Apparatus for electrospray ionization and method for electrospray ionization using the same

An apparatus for electrospray ionization may include: a platform including an inlet port, a first channel connected to the inlet port, a second channel connected to the first channel, and an outlet port connected to the second channel; a nebulizer provided in the first channel and configured to spray inert gas to a sample sprayed into the first channel through the inlet port; and a focusing lens provided in the second channel and configured to focus ions produced from the sprayed sample toward the outlet port.

TECHNICAL FIELD

Embodiments relate to an apparatus for electrospray ionization (ESI) and a method for ESI using the same.

BACKGROUND ART

In the field of bioscience and medicine, a systematic analysis of disease-related proteins is required for treatment and prevention of diseases. With the advancement in basic researches for drug discovery in molecular biology and genomics, the territory of drug discovery is changing rapidly and new methods are being developed for drug discovery as exemplified by the genomic drug discovery.

Also, in the field of bioscience and medicine including development of new drugs, identification of materials exhibiting physiological activities for specific diseases or under specific conditions is required. Since those biologically active substances are mostly proteins, elucidation of structures and functions of the proteins is of crucial importance.

Since the analysis of proteins is very complicated because of their various characteristics associated with molecular weight, isoelectric point (pI), hydrophilic or hydrophobic nature, or the like, it is needed to first separate the proteins and identify them based on mass spectrometry, bioinformatics, etc. Considering that the proteins related with diseases exist in relatively lower quantities than other proteins, high-performance protein separation techniques and low detection limits for the separated proteins are needed.

The electrospray ionization (ESI) technique was first introduced in 1984. Thermally unstable biochemical substances such as proteins, peptides and sugars are unsuited for structural analysis and characteristic study by gas chromatography-mass spectrometry (GC-MS).

For separation and fractionation of those thermally unstable biochemical substances, high-performance liquid chromatography (HPLC) is widely employed. For structural analysis of various biochemical substance separated by HPLC as well as qualitative and quantitative analysis, the biochemical substances dissolved in a solution are converted into charged ions by means of ESI and then injected into a mass spectrometer. Then, the mass spectrometer performs structural analysis through mass measurement of the injected ions using mass spectrometry (MS) spectrums and tandem spectrums (MS/MS spectrums).

The most frequently employed method for sample ionization by ESI is to inject the biochemical substances such as proteins or peptides eluted using a microsyringe pump or an HPLC pump capable of microflow rate control into a capillary emitter having an inner diameter of several to tens of micrometers (e.g. about 1 μm to about 20 μm). Then, by directly injecting heated nitrogen gas (e.g., to about 100° C. to about 300° C.) to the outlet port of the capillary emitter while applying a high voltage thereto, desolvation of the droplet formed by the electrospray is facilitated and ionization is enhanced.

However, since the nitrogen gas is injected directly to the capillary emitter, the capillary emitter may be shaken or the ions produced by the electrospray may be diffused. As a result, the movement of charged ions through the inlet port of the mass spectrometer may be affected.

DISCLOSURE OF INVENTION

Technical Problem

An aspect of the present invention is directed to providing an apparatus for electrospray ionization (ESI) capable of focusing ions produced by ESI to minimize diffusion thereof while taking advantage of a nebulizer using heated nitrogen gas, and a method for ESI using the same.

Solution to Problem

According to an embodiment, an apparatus for electrospray ionization (ESI) may include: a platform including an inlet port, a first channel connected to the inlet port, a second channel connected to the first channel, and an outlet port connected to the second channel; a nebulizer provided in the first channel and configured to spray inert gas to a sample sprayed into the first channel through the inlet port; and a focusing lens provided in the second channel and configured to focus ions produced from the sprayed sample toward the outlet port.

According to an embodiment, a method for ESI may be performed using the apparatus for ESI and may include: spraying the sample into the first channel through the inlet port; spraying the inert gas to the sprayed sample using the nebulizer; and focusing the ions produced from the sprayed sample toward the outlet port using the focusing lens.

Advantageous Effects of Invention

An apparatus for electrospray ionization (ESI) according to an aspect of the present invention may be used as an ESI source kit for liquid chromatography-mass spectrometry (LC-MS). In that case, the apparatus for ESI may be used for structural analysis and biochemical study of protein mixtures extracted from human blood or cells and peptide mixtures acquired from enzymatic processes in proteomic researches.

In particular, when applied for structural analysis or qualitative and quantitative analysis of various proteins related with human diseases, the apparatus for ESI exhibits improved ionization efficiency and lower limit of detection (LOD) for mass spectrometry as compared to the conventional ESI techniques. Accordingly, it may be utilized for exploration of biomarkers related with human diseases and top-down proteomic researches allowing structural study in protein level.

