance of the high charge peak clusters is much higher than that of the low charge peak clusters. Average charge number q of cytochrome c_av＝15.7+。

FIG. 3c shows the reaction solution containing 5mM NaNO₃As a result of detection of cytochrome c by the highly charged reagent. It can be seen from the figure that the abundance of the high charge peak clusters is much higher than that of the low charge peak clusters. Number of average charges q of cytochrome c_av＝14.5+。

FIG. 3d shows the result of cytochrome c assay using 5mM NaTFA as a highly charged reagent. When using NaTFA, the spectrum is dominated by the NaTFA cluster. But a bimodal cytochrome c distribution can still be determined. The high charge peak cluster takes a 15+ peak as a main peak, and the low charge peak cluster takes a 9+ peak as a main peak. Average number of charges q of cytochrome c_av＝13.9+。

FIG. 4 investigates two weak acid anions, including HS^-And Ac^-. When 5mM NaHS was used, a high abundance of NaHS clusters was observed in the spectra (fig. 4a), and two peak clusters of high and low charge were observed. Wherein the high charge peak cluster takes 15+ as a main peak, and the low charge peak cluster takes 9+ as a main peak. Average charge number q of cytochrome c_av11.5 +. When 5mM NaAc was used, a high abundance of NaAc clusters was observed in the spectra (fig. 4b), with one cluster observed with a 9+ dominant peak. Average charge number q of cytochrome c_av＝8.9+。

From the above results, it can be seen that when four kinds of strong acid sodium salts are added to the sample, the charge state of cytochrome c is close to that obtained with NaCl. In contrast, when two weak acid sodium salts were added to the sample, the charge state of cytochrome c was similar to that in the absence of the salt. The cations of these salts, i.e. Na +, are identical. The only difference between them is their anion. It can be concluded that the strong acid anion significantly increases the charge state of cytochrome c, while the weak acid anion has little effect.

Example 3: use of other chloride salts for the production of highly charged ions of cytochrome c

To examine the influence of salt cation on the detection effect, we varied the cation species on Li based on NaCl⁺，K⁺，Cs⁺，NH₄ ⁺，Mg²⁺，Ca²⁺，Fe³⁺And La³⁺Examined for the generation of highly charged ions of cytochrome c.

FIG. 5 is a mass spectrum of the result of detection of cytochrome c by the above-mentioned high-charge reagent, and the charge state of cytochrome c is summarized in FIG. 6. LiCl, KCl, CsCl and NH₄The concentration of the four salts of Cl was 5mM, and the mass spectra were very similar. A double peak is observed, wherein the high charge peak clusters are higher than the low charge peak clusters. Total q of four salts of cytochrome c_av14.8+, 14.4+, 14.9+ and 15.0+, respectively. MgCl₂And CaCl₂Was 2.5 mM. Using this concentration to make Cl in the sample^-The concentration of (2) was the same as that of the sample of 5mM NaCl. The mass spectra obtained for the two salts are very similar to those obtained with the four salts described above. A double peak is observed, wherein the high charge peak clusters are higher than the low charge peak clusters. Total q of the two salts_avRespectively 15.1+ and 15.2 +.

FeCl₃And LaCl₃Is 1.67mM, so that Cl is present in both samples^-Is 5 mM. Using FeCl in comparison with the other six salts₃And LaCl₃The charge state of the obtained cytochrome c was slightly higher. Although a bimodal charge distribution is still observed, the high charge peak clusters are significantly higher than the low charge peak clusters. The high charge peak clusters range from 11+ to 21+, centered at 17+, while the low charges range from 8+ to 10+, centered at 9 +. Total q of the two salts_av16.3+ and 16.1+ respectively.

Various cation species are studied, including strong and weak base cations, and cations having different charges are also contemplated. Overall, a significant increase in the charge state of cytochrome c was observed on all of these chloride salts compared to the results without the addition of the high charge agent. No significant difference in charge state was observed between the different cationic species. There is only a slight difference in the charge state obtained between cations having different charges. The 3+ charged cation gives the highest charge state of cytochrome c. The 2+ charged cations create a medium charge state. The lowest charge state is observed on the 1+ charge cation. This slight difference may be due to the reduced concentration of the cation itself. Little difference in the charge state of cytochrome c is observed between strong and weak base cations. By studying these cations, it was concluded that the cationic species had little effect on the charge state of cytochrome c.

