JP2019060824A - Electrospray stabilization auxiliary solvent introduction device for improved nano lc-ms - Google Patents

Abstract

To provide a method and a device for nano electrospray ionization for continuously carrying out stable, high-sensitivity analysis in analysis by a nano LC-ESI-MS method.SOLUTION: A nano electrospray ionization method includes: a step of introducing a separation solvent containing a sample into a column-integrated needle filled with a filler for liquid chromatography, and spraying a separated sample component toward a sample introduction orifice of a mass spectrometer from the tip of the needle; and a step of introducing an organic solvent-containing gas into a capillary for supplying gas, and supplying the organic solvent-containing gas to the tip of the column-integrated needle from the tip of the tube, where the two steps are simultaneously performed.SELECTED DRAWING: Figure 1

Description

  The present invention relates to a method and apparatus for nanoelectrospray ionization at MS interface of micro flow LC-MS. In particular, the present invention relates to a nanoelectrospray ionization method and apparatus for continuously performing stable high sensitivity analysis for nano LC-MS / MS analysis of RNA.

  In recent years, it has been clarified that various low molecular weight RNAs are deeply involved in life activities and diseases such as cancer caused by their abnormalities. In general, for analysis of small RNAs, a method is employed in which analysis is carried out by molecular biological methods after conversion to cDNA using reverse transcriptase, but this method is characterized by the function of small RNAs There is a drawback that analysis or quantitative analysis of chemical modifications important for expression can not be performed.

To address this problem, methods have been developed to identify small RNAs present in biological samples by direct mass spectrometry and analyze their chemical structures and functions. In this method, the target low molecular weight RNA is cleaved with RNAse, and the generated RNA oligomer is separated by liquid chromatography (LC) and ionized by electrospray ionization (ESI), and high performance mass spectrometry ( Based on the MS / MS spectrum obtained by the MS) analysis, the type, amount of the small RNA, chemical structure and the like are analyzed. That is, in this analysis, called nano-LC-ESI-MS method, for analyzing the components of the fmol level ⁽¹⁰ -15 mol), the nucleotide fragment of the sample at 50～500Nl / min about ultrafine flow LC It is necessary to separate and ionize it as nanodroplets for mass spectrometry. Furthermore, unlike the case of proteins and peptides, in the analysis of phosphate-rich low molecular weight RNA, it is required to mass-analyze hydrophilic nucleotides having (−) charge in “negative mode”. In the MS method, measurement in the “negative mode” is an order of magnitude lower in sensitivity than analysis in the “positive mode” in which peptides and the like are analyzed.

  In the conventional nano LC-ESI-MS method having the above limitations, hydrophilic nucleotides with (-) charge are eluted from the LC column under conditions of low organic solvent concentration during separation, so There is a problem that the surface tension is increased, so that ionization can not be performed efficiently by the nanoelectrospray ionization method, and the analysis sensitivity is significantly reduced. That is, when the nanoelectrospray ionization method is performed with an LC separation solution having a low organic solvent concentration, droplets are formed at the tip of the needle that sprays the separation solution as nanodroplets, and the spraying becomes intermittent, or spraying Could stop the analysis itself.

  To solve these problems, for analysis down to the microliter level (flow rate is μl / min level), use a double tube needle with a “sheath tube” that encloses in parallel the needle that sprays the separation liquid, A method has been reported in which the formation of droplets at the tip of the needle is prevented by supplying an organic solvent (sheath solution) to the LC separation solution sprayed from the sheath tube (Non-patent Document 1). However, although the above-mentioned technology is an effective method for analysis down to microliter level, it has not been applied to analysis of nanoliter level (flow rate is nl / min level).

  The inventors of the present invention have developed high performance nanoelectrospray ionization methods and devices in Patent Document 1, but there is a demand for development of methods and devices that can analyze more stably and with high sensitivity.