MODE FOR THE INVENTION

Exemplary embodiments now will be described more fully hereinafter with reference to the accompanying drawings, in which exemplary embodiments are shown. This disclosure may, however, be embodied in many different forms and should not be construed as limited to the exemplary embodiments set forth therein. Rather, these exemplary embodiments are provided so that this disclosure will be thorough and complete, and will fully convey the scope of this disclosure to those skilled in the art. In the description, details of well-known features and techniques may be omitted to avoid unnecessarily obscuring the presented embodiments.

The terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of this disclosure. As used herein, the singular forms “a”, “an” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, the use of the terms a, an, etc. does not denote a limitation of quantity, but rather denotes the presence of at least one of the referenced item. The use of the terms “first”, “second” and the like does not imply any particular order, but they are included to identify individual elements. Moreover, the use of the terms first, second, etc. does not denote any order or importance, but rather the terms first, second, etc. are used to distinguish one element from another. It will be further understood that the terms “comprises” and/or “comprising” or “includes” and/or “including” when used in this specification, specify the presence of stated features, regions, integers, steps, operations, elements, and/or components, but do not preclude the presence or addition of one or more other features, regions, integers, steps, operations, elements, components, and/or groups thereof.

In the drawings, like reference numerals denote like elements. The shape, size and regions, and the like, of the drawing may be exaggerated for clarity.

FIG. 1is an exploded perspective view of an apparatus for electrospray ionization (ESI) according to an embodiment, andFIG. 2is a perspective view showing an assembled state of the apparatus for ESI illustrated inFIG. 1.

Referring toFIG. 1andFIG. 2, an apparatus for ESI may comprise: a platform10comprising a first block11and a second block12; and a nebulizer20and a focusing lens30provided in the platform10. In an embodiment, the apparatus for ESI may further comprise a rubber ring40provided between the nebulizer20and the first block11of the platform10to prevent gas leakage.

The platform10serves as a body to fix the position of the nebulizer20and the focusing lens30in the apparatus for ESI. The platform10may be made of any processable material. For example, the platform10may be formed of acryl. The platform10may have a cylindrical shape, and the first block11and the second block12may also have cylindrical shapes. However, this is only exemplary, and the platform10may have other appropriate shapes in other embodiments.

The first block11of the platform10may have an inlet port110. The inlet port110is the portion where a liquid sample is injected into the platform10for ESI. The inlet port110may completely penetrate the first block11. The sample may be various biochemical substances in liquid state, including proteomes, peptides or other macro-molecules. The sample may be injected into the platform10while a high voltage is applied for ESI.

The inlet port110may be formed to be connected to, for example, a capillary emitter or a nanoflow capillary column through which the sample is transferred. For example, the inlet port110may be formed such that a 1/16-inch male nut and/or a 1/16-inch ferrule available from Upchurch Scientific, Inc., which is commonly used to join tubings when forming a flow pathway in high-performance liquid chromatography (HPLC), may be coupled therewith. However, the type of the inlet port110is not limited thereto, but may be determined appropriately depending on the shapes and types of the connection parts.

The second block12of the platform10may have a first channel121, a second channel122and an outlet port123. Through, for example, a tubing equipped at the inlet port110of the first block11, the sample may be injected into the first channel121. The first channel121is a space for fixing the nebulizer20, and the second channel122is a space for fixing the focusing lens30. The first channel121, the second channel122and the outlet port123may completely penetrate the second block12.

In an embodiment, the second block12may have one or more first hole(s)124connected from outside to the first channel121for injection of inert gas to the nebulizer20. The first hole124may be formed such that a ⅛-inch male nut and/or a ⅛-inch ferrule available from Upchurch Scientific, Inc. may be coupled therewith, but without being limited thereto. And, in an embodiment, the second block12may have a second hole125connected to the second channel122for electrical connection of the focusing lens30with outside.

By positioning the nebulizer20and the focusing lens30respectively in the first channel121and the second channel122of the second block12and then coupling the second block12with the first block11using, for example, a bolt, the apparatus for ESI according to an embodiment may be set up. Details about the shapes of the first block11and the second block12and the coupling of the first block11and the second block12will be described later referring toFIGS. 3,4,5and6.

As described, the nebulizer20may be located in the first channel121of the second block12. When the sample is sprayed into the first channel121through, for example, a tubing equipped at the inlet port110, the sample may become droplets with a size of tens to hundreds of micrometers. As the nebulizer20sprays heated inert gas to the area where the sample is sprayed, solvents are removed from the droplets (desolvation) and only sample ions remain. For example, the nebulizer20may be configured to spray nitrogen gas heated to about 100° C. to about 300° C., but without being limited thereto.

