# Patent

## Title
Nano-electrospray ionization technique and device

## Abstract

Nano-electrospray ionization techniques include the introduction of a separation solvent containing a sample to a column-integrated needle having a column filled with a resin for liquid chromatography. The separated sample components are sprayed from the tip of the column-integrated needle toward a sample introduction orifice of a mass spectrometer. An organic solvent is simultaneously introduced to a solvent-supplying needle. The organic solvent is supplied from the tip of the solvent-supplying needle to the tip of the column-integrated needle.

## Background
CROSS-REFERENCE TO RELATED APPLICATIONS
This application is a continuation of U.S. patent application Ser. No. 12/999,434 filed on 16 Dec. 2010, which is a U.S. Nationalization of PCT International Application No. PCT/JP2010/059732 filed on 2 Jun. 2010, which claims priority to Japanese Patent Application No. 2009-137085 filed on 8 Jun. 2009. Each of the foregoing applications is hereby incorporated by reference in their entirety.


TECHNICAL FIELD
Embodiments relate to nano-electrospray ionization techniques for a mass spectrometer (MS) interface of a nano-flow liquid chromatography-mass spectrometer (LC-MS).
BACKGROUND
Since the discovery of RNA interference, it has been clarified that various types of small RNAs are deeply involved in vital activities and diseases caused by abnormalities thereof, such as cancers. Usually, a small RNA is analyzed by being converted to a cDNA with an enzyme, so-called reverse transcriptase, and then measured by a molecular biological method. Such a method has the disadvantages that analysis of chemical modification, which is important for expression of function, and quantitative measurement of the small RNA are impossible.
In known nano-LC-ESI-MS methods having the above-described restrictions, since a hydrophilic nucleotide having (−) charge is eluted from an LC column during a separation process under a low organic solvent concentration, the surface tension of the separate solution is increased. Therefore, the efficiency of ionization in a nano-electrospray ionization technique is decreased, resulting in a problem of a significant decrease in analysis sensitivity. That is, in the nano-electrospray ionization technique in which an LC separation solution having a low organic solvent concentration is used, droplets are generated at the tip of a needle for spraying the separation solution and cause clogging, which makes the spraying intermittent or discontinuous and also the analysis itself impossible.
Against these problems regarding analysis in the micro order (i.e., a flow rate of a micro-litter (μL/min) level), methods have been developed for preventing the formation of droplets at the needle tip, wherein a needle having a double-tube structure in which a “sheath tube” parallel surrounds a needle for spraying separation solution is used. The sheath tube supplies an organic solvent (sheath solution) to the LC separation solution to be sprayed (Journal of Chromatography A, 870, (2000), 413-424).
Though the above-mentioned technology is an effective method for the analysis at the micro-level, the technology has not reached a level for being applied to analysis at the nano-level (i.e., a flow rate at a nano-litter (nL/min) level). This is because that it is technically difficult to form a double-tube structure including a micro sheath tube for suitably supplying a sheath solution to a micro needle having an inner diameter of about 75 to 200 μm used in the nano-LC-ESI-MS method.
SUMMARY
According to aspects of the embodiments described herein, nano-electrospray ionization methods enable significantly high accurate mass spectrometry by stably ionizing sample components by an LC-ESI-MS method at the nano-level.
Embodiments described herein result in the prevention of the generation of droplets at the tip of a needle, which previously has made the analysis sensitivity decrease. Thus, ions can be stably formed with high efficiency, and thereby the accuracy of mass spectrometry is significantly increased by externally supplying an appropriate amount of an organic solvent to the tip of a column-integrated needle for introducing a sample with a separation solvent, separating the sample into each component, and spraying it.