Example 4: effect of Weak acid additives on the invention

The presence of a certain amount of weak acid in the sample solution is critical for the effect of the anion. Higher concentrations of acid in the sample enhance the effect of strong acid anions on the increase in charge state. When no acid was added to the sample, almost no increase in charge state was observed (fig. 7a and b). A single peak of cytochrome c was observed when no weak acid additive was added to the sample (fig. 7 a). The clusters of peaks ranged from 7+ to 12+, centered at 8 +. q. q.s_avIs 8.9 +. When 5mM NaCl was added to the sample, the spectrum was dominated by NaCl clusters (FIG. 7 b). Still a single peak of cytochrome c can be obtained. It ranges from 7+ to 9+, centered at 8 +. q. q.s_avIs 7.7+, even lower than without NaCl.

Addition of 0.1% HAc to the sample restored Cl^-Action (fig. 7c and 7 d). When 0.1% HAc was added to the sample, a double peak was observed. The low charge peak cluster is taken as a main peak. The peak clusters range from 7+ to 10+, centered at 9 +. The abundance of the high charge peak clusters is rather low. It ranges from 13+ to 20+ with 18+ as the main peak. Whole q_avAnd 9.7 +. When the sample was further added with 5mm nacl, the abundance of the high charge peak clusters increased significantly. However, the low charge peak clusters still have higher abundance. Whole q_avIs 13.5+, which is much higher than without NaCl. Further improveThe concentration of the weak acid additive, the results of fig. 2 can be obtained.

The effect of acid was more pronounced in the experiments with myoglobin (holo-Mb) containing a heme prosthetic group, as shown in fig. 8. Very small amounts of HAc additive can induce the effect of NaCl. When 0.001% HAc was added to the sample, the mass spectrum consisted mainly of a cluster of holo-Mb peaks (fig. 8 a). It ranges from 8+ to 15+ with a 12+ dominant peak, representing a folded structure. q. q.s_avIs 11.6 +. Myoglobin (Apo-Mb) which has lost the heme prosthetic group is also observed, but it has only a very low abundance. The addition of 5mM NaCl to the sample resulted in a significant increase in the charge state (FIG. 8 b). The mass spectrum is mainly composed of Apo-Mb peak clusters. It ranges from 12+ to 25+ with a major peak at 19+, representing an unfolded structure with complete loss of the heme prosthetic group. q. q.s_av18.5+ which is significantly higher than without NaCl. Although holo-Mb is still observed, its abundance is very low.

Further increasing the concentration of HAc, the charge state will continue to increase. When 0.01% HAc was added to the sample, the mass spectrum consisted mainly of clusters of Apo-Mb peaks (fig. 8 c). It ranges from 11+ to 29+ with a 17+ dominant peak. q. q.s_avIs 18.9 +. Holo-Mb was also observed, but very low abundance. The addition of 5mM NaCl to the sample increased the charge state of Mb to a higher level (FIG. 8 d). The Holo-Mb was completely removed and only apo-Mb was observed. It ranges from 12+ to 29+ with a 20+ dominant peak. q. q.s_avIncreasing to 19.7 +.

The invention utilizes the interaction of anions of strong acid salts and protein molecules, and researches show that the anions of strong acids can obviously increase the charge state of the protein under acidic conditions, but the anions of weak acids cannot. From the above experimental results, it can be concluded that a key factor for making them different in result is the hydrolysis of weak acid anions in acidic solution. Hydrolysis of the weak acid anion results in a decrease in its concentration, thereby reducing the effect of the weak acid anion on protein structure.

The pH of the solution changes during ESI, and at the positive electrode, the pH drops due to oxidation reactions in the nanospray tips, such as 2H₂O→4H⁺+4e^-+O₂. For an unbuffered solution, the pH may drop from a near neutral pH to a pH of 3.43. Furthermore, during ESI, due to evaporation of the solution and H in the droplets⁺Also leads to a decrease in pH. The effective pH of the droplets during ion formation may be 1-3 pH units lower than the pH of the solution prior to ESI. The decrease in pH exacerbates the hydrolysis of the weak acid anion in the solution, further decreasing the concentration of the weak acid anion. As a result, the influence of weakly acidic anions is attenuated to a lower level.

