WO2015092494A1

WO2015092494A1 - Method of conducting electrospray ionization and method of analyzing an analyte utilizing the same

Info

Publication number: WO2015092494A1
Application number: PCT/IB2013/061224
Authority: WO
Inventors: Ravi Krovvidi
Original assignee: Agilent Technologies, Inc.
Priority date: 2013-12-20
Filing date: 2013-12-20
Publication date: 2015-06-25

Abstract

The present invention relates to a method of conducting electrospray ionization (ESI), a method of analyzing an analyte utilizing the same and the use of a supercharging reagent in electrospray ionization (ESI) of an analyte for improving an overall charge state of the analyte. The method of conducting electrospray ionization (ESI)comprises the steps of: providing an ESI device comprising a first inlet, a second inlet, an outlet and two opposing electrodes; introducing an analyte between the two opposing electrodes through the first inlet of the ESI device; applying voltage to the two opposing electrodes; wherein the method further comprises the step of introducing a supercharging reagent between the two opposing electrodes of the ESI device through the second inlet, wherein the supercharging reagent is introduced separately from the analyte, but the supercharging reagent comes into contact with the analyte within the ESI device.

Description

METHOD OF CONDUCTING ELECTROSPRAY IONIZATION AND METHOD OF ANALYZING AN ANALYTE UTILIZING THE SAME

BACKGROUND ART

[0001 ] The present invention relates to a method of conducting electrospray ionization (ESI), a method of analyzing an analyte utilizing the same and the use of a supercharging reagent in electrospray ionization (ESI) of an analyte for improving an overall charge state of the analyte.

[0002] Electrospray ionization (ESI) is a desorption ionization technique, which typically involves the introduction of an analyte (i.e. a compound or mixture of compounds to be analyzed), typically in dissolved form, in an ESI device having two opposing electrodes. Upon application of voltage, a Taylor cone is formed at the inlet where the analyte is introduced. From this Taylor cone, droplets of the analyte are dispersed by electrospray in the form of a jet, plume or fine aerosol. Due to the voltage applied, ions of the analyte are formed, which involves solvent evaporation from the analyte droplet, thereby decreasing the initial droplet size. As a result the charge/surface area ratio increases until it reaches its Rayleigh limit, where the electrostatic repulsion of like charges becomes more powerful than the surface tension holding the droplet together. At this point the droplet undergoes Coulomb fission whereby the droplet explodes under formation of many smaller, more stable droplets. Coulomb fission may occur repeatedly due to a continuous solvent evaporation.

[0003] Based on these principles, ESI is a soft ionization technique and is thus in particular suitable for the analysis of large biological molecules, such as proteins or oligonucleotides, which cannot be vaporized without decomposition by conventional ionization techniques, such as electron ionization (El) or chemical ionization (CI), by mass spectrometry (MS). Thus, ESI coupled with MS is widely used for the analysis of large biopolymers. In addition, ESI can be coupled with a precedent separation technique, such as liquid chromatography (LC), which allows the analysis of complex samples containing a variety of compounds. A further advantage of ESI resides in the formation of multiply charged analyte molecules, which - in view of the fact that the mass-to-charge ratio is typically detected in an MS analyzer - allows the analysis over a broad mass range.

[0004] On the other hand, ESI-MS often suffers from a reduced sensitivity which is disadvantageous in both qualitative analysis and quantitative analysis in terms of a low signal-to-noise ratio. In order to increase the sensitivity, ESI-MS measurements are typically carried with a low resolution which may not allow the resolution of isotopes of the analytes, which is particularly undesired if for instance radiolabeled biomolecules are to be analyzed.

DISCLOSURE

[0005] It is an object of the invention to provide an improved ESI-MS technique allowing an improved sensitivity, while maintaining a sufficient mass resolution. The object is solved by the independent claims. Further embodiments are shown by the dependent claims.