According to an aspect of the nano-electrospray ionization methods, a separation solvent containing a sample is introduced to a column-integrated needle having a column filled with a resin for liquid chromatography. The separated sample components are sprayed from the tip of the column-integrated needle toward a sample introduction orifice of a mass spectrometer. Simultaneously, the organic solvent is introduced to a solvent-supplying needle and the organic solvent is supplied from the tip of the solvent-supplying needle to the tip of the column-integrated needle.
In the nano-electrospray ionization methods, the sample generally contains a small RNA.
Furthermore, in the nano-electrospray ionization methods, the flow rates of the separation solvent containing the sample and the organic solvent can each be, for example, from 50 to 500 nL/min.
Furthermore, in the nano-electrospray ionization methods, the organic solvent can be methanol, acetonitrile, or a volatile acid mixture thereof.
Furthermore, in the nano-electrospray ionization methods, the organic solvent can be mixed with nitrogen gas, and then the mixture is introduced to the solvent-supplying needle.
Furthermore, in an aspect of the nano-electrospray ionization methods, the flow rate of the nitrogen gas can be from 0.4 to 40 mL/min.
In addition, a nano-electrospray ionization device includes a column-integrated needle having a column filled with a resin for liquid chromatography and a needle is arranged to be in contact with or close to a sample introduction orifice of a mass spectrometer. A solvent-supplying needle is held on first three-dimensional positioning means that is positioned on a base table on which the column-integrated needle is positioned. The tip of the solvent-supplying needle is arranged to be in contact with or close to the tip of the column-integrated needle by driving the positioning means.
In the nano-electrospray ionization device, the column-integrated needle can be held on a second three-dimensional positioning means positioned on the base table.
Furthermore, in the nano-electrospray ionization device, the first three-dimensional positioning means can be positioned on the second three-dimensional positioning means.
Furthermore, in the nano-electrospray ionization device, the base table can be positioned near the mass spectrometer.
In another aspect, a method for performing mass spectrometry of a biological sample includes discharging, from a first needle, first droplets of a biological sample that contains nucleotide fragments. The droplets are discharged toward an orifice of a mass spectrometer. Second droplets of an organic solvent are discharged from a second needle, such that second droplets mix with the first droplets. A mixture of the first droplets and the second droplets is received at the orifice of the mass spectrometer.
According to this aspect, discharging the second droplets substantially prevents accumulation of liquid at a tip of the first needle that would otherwise disrupt the discharge of the first droplets from the first needle.
The method of performing mass spectrometry can include spectrometrically determining at least one of a type, an amount, and a chemical structure of small RNA, and the mass spectrometry can be performed as part of a method of diagnosing a disease or disorder.
In an aspect of the embodiments described herein, methods relate to the analysis of the chemical structure and function of a small RNA by directly identifying the small RNA present in a biological specimen by mass spectrometry. The small RNA as an object is cleaved with an RNase. The resulting nucleotide fragments are ionized by an electrospray ionization technique (ESI technique) while being separated by liquid chromatography (LC). The type, amount, chemical structure, and so on, of the small RNA are analyzed on the basis of an MS/MS spectrum obtained by high-performance mass spectrometry (MS) method. That is, in this so-called nano-LC-ESI-MS method, since a component in an amount of the order of fmol (10′15 mol) is analyzed, a nucleotide fragment that is to be applied to mass spectrometry as a sample is separated by LC at an ultra-low flow rate of about 50 to 500 nL/min and then ionized as nanodroplets. Furthermore, in analysis of a phosphate group-rich small RNA, a hydrophilic nucleotide is applied, having (−) charge to mass spectrometry in the “negative mode”.
According to embodiments described herein, an ultra small amount of a low-molecular-weight component can be stably ionized, and significantly high-accurate analysis can be achieved. This makes it possible to analyze a small RNA in detail and with excellent reliability, and makes it possible to highly contribute to the application of the substance to, for example, medical treatments or drugs.