FIG. 9 shows a mass spectrum of cytochrome c obtained using chloroacetic acid (MCA) and HCl. The initial pH of both samples remained the same as the initial pH of 1% HAc (pH 2.74). The molarity of 1% HAc was 174 mM. The acidity coefficient (pKa) of the HAc was 4.74. Compared to HAc, the pKa of MCA is much lower, 2.86. The concentration of MCA was only 3.9mM (pH 2.76). A double peak was observed (fig. 9a), with a significant increase in the abundance of the high charge peak cluster compared to the results obtained with HAc (fig. 2 a). Whole q_avIncreasing to 12.2 +. When HCl was used, the charge state of cytochrome c increased to an even higher level (fig. 9 b). The pKa of HCl is-8.00. The HCl concentration was 1.7mM (pH 2.75). A double peak is observed, with the high charge peak clusters being more abundant than the low charge peak clusters. Whole q_avIncreasing to 15.1 +.

Although the initial pH of the three samples with HAc, MCA and HCl were the same, the charge state of the obtained cytochrome c was significantly different. The charge state of cytochrome c increases with the acidity of the acid used. The acidity of the HAc is the weakest. The resulting population q_avIs the lowest. In contrast, HCl is the most acidic. Obtained whole q_avIs the highest. During the desolvation process, the pH of the spray droplets decreases. The decrease in pH exacerbates the hydrolysis of the weak acid anion, resulting in a decay in the weak acid anion concentration.

HAc is the weakest of the three acids, and its hydrolysis is the most severe, with the lowest final concentration. Thus, Ac^-The influence on the conformation of cytochrome c molecules is the weakest. The acidity of MCA is much stronger than HAc. MCA in contrast to HAc^-Is hydrolyzed without Ac^-Is serious. MCA^-To end ofAt a concentration much higher than Ac^-The concentration of (c). MCA^-Has stronger influence on the conformation of cytochrome c molecules than Ac^-Resulting in a more unfolded conformation of the cytochrome c molecule and hence an increased charge state. HCl is a strong acid and hydrolysis does not occur. Cl in the liquid droplets^-Is much higher than Ac^-And MCA^-The concentration of (c). In Cl^-Under the influence of (3), the conformation of cytochrome c molecule is more spread. The charge state of cytochrome c is therefore the highest.

The situation was similar when NaCl was added to cytochrome c samples buffered with 1% HAc. No change in pH was observed, whether or not NaCl was added to the sample, as shown in Table 1. The initial pH values of the four samples were the same. However, when NaCl was added to the sample, the charge state of cytochrome c increased significantly. Cl in the sample^-Induces unfolding of the cytochrome c molecule and results in an increase in the charge state of cytochrome c.

TABLE 1

The method can be realized on a common Nano-ESI ion source without improvement on the device or change of the ion source condition. Other methods such as PR-nESI-based methods require a high-voltage power supply with a positive and negative bidirectional output function, a high-charge reagent with a certain concentration, and then a certain voltage switching operation is performed to generate high-charge ions, but the high-charge signal can only last for a short time.

The low concentrations of the highly charged agent and the weak acid additive required by the present invention allow the protein to retain its native folded structure in solution. The concentration of the strong acid salt in the solution is 1-10mM (10)^-3mol/L), the concentration of the weak acid additive is generally between 0.001% and 1% (v/v), which does not affect the natural structure of the protein in solution. In the prior art, reagents such as strong acid, strong base and the like are generally used in the method based on the common nano-ESI, so that protein is unfolded or even inactivated in a solution, and the maintenance of an instrument is not facilitated; the concentration of m-Nitrobenzyl alcohol (m-NBA), sulfolane or dimethyl sulfoxide (DMSO) used in the method based on Supercharging regents is 1-5% (v/v), and the concentration is far greater than that of the additive used by people, and almost reaches the limit of solubility.