[0006] In particular the present invention aims at improving the electrospray ionization, in contrast to an optimization of the mass spectrometry, as it is conventionally tried. The present inventor has surprisingly found that by introducing a supercharging reagent within an ESI device, the overall charge state of an analyte, in particular large biological molecules, such as proteins, peptides or oligonucleotides, can be enhanced thereby improving the signal-to-noise ratio (at lower m/z ratios) and the mass resolution. Without wishing to be bound to any theory, it is assumed that supercharging reagents having a comparatively high surface tension exhibit an enhanced charging of the analyte molecules, consistent with the Rayleigh equation. In addition, the supercharging reagent may also affect the droplet size of the analyte and thus the desolvation process.

[0007] According to an embodiment of the present invention, a method of conducting electrospray ionization (ESI) is provided which comprises the steps of: providing an ESI device comprising a first inlet, a second inlet, an outlet and two opposing electrodes; introducing an analyte between the two opposing electrodes through the first inlet of the ESI device; applying voltage to the two opposing electrodes; wherein the method further comprises the step of introducing a supercharging reagent between the two opposing electrodes of the ESI device through the second inlet, wherein the supercharging reagent is introduced separately from the analyte, but the supercharging reagent comes into contact with the analyte within the ESI device.

[0008] According to another embodiment of the present invention, a method of analyzing an analyte is provided which comprises the steps of: conducting electrospray ionization; and subsequently conducting mass spectrometry analysis; wherein the electrospray ionization is conducted as defined above.

[0009] According to still another embodiment of the present invention, a use of a supercharging reagent in electrospray ionization (ESI) of an analyte for improving an overall charge state of the analyte is provided, wherein the supercharging reagent is introduced separately from the analyte into an ESI device.

[0010] In the following, further exemplary embodiments of the method of conducting electrospray ionization, the method of analyzing an analyte and the use of a supercharging reagent in electrospray ionization will be explained.

[001 1 ] It should be noted that features described in connection with one exemplary embodiment or exemplary aspect may be combined with any other exemplary embodiment or exemplary aspect, in particular features described with any exemplary embodiment of the method of conducting electrospray ionization may be combined with any exemplary embodiment of the method of analyzing an analyte or with any exemplary embodiment of the use of a supercharging reagent in electrospray ionization and vice versa, unless specifically stated otherwise.

[0012] Where an indefinite or definite article is used when referring to a singular term, such as "a", "an" or "the", a plural of that term is also included and vice versa, unless specifically stated otherwise.

[0013] The expression "comprising", as used herein, includes not only the meaning of "comprising", "including" or "containing", but may also encompass "consisting essentially of" and "consisting of".

[0014] The term "supercharging reagent", as used herein, has to be distinguished from a "sheath liquid", as sometimes used in ESI-MS, in particular in HPLC/ESI-MS or CE/ESI-MS. The supercharging reagent may be in particular miscible with an LC mobile phase and enhances the overall charge state of an analyte, whereas a sheath liquid is poorly miscible with the LC mobile phase and functions as the terminal buffer reservoir in CE/ESI-MS analysis thereby introducing additional source of chemical background.

[0015] In the method of conducting electrospray ionization (ESI), an ESI device is provided that comprises a first inlet, a second inlet, an outlet and two opposing electrodes. An analyte is introduced between the two opposing electrodes through the first inlet of the ESI device and a supercharging reagent is introduced between the two opposing electrodes of the ESI device through the second inlet. As will be described in further detail below, the supercharging reagent and the analyte may be introduced by means of a microfluidic chip based setup; i.e. in that case the microfluidic chip based setup may comprise the first inlet and the second inlet and the ESI device may comprise the microfluidic chip based setup.

[0016] In the method of conducting electrospray ionization (ESI), the supercharging reagent is introduced separately from the analyte, which may in particular allow an appropriate adjustment of the amount of supercharging reagent. The term "separately" as used herein may in particular denote that the analyte and the supercharging reagent are introduced through separate inlets (i.e. the first and the second inlet) of the ESI device, even though these inlets may be comprised in the same member or part of the ESI device, such as a microfluidic chip based setup, as described above.