DESCRIPTION OF THE FIGURES
FIG. 1 is a block diagram showing a constitution of a common LC-ESI-MS;
FIG. 2 is a block diagram showing a constitution of an LC-ESI-MS in a nano-electrospray ionization technique;
FIG. 3 is a block diagram showing a constitution of an LC-ESI-MS in a nano-electrospray ionization technique;
FIG. 4 is a graph showing a change (gradient) in organic solvent concentration in separation solvent in a nano-LC-ESI-MS method;
FIG. 5 is a diagram showing a distance between needles according to embodiments described herein;
FIG. 6 is a front view of a nano-electrospray ionization device;
FIG. 7 is a side view of the nano-electrospray ionization device;
FIG. 8A is a chromatogram of RNase T1 digestion products of human 5s-rRNA determined by a nano-LC-ESI-MS method;
FIG. 8B is a chromatogram of RNase T1 digestion products of human 5 s-rRNA determined by a conventional nano-LC-ESI-MS method;
FIG. 9A is a chromatogram of trypsin digestion products of human serum albumin determined by a nano-LC-ESI-MS method; and
FIG. 9B is a chromatogram of trypsin digestion products of human serum albumin determined by a conventional nano-LC-ESI-MS.



DETAILED DESCRIPTION
Nano-electrospray ionization techniques according to embodiments described herein are nano-LC-ESI-MS methods for analyzing ultra small amounts of components with low molecular weights, wherein an organic solvent is supplied to the tip of an ESI needle, which sprays a sample separated by LC and ionizes the sample, from a needle other than the ESI needle.
In general, in a nano-LC-ESI-MS method, an ESI needle integrated to a column for LC can be used for minimizing, in particular, the post-column dead volume. The column-integrated needle is one in which a reversed-phase separation silica gel column having a tapering structure is filled with a resin for liquid chromatography and can immediately spray the sample components separated by the resin from the tapering column tip and form droplets of the components. The inner diameter of the narrowed tip can be designed to be about 5 to 15 μm.
In addition, the samples used in the nano-electrospray ionization techniques are not particularly limited as long as it can be used in a common nano-LC-ESI-MS method, and examples thereof include those containing, for example, protein, DNA, or RNA and mixtures thereof. According to an embodiment, a sample containing a small RNA, which have been difficult to analyze by conventional methods, can be processed. Examples of the small RNA include functional non-coding RNA (ncRNA) of about 20 to 30 nucleotides contained in a biological sample, such as microRNA or siRNA. These samples can be used for analysis by being dissolved in a buffer or salt solvent.
FIG. 1 is a schematic diagram showing a constitution of a common nano-LC-ESI-MS. In FIG. 1, the nano-LC-ESI-MS includes sample-separating means 12 and mass-analyzing means 14.
The sample-separating means 12 includes a separation solvent 18, a separation solvent delivery pump 20, an injector 22, a gradient generator 24, a spray electrode 26, and a column-integrated needle 28, and these elements are properly connected to one another with flow paths. The mass-analyzing means 14 includes a sample introduction orifice 30 and a mass spectrometer 32 connected to the orifice 30. The orifice 30 is arranged so as to open toward the direction directed by the tip of the column-integrated needle 28.
In the sample-separating means 12, an analysis sample appropriately dissolved in, for example, water is sent to the gradient generator 24 by a flow of a separation solvent introduced by the injector 22 and is delivered by the separation solvent delivery pump 20 at a predetermined flow rate. The gradient generator 24 adjusts the composition and concentration of the separation solvent according to the elution characteristics of the sample components and more precisely controls the subsequent separation of the sample components in the column-integrated needle 28. In the column-integrated needle 28 having a column filled with a resin for liquid chromatography, the sample introduced with the separation solvent is separated to each component depending on the difference in hydrophilicity to the separation solvent adjusted by the gradient generator 24 and the resin. Furthermore, the column-integrated needle 28 is applied with a voltage of about −1 to −2 kV by the spray electrode 26, and the sample components separated in the column are sprayed from the tip of the column-integrated needle as charged tiny droplets in which a large number of separation solvent molecules are associated to an ion. The sample components in a droplet form are ionized by the ions inside the droplets and are introduced to the mass-analyzing means 14 through the sample introduction orifice 30 positioned in the direction directed by the needle tip.
FIG. 2 is a schematic diagram showing a constitution in which a nano-electrospray ionization technique is introduced to the constitution of the above-described nano-LC-ESI-MS.