If there is a void space at the portion where the nebulizer20is coupled with the second block12, movement of gas may be non-uniform. Accordingly, in order to minimize non-uniform gas movement, the outside portion of the nebulizer20may be sealed with Teflon, silicone or other suitable materials, so that no space is formed between the nebulizer20and the inside surface the second block12. Further, in an embodiment, the rubber ring40may be inserted between the nebulizer20and the first block11to prevent gas leakage.

The nebulizer20may be made of stainless steel. And, the nebulizer20may have a cylindrical shape. However, this is only exemplary, and the nebulizer20may be made of other suitable materials and/or have other appropriate shapes.

The focusing lens30may be located in the second channel122of the second block12. The focusing lens30is a device for focusing the sample ions generated when the sample sprayed into the first channel121meets the heated inert gas sprayed by the nebulizer20toward the outlet port123. That is to say, the focusing lens30may serve to prevent spreading of the multiple charged ions of the biochemical substance produced by ESI.

In an embodiment, the focusing lens30may have a conical shape for efficient collection of diffused ions. When the focusing lens30has a conical shape, the portion where the ions are injected may be relatively wider for easier collection of the diffused ions, and the portion where the ions are discharged may be relatively narrower for easier focusing of the ions. For example, the focusing lens30may have a diameter of about 7 mm at the inlet portion and a diameter of about 2 mm at the outlet portion. The focusing lens30may be made of stainless steel.

However, the material and shape of the focusing lens30are not limited to those described above. The focusing lens30may be made of other conducting materials such as metal and may have other shapes allowing focusing of the ions.

The focusing lens30may focus the sample ions using a potential gradient. The potential gradient formed by the focusing lens30may be selected adequately depending on the polarity of the sample ions produced by ESI. For example, if the sample ions are positively (+) charged ions, a positive (+) potential gradient may be formed at the focusing lens30. And, if the sample ions are negatively (−) charged ions, a negative (−) potential gradient may be formed at the focusing lens30. It may be configured such that a voltage applied to the focusing lens30may be controllable.

For this, the focusing lens30may be electrically connected to an external power supply (not shown). For example, a conducting wire may be connected between the focusing lens30and the external power supply through the second hole125of the second block12. For example, the end portion of the conducting wire may be drawn by about 2 mm to about 3 mm inward the second channel122of the second block12and fixed, and then the focusing lens30may be inserted in the second channel122, so as to connect the conducting wire to the focusing lens30. The presence of impurities in the second block12may cause difficulties in positioning the focusing lens30, and thus, the second block12may be cleaned to remove the impurities before connecting the conducting wire to the focusing lens30.

By providing the nebulizer20with a cylindrical shape in the cylindrical platform10as describe above, a uniform nitrogen gas flow may be provided and the nitrogen gas flow may be focused toward the capillary emitter. Since the nitrogen gas is distant from the capillary emitter, shaking of the capillary emitter by the nitrogen gas flow may be minimized. Further, since the voltage-controllable focusing lens30for preventing diffusion of and focusing the ions produced by ESI is provided before the capillary emitter, the ionized samples may be easily focused and injected to a mass spectrometer.

FIG. 3is a plan view of the first block11of the platform10of an apparatus for ESI according to an embodiment, andFIG. 4is a side cross-sectional view of the first block11of the platform10of an apparatus for ESI according to an embodiment.

Referring toFIGS. 3 and 4, the first block11may be in the form of a cylinder with a diameter R1of about 42 mm and a thickness D1of about 15 mm. The first block11may have the inlet port110for coupling and fixing with, for example, a capillary emitter or a nanoflow capillary column.

The inlet port110may have a spiral shape with an outer diameter r11of about 6.2 mm, an inner diameter r12of about 1.8 mm and a depth d11of about 10 mm, so that a 1/16-inch male nut or a 1/16-inch ferrule may be coupled therewith. The remaining portion of the inlet port110except for the spiral structure may have a depth d12of about 5 mm. The inlet port110may completely penetrate the first block11.

The first block11may have one or more coupling port(s)111for connection with the second block12(seeFIGS. 5 and 6). For example, the first block11may have four coupling ports111located at four apices of a square. The first block11may be coupled with the second block12by inserting a bolt in each coupling port111. Each coupling port111may have a diameter r13of about 4 mm. However, the shape and number of the coupling ports111may be different from those described above. Also, the first block11and the second block12may be coupled differently, not using the coupling ports111.