[0017] In the method of conducting electrospray ionization (ESI), the supercharging reagent comes into contact with the analyte within the ESI device. In particular, the supercharging reagent and the analyte may not come into contact with each other (or they may not be mixed with each other) prior to an optional separation means, such as a liquid chromatography column (e.g. a pre-column mixing). Rather, if an optional separation means, which will be described in further detail below, is used, the analyte after having passed such separation means may come into contact with (or mixed with) the supercharging reagent, e.g. a post- column mixing. Thereby, the supercharging reagent may not interfere with the optional separation process of the analyte.

[0018] In the method of conducting electrospray ionization (ESI), a voltage is applied to the two opposing electrodes.

[0019] In an embodiment, upon applying voltage to the two opposing electrodes, a Taylor cone comprising the analyte is formed, in particular at the first inlet. If the analyte is introduced by means of a microfluidic chip based setup, a Taylor cone may be formed at an outlet of the microfluidic chip based setup, where the analyte is dispensed (or released) between the two opposing electrodes.

[0020] In an embodiment, the supercharging reagent is introduced prior to the Taylor cone. This may be accomplished for instance by using a microfluidic chip based setup, as will be described in further detail below. In this embodiment, the supercharging reagent may come into contact with the analyte before the analyte enters the Taylor cone. Thus, the analyte mixed with the supercharging reagent may enter the Taylor cone and the analyte mixed with the supercharging reagent may be present within the Taylor cone. In particular, the Taylor cone may comprise or consist of (or may be formed by) the analyte mixed with the supercharging reagent. [0021 ] In an embodiment, the supercharging reagent and/or the analyte is introduced by means of a microfluidic chip based setup, in particular both the supercharging reagent and the analyte are introduced by means of the microfluidic chip based setup. The microfluidic chip based setup may comprise at least one inlet, in particular at least two inlets, such as the first inlet and the second inlet of the ESI device, a microfluidic capillary and at least one outlet, where the supercharging reagent and/or the analyte may be dispensed (or released) between the two opposing electrodes of the ESI device. In particular, the ESI device may comprise the microfluidic chip based setup. The microfluidic capillary may in particular provide fluid communication between the at least one inlet of the microfluidic chip based setup and the outlet of the microfluidic chip based setup. The microfluidic capillary may in particular have a diameter in the range of from 50 to 100 pm, such as about 75 pm. An example of a microfluidic chip based setup suitable for use in the present invention includes the Agilent HPLC-Chip G4240-61001 , provided by Agilent Technologies. Further details thereof are available under www.agilent.com which shall be incorporated herein by reference.

[0022] When both the supercharging reagent and the analyte are introduced by means of the microfluidic chip based setup, the supercharging reagent may come into contact with the analyte within (inside) the microfluidic chip based setup, in particular the supercharging reagent and the analyte may be mixed with each other within (inside) the microfluidic chip based setup. Thereby, the supercharging reagent may come into contact with the analyte before entering the Taylor cone, as further described above.