The nano-LC-ESI-MS shown in FIG. 2 includes solvent-supplying means 36, in addition to the sample-separating means 12 and the mass-analyzing means 14. The solvent-supplying means 36 includes an organic solvent 38, an organic solvent delivery pump 40, and a solvent-supplying needle 42, and the solvent-supplying needle 42 is positioned in such a manner that the direction of the tip of the solvent-supplying needle 42 points the tip of the column-integrated needle 28 of the sample-separating means 12.
In this constitution, the process in which a sample is introduced, separated, and then ionized for mass spectrometry is the same as that in the constitution (FIG. 1) of the above-described common nano-LC-ESI-MS.
However, in this constitution, the organic solvent 38 is delivered and introduced to the solvent-supplying needle 42 at a predetermined flow rate by the organic solvent delivery pump 40, and at the same time of the above-mentioned process, the solvent-supplying means 36 supplies the organic solvent 38 to the tip of the column-integrated needle 28 of the sample-separating means 12 from the tip of the solvent-supplying needle 42.
In another constitution shown in FIG. 3, the solvent-supplying means 36 further includes a nitrogen gas tank 44. That is, in the constitution shown in FIG. 3, the organic solvent 38 is delivered by the organic solvent delivery pump 40 and is confluent with nitrogen gas delivered from the nitrogen gas tank 44, and the solvent-supplying means 36 supplies the organic solvent 38 to the tip of the column-integrated needle 28 from the solvent-supplying needle 42 as a mixture with the nitrogen gas.
The separation solvent can be a mobile phase that is usually used in liquid chromatography (LC), and, for example, a mixture system of water or a salt solution and an organic solvent (for example, a methanol solution, an acetonitrile solution, a formic acid aqueous solution, or a triethylamine acetate aqueous solution) can be used.
In a conventional nano-LC-ESI-MS method, since minor components contained in a sample are closely separated depending on hydrophilicity (solubility) to the organic solvent in the separation solvent, in the gradient generator 24, the composition ratio of the separation solvent is usually adjusted so as to have a gradient in which the organic solvent concentration is gradually increased from a low concentration to a high concentration. An ideal gradient line (concentration gradient of the organic solvent in the separation solvent) on this adjustment is that shown on the upper side of FIG. 4.
However, in actual measurement, in the stage where the concentration of the organic solvent in the separation solvent is low in the above-mentioned gradient, that is, in the initial stage of gradient analysis, the organic solvent concentration is low, such as several percent or less, and the droplets generated by spraying at this point are very large compared to those of the organic solvent only. Therefore, the analysis solvent in the initial stage remains at the tip of the column-integrated needle 28, due to the surface tension, and are frequently formed into droplets. Since the tip of the column-integrated needle used in nano-analysis is significantly small, if the droplets are generated, the spraying of sample components becomes intermittent (condition A) or is completely inhibited (condition B), as shown on the lower side of FIG. 4. As a result, normal analysis cannot be continued.
Accordingly, in the constitutions shown in FIGS. 2 and 3, the organic solvent is supplied to the tip, which serves as an opening for spraying the sample components, of the column-integrated needle so as to synchronize with the spraying of the sample components from the column-integrated needle, and thereby the separated sample components are diluted with the organic solvent not to remain at the opening for spraying. Thus, the generation of the droplets that prevent spraying is inhibited.
In the nano-LC-ESI-MS method including the nano-electrospray ionization shown in FIGS. 2 and 3, the sample to be introduced can advantageously be in a solution form, according to need, in which the sample is dissolved in an appropriate solvent such as water, ethanol, or acetone.
Furthermore, the flow rates of the separation solvent and the sample in the column-integrated needle 28 depend on the components of the sample and the type of the separation solvent, but can generally be 50 to 500 nL/min. In addition, the flow rate of the organic solvent supplied from the solvent-supplying needle 42 to the tip of the column-integrated needle 28 can also generally be 50 to 500 nL/min, in accordance with that of the sample solution, in order to avoid extreme dilution of the column elution solution.
Furthermore, the solvent-supplying needle 42 is not particularly limited in the material and the shape as long as it is tubular so that an organic solvent can be supplied at a very small flow rate as described above. Like the column-integrated needle, an ESI needle having a tip inner diameter of 5 to 15 μm can be advantageously used.