The high-charge reagent and the weak-acid reagent used in the invention are common and easily available, and have low toxicity. The high-charge reagents (strong acid salts: such as sodium chloride (NaCl) and potassium chloride (KCl)) and weak acid additives (such as Formic Acid (FA) and acetic acid (HAc)) are common chemical reagents in laboratories, and most of the reagents are low-toxicity or even non-toxic and have small harm to human bodies.

The method for increasing the electrospray charge number of the protein molecule based on the strong acid salt provided by the embodiment of the application is described in detail above. The above description of the embodiments is only for assisting in understanding the method of the present application and its core ideas; meanwhile, for a person skilled in the art, according to the idea of the present application, there may be variations in the specific embodiments and the application scope, and as described above, the content of the present specification should not be construed as a limitation to the present application.

As used in the specification and claims, certain terms are used to refer to particular components. As one skilled in the art will appreciate, manufacturers may refer to a component by different names. This specification and claims do not intend to distinguish between components that differ in name but not function. In the following description and in the claims, the terms "include" and "comprise" are used in an open-ended fashion, and thus should be interpreted to mean "include, but not limited to. "substantially" means within an acceptable error range, and a person skilled in the art can solve the technical problem within a certain error range to substantially achieve the technical effect. The description which follows is a preferred embodiment of the present application, but is made for the purpose of illustrating the general principles of the application and not for the purpose of limiting the scope of the application. The protection scope of the present application shall be subject to the definitions of the appended claims.

It is also noted that the terms "comprises," "comprising," or any other variation thereof, are intended to cover a non-exclusive inclusion, such that a good or system that comprises a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such good or system. Without further limitation, an element defined by the phrase "comprising an … …" does not exclude the presence of other like elements in a commodity or system that includes the element.

It should be understood that the term "and/or" as used herein is merely one type of association that describes an associated object, meaning that three relationships may exist, e.g., a and/or B may mean: a exists alone, A and B exist simultaneously, and B exists alone. In addition, the character "/" herein generally indicates that the former and latter related objects are in an "or" relationship.

The foregoing description shows and describes several preferred embodiments of the present application, but as aforementioned, it is to be understood that the application is not limited to the forms disclosed herein, but is not to be construed as excluding other embodiments and is capable of use in various other combinations, modifications, and environments and is capable of changes within the scope of the application as expressed herein, commensurate with the above teachings, or the skill or knowledge of the relevant art. Rather, modifications and variations may occur to those skilled in the art without departing from the spirit and scope of the application, which should be determined from the following claims.

Claims

1. A method for improving the electrospray charge number of protein molecules based on a strong acid salt is characterized in that a nano-spray ion source is adopted to carry out ionization operation on a protein solution, and the method is characterized in that the protein solution is added with the strong acid salt and a weak acid additive.

2. The method for enhancing the electrospray charge number of a protein molecule according to claim 1, wherein said strong acid salt is one or more of chloride, bromide, iodide, nitrate trifluoroacetate and trichloroacetate.

3. The strong acid salt based method for increasing the electrospray charge number of a protein molecule according to claim 1, wherein said weak acid additive is one or more of formic acid, acetic acid, oxalic acid, citric acid and lactic acid.

4. The method for enhancing electrospray charge number of protein molecules based on a strong acid salt according to claim 1 or 2, characterized in that the concentration of said strong acid salt added in said protein solution is 1-10 mM.

5. A strong acid salt based method for increasing the electrospray charge number of protein molecules according to claim 1 or 3, characterized in that said weak acid additive is added in a volume fraction of 0.001-1% in said protein solution.

6. The method of claim 2, wherein the cation species of the strong acid salt is not limited.

7. The method of claim 1, wherein the average charge of protein cytochrome c is 14.4+ to 19.7 +.

8. The method of claim 7, wherein the average charge of the ionized protein cytochrome c is 18.5+ to 19.7+ when the protein is myoglobin containing a heme prosthetic group.

9. The method for enhancing electrospray charge number of protein molecules according to claim 4, wherein said salt is added at a concentration of 5 mM.

10. The method of claim 5, wherein the weak acid additive is present in an amount of 1% by volume.