FIG. 5is a plan view of the second block12of the platform10of an apparatus for ESI according to an embodiment, andFIG. 6is a side cross-sectional view of the second block12of the platform10of an apparatus for ESI according to an embodiment.

Referring toFIGS. 5 and 6, the second block12may be in the form of a cylinder with a diameter R2of about 42 mm and a thickness D2of about 35 mm. The diameter R2of the second block12may be the same as the diameter R1of the first block11. The second block12may have the first channel121, the second channel122and the outlet port123. The first channel121, the second channel122and the outlet port123may be sequentially connected and may completely penetrate the second block12.

The first channel121may be a cylindrical space formed with a depth d21of about 15 mm from the surface of the second block12. The first channel121may have a cross-sectional diameter r21of about 12 mm. The first channel121is a space for providing the nebulizer. The shape and size of the first channel121described are only exemplary, and may be determined appropriately according to the nebulizer used.

In an embodiment, the second block12may further have one or more gas circulation chamber(s)120connected to the first channel121and provided outside the circumference of the first channel121. The gas circulation chamber120is provided to allow smoother movement of the inert gas sprayed by the nebulizer in the first channel121, and may be formed with a thickness d0of about 2 mm and a length l0of about 10 mm outward the first channel121.

The second channel122may be connected with the first channel121at the end portion of the first channel121. The second channel122may be a cylindrical space formed with a depth d22of about 10 mm from the portion where the first channel121ends. The cross-sectional diameter r22of the second channel122may be smaller than the cross-sectional diameter r21of the first channel121. For example, the second channel122may have a cross-sectional diameter r22of about 8 mm.

The outlet port123may be connected to the second channel122at the end portion of the second channel122. The outlet port123may be a space formed with a depth d23of about 10 mm from the portion where the second channel122ends. The outlet port123may have a two-stage structure with a cross-sectional diameter r231of about 1 mm at a predetermined portion close to the second channel122and a cross-sectional diameter r232of about 3 mm at the remaining portion close to the surface of the second block12. However, the shape of the outlet port123is not limited thereto. For example, in another embodiment, the outlet port123may have a cylindrical shape with one cross-sectional diameter.

In an embodiment, the second block12may have the one or more first hole(s)124for connection of the first channel121with outside. For example, the one or more first hole(s)124may be formed to penetrate the second block12from the side surface of the second block12to the first channel121. The one or more first hole(s)124is the portion for injecting the inert gas to the nebulizer, which will be provided in the first channel121. In case the second block12has the gas circulation chamber120, each of the first holes124may be connected to the gas circulation chamber120.

Also, in an embodiment, the second block12may have the second hole125for connection of the second channel122with outside. For example, the second hole125may be formed to penetrate the second block12from the side surface of the second block12to the second channel122. The second hole125is the portion for electrical connection with the focusing lens, which will be provided in the second channel122. The focusing lens may be electrically connected with the external power supply by inserting a conductor in the second hole125.

Further, in an embodiment, the second block12may have one or more coupling port(s)126for connection with the first block11. For example, the second block12may have four coupling ports126located at four apices of a square. The first block11may be coupled with the second block12by inserting a bolt in each coupling port126. Each coupling port126may have a diameter r26of about 4 mm. However, the shape and number of the coupling ports126are not limited thereto.

In an embodiment, the first hole124and the second hole125may have spiral shapes with outer diameters r241r251of about 6.2 mm, inner diameters r242, r252of about 1.8 mm and depths d24, d25of about 10 mm from the side surface of the second block12. In the embodiment illustrated inFIGS. 5 and 6, the first hole124and the second hole125have the same spiral shape. However, this is only exemplary, and the first hole124and the second hole125may have different structures.

In the first block11and the second block12described referring toFIGS. 3,4,5and6, the inlet port110of the first block11and the first hole124and the second hole125of the second block12are formed such that a tubing may be coupled therewith using, for example, a 1/16-inch male nut or a 1/16-inch ferrule available from Upchurch Scientific, Inc. Further, a 1/16-inch sleeve with an inner diameter of about 380 μm, which is available from Upchurch Scientific, Inc., may be used to prevent gas leakage during assemblage of the connection parts. However, these are only exemplary, and the configuration of the inlet port110, the first hole124and the second hole125may be different depending on the type of the connection parts.

FIG. 7is a schematic view illustrating connection of an apparatus for ESI according to an embodiment to an introducing part of a mass spectrometer. For the brevity of explanation, details of the features that may be easily understood by those skilled in the art from the existing art will be omitted.