In addition, the organic solvent supplied from the solvent-supplying needle 42 may be any solvent that can be applied to a common liquid chromatography and can be methanol, acetonitrile, or a volatile acid (for example, formic acid or acetic acid) mixture thereof.
Furthermore, as shown in FIG. 3, when the organic solvent is mixed with nitrogen gas, the flow rate of the nitrogen gas can be, for example, 0.4 to 40 mL/min. The solvent-supplying needle in this case has an inner diameter of 30 to 75 μm, and the flow rate of the organic solvent mixed with the gas can be, for example, 50 to 500 nL/min. By supplying the nitrogen gas together with the organic solvent, the removal of the solvent from the sample components in the droplet form is accelerated, and the components are more efficiently ionized.
Furthermore, the column-integrated needle can be arranged close to the sample introduction orifice of the mass spectrometer in a degree capable of introducing the ionized sample components and in such a manner that the needle tip faces the orifice. In addition, the solvent-supplying needle is arranged to be in contact with or close to the column-integrated needle in such a manner that the tip thereof faces the column-integrated needle. The distance (distance indicated by “a” in FIG. 5) between the column-integrated needle and the solvent-supplying needle can be, for example, about 0 to 100 μm. A distance of 0 μm refers to that the tip of the solvent-supplying needle is in contact with the column-integrated needle. A distance of 100 μm is a rough target of the distance so that the organic solvent droplets supplied from the tip of the solvent-supplying needle reach the column-integrated needle, and a more suitable distance can be appropriately adjusted within a range that the organic solvent droplets can move between the needles.
Furthermore, in usual, in the electrospray ionization using a column-integrated needle (ESI needle), droplets that prevent the sample components from being sprayed from the tip of the needle tend to be generated from a position 1 to 2 mm behind the opening for spraying. Therefore, the tip of the solvent-supplying needle can advantageously be arranged about 1 to 2 mm behind the tip of the column-integrated needle (distance indicated by “b” in FIG. 5). By arranging each needle as in above, the generation of droplets can be more reliably avoided, and normal ionization can be continued.
From the standpoint of determining needle arrangement, the nano-electrospray ionization techniques can use a nano-electrospray ionization device having positioning means for adjusting the arrangement of the column-integrated needle and the solvent-supplying needle.
That is, nano-electrospray ionization devices have a structure in which the tip of the column-integrated needle is arranged to be in contact with or close to the sample introduction orifice of the mass spectrometer and the tip of the solvent-supplying needle can be arranged to be in contact with or close to the tip of the column-integrated needle.
The nano-electrospray ionization devices will be described according to embodiments below, but are not limited thereto.
Embodiment 1
An embodiment of the nano-electrospray ionization devices is shown in FIG. 6 (plan view) and FIG. 7 (side view). In FIGS. 6 and 7, the device is constituted of a base table 102, column-integrated needle holding means 104, solvent-supplying needle holding means 106, three-dimensional positioning means 108. The column-integrated needle holding means 104 and the three-dimensional positioning means 108 are positioned on the base table 102, and the solvent-supplying needle holding means 106 is positioned on the three-dimensional positioning means 108.
The column-integrated needle holding means 104 can make the direction directed by the tip of the column-integrated needle held by the holding means 104 face the sample introduction orifice of the mass spectrometer.
The three-dimensional positioning means 108 is movable in the three-dimensional directions (X, Y, and Z directions) with respect to the column-integrated needle. The tip of the solvent-supplying needle held by the solvent-supplying needle holding means 106 can be arranged to be in contact with or close to the tip of the column-integrated needle by operating the means 108.
Embodiment 2
In another embodiment, the column-integrated needle holding means is held on second three-dimensional positioning means positioned on the base table. The second three-dimensional positioning means can be positioned on the base table independently from the first three-dimensional positioning means having the solvent-supplying needle holding means, or can be positioned on the base table in such a manner that the first three-dimensional positioning means and the column-integrated needle holding means are positioned on the second three-dimensional positioning means. In this embodiment, the second three-dimensional positioning means operates the tip of the column-integrated needle in the three-dimensional directions with respect to the sample introduction orifice of the mass spectrometer, and the first three-dimensional positioning means operates the tip of the solvent-supplying needle in the three-dimensional directions with respect to the tip of the column-integrated needle. Thus, the needles can be arranged to be in contact with or close to the respective object.