[0023] In an embodiment, the supercharging reagent comes into contact with the analyte within a microfluidic capillary of the microfluidic chip based setup. As described above, the microfluidic capillary may in particular provide fluid communication between the at least one inlet of the microfluidic chip based setup, in particular the at least two inlets thereof, such as the first inlet and the second inlet of the ESI device, and the outlet of the microfluidic chip based setup. The microfluidic capillary may in particular have a diameter in the range of from 50 to 100 pm, such as about 75 pm. [0024] In an embodiment, the microfluidic chip based setup comprises a (fluidic) valve, such as a 6-port valve, for instance a 6-port valve as included in the Agilent HPLC-Chip G4240-61001 . The (6-port) valve may in particular serve for introducing and mixing the analyte and the supercharging reagent. The (6-port) valve may in particular comprise the microfluidic capillary, referred to in the foregoing. The (6- port) valve in particular comprises a stator having ports and a rotor having grooves and channels (conduits). The (6-port) valve can be opened for inserting a chip in between the stator and the rotor automatically. The analyte may be introduced into the (6-port) valve through a first port (for instance port #2 of the 6-port valve as included in the Agilent HPLC-Chip G4240-61001 ) and conducted via grooves and channels to a third port (for instance port #6 of the 6-port valve as included in the Agilent HPLC-Chip G4240-61001 ). The supercharging reagent may be introduced into the (6-port) valve through a second port (for instance port #4 of the 6-port valve as included in the Agilent HPLC-Chip G4240-61001 ) and conducted via grooves and channels to the third port. In the third port, the analyte and the supercharging reagent may be mixed with each other and the mixed solution may be conducted via a microfluidic capillary, such as the microfluidic capillary, referred to in the foregoing, for instance a low volume fused silica capillary, to a fourth port (for instance port #3 of the 6-port valve as included in the Agilent HPLC-Chip G4240-61001 ). From the fourth port, the mixed solution may exit the (6-port) valve and it may be conducted through a channel to a spray tip of the microfluidic chip, where it may be sprayed as ESI spray. The remaining ports of the (6-port) valve (for instance ports #1 and #5 of the 6-port valve as included in the Agilent HPLC-Chip G4240-61001 ) may be blocked, for instance by means of plumbings. By such an arrangement, a particularly efficient way of mixing the supercharging reagent and the analyte within the ESI device can be achieved.

[0025] In an embodiment, the supercharging reagent comprises, in particular consists of, at least one polar solvent, in particular at least one polar aprotic solvent. Such solvents have a comparatively high surface tension and may thereby exhibit an enhanced charging of analyte molecules during electrospray ionization. The supercharging reagent may be in particular miscible with a mobile phase, used in an optional precedent separation step.

[0026] In an embodiment, the supercharging reagent comprises, in particular consists of, at least one selected from the group consisting of dimethyl sulfoxide, dimethylformamide, thiodiglycol, dimethylacetamide, N-methyl-2-pyrrolidone and N- methylpyrrolidine. In particular, the supercharging reagent may comprise, in particular consist of, dimethyl sulfoxide (DMSO).

[0027] In an embodiment, the supercharging reagent is comprised in an aqueous solution. The water used in the aqueous solution may be in particular suitable for analytical purposes, such as de-ionized water, in particular ultrapure water (e.g. Milli-Q^® water). In particular, a proportion of the supercharging reagent in the aqueous solution may be from 1 to 50 vol-%, in particular from 3 to 45 vol-%, in particular from 5 to 40 vol-%, in particular from 7.5 to 35 vol-%, in particular from 10 to 30 vol-%.

[0028] In an embodiment, the analyte is introduced in dissolved form, i.e. the analyte is dissolved in an appropriate solvent, such as water or a solvent or mobile phase, used in an optional precedent separation step.

[0029] In an embodiment, the analyte comprises at least one selected from the group consisting of proteins, peptides, deoxyribonucleic acid, ribonucleic acid and polysaccharides. The methods according to the present invention are in particular suitable for the ionization and analysis of large biological molecules, such as proteins, peptides, deoxyribonucleic acid, ribonucleic acid and polysaccharides. In particular, the analyte may comprise at least one protein or peptide, in particular a protein or peptide mixture. [0030] In an embodiment, the first inlet of the ESI device is coupled either directly or indirectly to a separation means and the analyte is introduced after having passed the separation means. This is in particular advantageous when the analyte comprises a mixture of compounds, such as a protein or peptide mixture. Depending on the amount of fluid provided by the separation means, such as the flow rate in liquid chromatography, it might be advantageous to indirectly couple the first inlet of the ESI device to the separation means, for instance via a splitting means which allows only a part of the fluid leaving the separation means to enter the ESI device through the first inlet.