Referring toFIG. 7, a capillary emitter2transferring a liquid sample may be connected to a micro-T3, for example, using a silica capillary having an outer diameter of about 360 μm. Between the capillary emitter2and the micro-T3, a microsyringe pump or a micro HPLC pump (not shown) for pumping the sample may be provided.

The micro-T3may be connected to a power supply4to apply a voltage to the sample transferred from the capillary emitter2. For example, the micro-T3may be electrically connected to the power supply4for application of a high voltage using a platinum (Pt) wire7. The power supply4may apply a voltage of about 1.5 kV to about 2.0 kV to the Pt wire7connected to the micro-T3for ESI. However, this voltage is only exemplary, and the amplitude of the voltage may be different depending on, for example, the sample composition and/or flow rate.

The sample to which the voltage is applied using the micro-T3may be injected into an apparatus1for ESI through, for example, a silica capillary. When the sample is sprayed into the apparatus1for ESI, heated inert gas may be sprayed to the area where the sample is sprayed so as to remove a solvent from sample droplets and allow only sample ions to remain. Then, the apparatus1for ESI may focus the ions using a focusing lens30and inject them to a mass spectrometer6. For example, the apparatus1for ESI may focus the ionized sample to an inlet orifice of the mass spectrometer6. The focusing lens30may be electrically connected to a power supply5. The power supply5may apply a voltage of about 50 V to about 300 V to the focusing lens30.

The mass spectrometer6is operated using the two power supplies4,5. The power supplies4,5are respectively electrically connected to the Pt wire7of the micro-T3and the focusing lens30of the apparatus1for ESI so as to apply a controllable high voltage. Ground electrodes of the power supplies4,5may be grounded to the inlet orifice of the mass spectrometer6where the ionized sample is injected.

The mass spectrometer6performs structural analysis of the sample through mass measurement of the sample ions injected from the apparatus1for ESI using mass spectrometry (MS) spectrums and tandem spectrums (MS/MS spectrums). Details about the configuration and operation of the mass spectrometer6will be omitted since they are well known to those skilled in the art.

The above configuration where the apparatus1for ESI is applied in the introducing part of the mass spectrometer6may be applied in an ESI source kit for liquid chromatography-mass spectrometry (LC-MS). In particular, the apparatus1for ESI may be used for proteomic researches employing HLPC and mass spectrometer.

FIG. 8compares a mass spectrometry spectrum obtained using an apparatus for ESI according to an embodiment with one obtained according to the related art.

FIG. 8shows the mass spectrometry spectrums of cytochrome c (about 12.4 kDa) as standard protein, obtained by diluting with a 50:50 (v/v) mixture of methanol containing about 0.2% formic acid and water (H2O) to a concentration of 10 fmol and performing ESI at a flow rate of about 0.5 μm/min using a microsyringe pump. A Fourier transform ion cyclotron resonance (FT-ICR) mass spectrometer operating at a magnetic field of about 15 T was used.

InFIG. 8, peaks610and620are those of the multiple charged ions of cytochrome C obtained according to the related art and according to an embodiment of this disclosure, respectively. In both cases, cytochrome C was injected into a capillary emitter at a rate of about 0.5 μm/min and ESI was carried out by applying a voltage of about 1.7 kV. However, in the case where the apparatus for ESI according to an embodiment was used, nitrogen gas was injected from the nebulizer at a rate of about 0.2 L/min to remove the solvent and a positive (+) voltage of about 50 V was applied to the focusing lens.

As seen fromFIG. 8, when the apparatus for ESI according to an embodiment was used, a detection sensitivity of about 2 times was exhibited for the [M+14H+]14+ion having a mass-to-charge ratio (m/z) of about 883.8 as compared to the related art case. The detection sensitivity of the multiple charged ions increased gradually as the number of hydrogen ions decreased, starting from the [M+16H+]16+ion formed by 16 hydrogen ions and having a mass-to-charge ratio of about 773.4. Overall, the apparatus for ESI according to an embodiment showed significantly improved ionization efficiency over the related art.

While the exemplary embodiments have been shown and described, it will be understood by those skilled in the art that various changes in form and details may be made thereto without departing from the spirit and scope of this disclosure as defined by the appended claims.

In addition, many modifications can be made to adapt a particular situation or material to the teachings of this disclosure without departing from the essential scope thereof. Therefore, it is intended that this disclosure not be limited to the particular exemplary embodiments disclosed as the best mode contemplated for carrying out this disclosure, but that this disclosure will include all embodiments falling within the scope of the appended claims.

INDUSTRIAL APPLICABILITY

Embodiments relate to an apparatus for electrospray ionization (ESI) and a method for ESI using the same.