The structures of the first and second three-dimensional positioning means are not particularly limited as long as they can respectively control the tips of the needles positioned thereon in the three-dimensional directions with respect to the respective object. The three-dimensional positioning means 108 exemplified in FIGS. 6 and 7 is a suitable structure. In the drawings, the three-dimensional positioning means 108 includes an X-axis frame 112 fitted with an X-axis feed screw 110 in the X-axis direction, a Y-axis frame 116 fitted with a Y-axis feed screw 114 in the Y-axis direction, and a Z-axis frame 120 fitted with a Z-axis feed screw 118 in the Z-axis direction. The frames have the respective stages (122, 124, and 126) that are movable in the corresponding axis directions. By assembling these frames to be each perpendicular to the base table 102, the needle held by the means can be moved in the three-dimensional directions (X, Y, and Z directions).
Furthermore, as exemplified in FIGS. 6 and 7, it is also possible to dispose a fixing stand 128 on the base table and to fix the entire devices on the base table to the mass spectrometer through the stand. The positioning of the column-integrated needle with respect to the sample introduction orifice can be more easily performed by fixing the entire devices to be close to the sample introduction orifice.
However, when the device is fixed to the mass spectrometer, a constitution can be employed in which the three-dimensional positioning means of the solvent-supplying needle is positioned at a position apart from the mass spectrometer, as in Embodiment 1, or in which the three-dimensional positioning means of the solvent-supplying needle and the three-dimensional positioning means of the column-integrated needle are assembled to be perpendicular to each other into a single unit, as in Embodiment 2. When the three-dimensional positioning means of the solvent-supplying needle is positioned on the base table at a position near the mass spectrometer, microvibration due to, for example, the turbo pump inside the mass spectrometer is propagated to the device, and the position of the solvent-supplying needle held by the positioning means may be gradually deviated.
EXAMPLES
Nano-electrospray ionization techniques and devices therefor will be specifically shown by examples below, but are not limited thereto.
Test Example 1
RNase T1 digestion products of human 5s-rRNA were analyzed using a nano-LC-ESI-MS (TOF) having a constitution shown in FIG. 2 in which the electrospray ionization device of the Embodiment 1 is connected. FIG. 8A shows the resulting chromatogram. Furthermore, FIG. 8B shows a chromatogram of the same sample by a common nano-LC-ESI-MS (TOF) having a constitution not having the solvent-supplying means as shown in FIG. 1.
The flow rate of the separation solvent containing a sample was 100 nL/min, and a mixture system of 100 nL/min of acetonitrile and 3.4 mL/min of nitrogen gas was supplied from the solvent-supplying means.
As shown in FIGS. 8A and 8B, in the measurement (FIG. 8B) by a conventional nano-LC-ESI-MS method, droplets were generated at the tip of the needle for spraying the sample solution during the electrospray ionization process, which made the spraying impossible soon after the starting of the test, and analysis data could not be obtained. On the other hand, in the measurement (FIG. 8A) by the nano-LC-ESI-MS method performed according to embodiments described herein, the spraying of the sample components from the column-integrated needle and the ionization stably progressed to give analysis data with high accuracy.
Test Example 2
Trypsin digestion products of human serum albumin were analyzed using the same system as that in Test Example 1. FIG. 9A shows the resulting chromatogram measured using the constitution having solvent-supplying means, and FIG. 9B shows the result by the constitution not having the solvent-supplying means.
The flow rate of the separation solvent containing a sample was 100 nL/min, and a mixture system of 100 nL/min of acetonitrile containing 0.1% formic acid and 3.4 mL/min of nitrogen gas was supplied from the solvent-supplying means.
As shown in FIGS. 9A and 9B, in the system (FIG. 9A) to which the nano-electrospray ionization technique and the device therefor according to embodiments described herein were applied, the analysis sensitivities for peptide components (in particular, YICENQDSISSK, FQNALLVR, and AVMDDFAAFVEK) in very small amounts were significantly high, compared to those in the system (FIG. 9B) to which the technique and the device were not applied.