[0031 ] In an embodiment, the separation means, referred to above, is a liquid chromatography column or a capillary electrophoresis capillary, in particular a high- performance liquid chromatography (HPLC) column. Such separation means may allow for an efficient separation of different compounds in the analyte and are therefore in particular advantageous when the analyte comprises a mixture of compounds, such as a protein or peptide mixture. [0032] In an embodiment, the second inlet is coupled either directly or indirectly to a supercharging reagent reservoir. As the name implies, a supercharging reagent reservoir serves for storing the supercharging reagent prior to its use. The size of the supercharging reagent reservoir is not particularly limited, as long as it can provide a sufficient amount of supercharging reagent required for the method of conducting electrospray ionization according to the present invention.

[0033] In an embodiment, the outlet of the ESI device is coupled either directly or indirectly to a mass spectrometry (MS) analyzer. Thereby, the "supercharged" analyte may be forwarded to a MS analyzer for subsequent analysis thereof. The means for coupling the outlet of the ESI device the MS analyzer is not particularly limited. For instance a chip cube interface may be used. A suitable example thereof includes the Agilent 1260 Infinity HPLC-Chip Cube Interface, provided by Agilent Technologies. Further details thereof are available under www.agilent.com which shall be incorporated herein by reference. Such a chip cube interface may accommodate the entire ESI device, and may in particular accommodate a microfluidic chip based setup suitable for introducing the supercharging reagent and/or the analyte. Thereby, a user-friendly setup may be provided allowing an easy and reliable coupling with an optional precedent separation means and a subsequent MS analyzer.

[0034] The type of MS analyzer is not particularly limited and various types thereof may be coupled to the outlet of the ESI device. In an embodiment, the MS analyzer is a time-of-flight (TOF) mass spectrometry analyzer, wherein the mass-to- charge ratio of an ion (such as the "supercharged" analyte) is determined by a time measurement. In particular, the time-of-flight mass spectrometry analyzer may be a quadrupole time-of-flight (QTOF) mass spectrometry analyzer.

[0035] In the method of analyzing an analyte, electrospray ionization is conducted as defined above, and subsequently a mass spectrometry analysis is conducted. The mass spectrometry analysis may be in particular conducted by use of an MS analyzer, as described above. In addition, the ESI device used for conducting electrospray ionization may be coupled either directly or indirectly to an MS analyzer, as described above. [0036] In an embodiment, the method of analyzing an analyte further comprises the step of separating the analyte by means of a separation means prior to the step of conducting electrospray ionization. This is in particular advantageous when the analyte comprises a mixture of compounds, such as a protein or peptide mixture.

[0037] In an embodiment, the step of separating comprises liquid chromatography or capillary electrophoresis, in particular high-performance liquid chromatography (HPLC). Such separation steps may allow for an efficient separation of different compounds in the analyte and are therefore in particular advantageous when the analyte comprises a mixture of compounds, such as a protein or peptide mixture. [0038] In an embodiment, the mass spectrometry analysis comprises time-of- flight (TOF) mass spectrometry, such as quadrupole time-of-flight (QTOF) mass spectrometry.

[0039] In an embodiment, the analyte comprises at least one selected from the group consisting of proteins, peptides, deoxyribonucleic acid, ribonucleic acid and polysaccharides, in particular at least one protein or peptide, in particular a protein or peptide mixture. As explained above, the methods according to the present invention are in particular suitable for the ionization and analysis of large biological molecules, such as proteins, peptides, deoxyribonucleic acid, ribonucleic acid and polysaccharides.

[0040] In the use of a supercharging reagent in electrospray ionization (ESI) of an analyte, the supercharging reagent is used in electrospray ionization of an analyte for improving an overall charge state of the analyte, wherein the supercharging reagent is introduced separately from the analyte into an ESI device. The term "an overall charge state of the analyte" as used herein in particular denotes the (average) number of charges per one analyte molecule. Without wishing to be bound to any theory, it is assumed that supercharging reagents having a comparatively high surface tension exhibit an enhanced charging of the analyte molecules, consistent with the Rayleigh equation, and the supercharging reagent may also affect the droplet size of the analyte and thus the desolvation process. Thereby, the overall charge state of the analyte may be improved, in particular increased or enhanced.