## Claims

1. A method for performing mass spectrometry analysis of a biological sample, the method comprising:
discharging, from a first needle, first droplets of the biological sample comprising nucleotide fragments, the first droplets being discharged toward an orifice of a mass spectrometer;
discharging, from a second needle, second droplets of a first organic solvent, such that the second droplets mix with the first droplets to form a mixture;
receiving the mixture of the first droplets and the second droplets at the orifice of the mass spectrometer; and
spectrometrically determining from the received mixture the identity of the biological sample with the mass spectrometer.

2. The method according to claim 1, wherein discharging the second droplets substantially prevents accumulation of liquid at a tip of the first needle that would otherwise disrupt the discharge of the first droplets from the first needle.

3. The method according to claim 1, before discharging the first droplets from the first needle, further comprising:
separating the nucleotide fragments using liquid chromatography; and
ionizing the nucleotide fragments using electrospray ionization.

4. The method according to claim 1, wherein discharging, from the first needle, the first droplets of the biological sample comprises discharging the first droplets at a flow rate in a range from about 50 nL/min to about 500 nL/min.

5. The method according to claim 1, wherein discharging, from the second needle, the second droplets of the first organic solvent comprises discharging the second droplets at a flow rate in a range from about 50 nL/min to about 500 nL/min.

6. The method according to claim 1, wherein the nucleotide fragments comprise RNA.

7. The method according to claim 6, wherein spectrometrically determining from the received mixture the identity of the biological sample further comprises spectrometrically determining from the received mixture whether the identity of the biological sample includes at least one member selected from the group consisting of a type of the RNA, an amount of the RNA, and a chemical structure of the RNA with the mass spectrometer.

8. The method according to claim 1, wherein discharging the first droplets further comprises discharging the first droplets including the biological sample and a second organic solvent each at a flow rate of about 50 nl/min to about 500 nl/min.

9. The method according to claim 8 wherein the second organic solvent comprises at least one of methanol, acetonitrile, or a volatile acid mixture thereof.

10. The method according to claim 1, wherein the first organic solvent comprises at least one of methanol, acetonitrile, or a volatile acid mixture thereof.

11. The method according to claim 1, further comprising mixing the first organic solvent with a nitrogen gas prior to introducing to the second needle.

12. The method according to claim 11, wherein mixing the first organic solvent with a nitrogen gas further comprises mixing the first organic solvent with the nitrogen gas, the nitrogen gas having a flow rate of 0.4 ml/min to 40 ml/min.

13. An apparatus for performing mass spectrometry analysis of a biological sample, the apparatus comprising:
a column-integrated needle in contact with or in proximity to a sample-introducing orifice of a mass spectrometer;
a solvent-feeding needle;
a tip portion of the solvent-feeding needle positioned in contact with or in proximity to a tip portion of the column-integrated needle, the tip portion of the solvent-feeding needle operably arranged to provide to the tip portion of the column-integrated needle a mixture of a first organic solvent and a nitrogen gas;
a gradient generator configured to regulate a concentration of a second organic solvent in a separation solvent to be introduced to the column-integrated needle, the separation solvent comprising the biological sample and the second organic solvent having a concentration gradient from a low concentration to a high concentration; and
a spray electrode configured to apply a voltage to the column-integrated needle to generate charged droplets;
the column-integrated needle being configured to provide a spray of the charged droplets to the sample-introducing orifice of the mass spectrometer; and
the mass spectrometer configured to determine from the charged droplets whether the biological sample includes at least one of a type of the biological sample, an amount of the biological sample, or a chemical structure of the biological sample.

14. The apparatus according to claim 13, wherein the column-integrated needle comprises a resin for liquid chromatography filling the column-integrated needle.

15. The apparatus according to claim 13, wherein the column-integrated needle is secured to a first three-dimensionally positioning unit provided on a base.

16. The apparatus according to claim 15, wherein the solvent-feeding needle is secured to a second three-dimensionally positioning unit provided on the first three-dimensionally positioning unit.

17. A method for performing mass spectrometry analysis of a biological sample, the method comprising:
providing a regulated concentration gradient of a first organic solvent in a separation solvent comprising the biological sample and the first organic solvent to be introduced to a LC-ESI (liquid chromatography-electrospray ionization) column-integrated needle;
discharging substantially simultaneously:
first droplets comprising the separation solvent from the LC-ESI column-integrated needle; and
second droplets from a solvent-supplying needle comprising a mixture of a second organic solvent and a nitrogen gas, a tip of the solvent-supplying needle positioned in contact with or in proximity to a tip of the LC-ESI column-integrated needle, such that the first droplets from the LC-ESI column-integrated needle and the second droplets from the solvent-supplying needle mix together to form a mixture;

receiving the mixture of droplets into an orifice of a mass spectrometer; and
spectrometrically determining from the received mixture at least one of a type of the biological sample, an amount of the biological sample, or a chemical structure of the biological sample.

18. The method of claim 17, wherein the biological sample comprises at least one member selected from the group consisting of a protein, DNA fragments, and RNA fragments.

19. The method of claim 17, the second droplets from the solvent-supplying needle further comprising:
substantially preventing accumulation of liquid at the tip of the column-integrated needle that would otherwise disrupt the discharge of the first droplets from the column-integrated needle.

20. The method of claim 17, wherein discharging the first droplets comprises discharging the first droplets at a flow rate in a range from about 50 nL/min to about 500 nL/min.

21. The method of claim 17, wherein discharging the second droplets comprises discharging the second droplets at a flow rate in a range from about 50 nL/min to about 500 nL/min.

22. The method according to claim 17, wherein mixing the second organic solvent with the nitrogen gas further comprises mixing the second organic solvent with the nitrogen gas, the nitrogen gas having a flow rate of 0.4 ml/min to 40 ml/min.