BRIEF DESCRIPTION OF DRAWINGS

[0041 ] Other objects and many of the attendant advantages of embodiments of the present invention will be readily appreciated and become better understood by reference to the following more detailed description of embodiments in connection with the accompanied drawing.

[0042] Figure 1 shows a fluidic valve of a microfluidic chip based setup for use in a method according to an embodiment of the present invention, particularly for use in high performance liquid chromatography (HPLC).

[0043] The illustration in the drawing is schematically. [0044] Referring now in greater detail to the drawing, Figure 1 depicts a fluidic valve 100 for a microfluidic chip based setup for use in a method according to an embodiment of the present invention. With regard to details concerning the method, reference is made to the above description.

[0045] The fluidic valve 100 may in particular serve for introducing and mixing an analyte and a supercharging reagent, as described above in detail. The fluidic valve 100 comprises a stator having a plurality of ports 1 -6, each appropriate for connecting a fluidic member or a fluidic conduit. The fluidic valve 100 furthermore comprises a rotor having grooves 101 -108. The rotor and the stator are stacked on top of one another perpendicular to the paper plane of Figure 1 . When the rotor is rotated relative to the stator, different fluidic coupling states between different ones of the ports 1 -6 and different one of the grooves 101 -108 may be adjusted, so that selectable sets of ports 1 -6 can be fluidically coupled by a respectively different one of the grooves 101 -108, as known by those skilled in the art. The fluidic valve 100 is configured so as to be operable to accommodate a microfluidic chip in between the stator and the rotor. Moreover, a capillary 150 (such as a low volume fused silica capillary) connecting ports 3 and 6 is provided. Additionally, a plurality of fluidic conduits 160-169 are foreseen for coupling several ones of the above-mentioned fluid features to one another.

[0046] In the shown switching state of Figure 1 , port 1 and port 5 are blocked. Port 2 is connected to a nano pump (not shown). Mixed solvents may be supplied via port 3. A supercharge reactant may be supplied via port 4. A mixture of supercharge reactant and analyte may be drained via port 6.

[0047] With the illustrated valve 100, the following flow characteristics can be achieved: [0048] The supercharge reactant may be introduced through port 4 and may then flow into the connected fluidic conduit 166 and through groove 108. The supercharge reactant continues to flow through connected conduit 168, groove 107, connected conduit 167, groove 104, connected conduit 162, groove 106, connected conduit 163, groove 105, connected conduit 161 and finally reaches port 6. [0049] The liquid chromatography analyte is introduced into the port 2, and flows via groove 102, connected conduit 164, groove 103, connected conduit 169, groove 101 , connected conduit 160, and reaches port 6.

[0050] Hence, the supercharge reactant and the analyte are mixed at port 6.

[0051 ] Port 6 and port 3 are fluidically connected to one another via capillary 150, so that the mixed solution flows from port 6 to port 3. The mixed solution exits from port 3 directly into the connected conduit 165. The latter leads to a spray tip of the microfluidic chip and sprays an ESI spray, in front of an orifice of a mass spectrometry device.

[0052] Embodiments of the invention can be partly or entirely embodied or supported by one or more suitable software programs, which can be stored on or otherwise provided by any kind of data carrier, and which might be executed in or by any suitable data processing unit. Software programs or routines can be preferably applied in or by a control unit, such as a processor.

[0053] While the present invention has been described in detail by way of specific embodiments or examples, the invention is not limited thereto and various alterations and modifications are possible, without departing from the scope of the invention.

Claims

A method of conducting electrospray ionization (ESI), the method comprising the steps of: providing an ESI device comprising a first inlet, a second inlet, an outlet and two opposing electrodes, introducing an analyte between the two opposing electrodes through the first inlet of the ESI device, applying voltage to the two opposing electrodes, characterized in that the method further comprises the step of introducing a supercharging reagent between the two opposing electrodes of the ESI device through the second inlet, wherein the supercharging reagent is introduced separately from the analyte, but the supercharging reagent comes into contact with the analyte within the ESI device.

The method according to claim 1 , wherein upon applying voltage to the two opposing electrodes a Taylor cone comprising the analyte is formed.

The method according to claim 2, wherein the supercharging reagent is introduced prior to the Taylor cone.

The method according to any one of the preceding claims, wherein the supercharging reagent and/or the analyte is introduced by means of a microfluidic chip based setup.

The method according to any one of the preceding claims, wherein the supercharging reagent and the analyte are introduced by means of a microfluidic chip based setup and the supercharging reagent comes into contact with the analyte within the microfluidic chip based setup.

The method according to claim 5, wherein the supercharging reagent comes into contact with the analyte within a microfluidic capillary of the microfluidic chip based setup.

7. The method according to claim 5 or 6, wherein the supercharging reagent comes into contact with the analyte within a valve (100) of the microfluidic chip based setup.

8. The method according to any one of the preceding claims, wherein the supercharging reagent comprises at least one polar solvent.

9. The method according to any one of the preceding claims, wherein the supercharging reagent comprises at least one polar aprotic solvent.

10. The method according to any one of the preceding claims, wherein the supercharging reagent comprises at least one selected from the group consisting of dimethyl sulfoxide, dimethylformamide, thiodiglycol, dimethylacetamide, N-methyl-2-pyrrolidone and N-methylpyrrolidine.

1 1 . The method according to claim 10, wherein the supercharging reagent comprises dimethyl sulfoxide.

12. The method according to any one of the preceding claims, wherein the supercharging reagent is comprised in an aqueous solution.

13. The method according to claim 12, wherein a proportion of the supercharging reagent in the aqueous solution is from 1 to 50 vol-%.

14. The method according to claim 13, wherein a proportion of the supercharging reagent in the aqueous solution is from 5 to 40 vol-%. 15. The method according to any one of the preceding claims, wherein the analyte is introduced in dissolved form.

16. The method according to any one of the preceding claims, wherein the analyte comprises at least one selected from the group consisting of proteins, peptides, deoxyribonucleic acid, ribonucleic acid and polysaccharides. 17. The method according to claim 16, wherein the analyte comprises at least one protein or peptide, in particular a protein or peptide mixture.

18. The method according to any one of the preceding claims, wherein the first inlet is coupled either directly or indirectly to a separation means and the analyte is introduced after having passed the separation means.

19. The method according to claim 18, wherein the separation means is a liquid chromatography column or a capillary electrophoresis capillary. 20. The method according to claim 19, wherein the separation means is a high- performance liquid chromatography (HPLC) column.

21 . The method according to any one of the preceding claims, wherein the second inlet is coupled either directly or indirectly to a supercharging reagent reservoir.

22. The method according to any one of the preceding claims, wherein the outlet is coupled either directly or indirectly to a mass spectrometry (MS) analyzer.

23. The method according to claim 21 , wherein the MS analyzer is a time-of-flight mass spectrometry analyzer.

24. A method of analyzing an analyte, the method comprising the steps of: conducting electrospray ionization, and subsequently conducting mass spectrometry analysis; wherein the electrospray ionization is conducted as defined in any one of claims 1 to 23.

25. The method according to claim 24, wherein the method further comprises the step of separating the analyte by means of a separation means prior to the step of conducting electrospray ionization.

26. The method according to claim 25, wherein the step of separating comprises liquid chromatography or capillary electrophoresis.

27. The method according to claim 26, wherein the step of separating comprises high-performance liquid chromatography (HPLC). 28. The method according to any one of claims 24 to 27, wherein the mass spectrometry analysis comprises time-of-flight mass spectrometry.

29. The method according to any one of claims 24 to 28, wherein the analyte comprises at least one selected from the group consisting of proteins, peptides, deoxyribonucleic acid, ribonucleic acid and polysaccharides, in particular at least one protein or peptide, in particular a protein or peptide mixture.

30. Use of a supercharging reagent in electrospray ionization (ESI) of an analyte for improving an overall charge state of the analyte, wherein the supercharging reagent is introduced separately from the analyte into an ESI device.