Therefore, with regard to nano LC-ESI-MS used for small RNAs and the like, a method for performing electrospray ionization continuously and in a stable manner was still in the search stage.
In addition, this method can be similarly used in proteomics analysis frequently performed in recent years. That is, even in proteomics analysis for proteins and polypeptides produced by enzyme digestion, the spray is often unstable due to changes in the shape of the tip of the column during analysis, and results with good reproducibility can be stably obtained. There was no big problem.

JP, 2010-281777, A

Huber, C. and Krajete, A., Journal of Chromatography A, 870: 413-424, 2000

  The present invention has been made in view of the above problems, and maintains nano LC-MS / MS analysis of RNA independently of instability factors such as the shape of the tip of the column, and stably high sensitivity analysis. It is an object of the present invention to provide a nanoelectrospray ionization method and apparatus for performing continuously.

  In order to achieve the above object, as a result of intensive studies by the present inventors, the sample is introduced together with the separation solvent, and the tip of the column-integrated needle that separates the sample into components and sprays it from the outside It has been found that stable high sensitivity analysis can be carried out continuously by supplying an organic solvent-containing gas, and the present invention has been completed.

That is, in one aspect, the present invention may be as follows.
[1] A separation solvent containing a sample is introduced into a column-integrated needle packed with a packing material for liquid chromatography, and a voltage is applied to the tip of the needle to separate the separated sample components from the tip of the needle Spraying and ionizing to the sample introduction orifice of the mass spectrometer;
Introducing an organic solvent-containing gas into the gas supply capillary and supplying the gas from the capillary tip to the column-integrated needle tip;
A nanoelectrospray ionization method comprising performing the two steps simultaneously.

  [2] The sample is a natural RNA having a base length of about 2 to 100 bases, a non-natural RNA, an enzyme-digested RNA oligomer, a natural or non-natural DNA, a DNA-RNA hybrid, an enzyme-digested DNA The method according to the above-mentioned [1], which comprises an oligomer, a naturally-occurring and non-naturally-occurring protein, a peptide produced by enzymatic digestion of a protein, or a synthetic polypeptide.

[3] The method according to [1] or [2] above, wherein the gas is nitrogen gas or air containing a vaporized organic solvent, and the organic solvent is an organic solvent having water solubility.
[4] The method according to the above [3], wherein the organic solvent having water solubility is alcohol, acetonitrile, acetone, methyl acetate, ethyl acetate, dichloromethane.

[5] The method according to any one of the above [1] to [4], wherein the inner diameter of the gas supply capillary is 0.2 to 1.0 mm.
[6] The method according to any one of the above [1] to [5], wherein the flow rate of the gas is 10 to 1500 mL / min.

[7] A nanoelectrospray ionization device for performing the method according to the above [1] to [6],
A column-integrated needle packed with a packing material for liquid chromatography and placed in contact with or in close proximity to a sample introduction orifice of a mass spectrometer;
A spray electrode connected to the column-integrated needle,
An organic solvent-containing gas supply means;
Equipped with
The organic solvent-containing gas supply means is
A gas supply unit,
An organic solvent reservoir,
And a gas supply capillary,
The gas supply unit is connected to the organic solvent reservoir via a flow path to supply a gas to the organic solvent reservoir.
The organic solvent reservoir is connected to the gas supply capillary via a flow path, and supplies an organic solvent-containing gas generated by supplying the gas to the organic solvent reservoir to the gas supply capillary. And
The gas supply capillary is disposed such that its tip is in contact with or close to the tip of the column-integrated needle.
A nanoelectrospray ionization device characterized in that.

  According to the present invention, the sensitivity and stability of the analysis do not depend on instability factors such as the tip shape of the chip-type column, and the extremely small amount of low molecular components are ionized extremely stably, and analysis of extremely high sensitivity is achieved. It can be carried out continuously. This makes it possible to stably carry out analysis of low-molecular-weight RNAs, which has hitherto been difficult and which is excellent in detail and reliability, and can greatly contribute to the application to the development of RNA medicine and the like.

  Moreover, proteomics analysis can not only prolong the life of the column, but also can contribute to proteomics research including drug development by reducing running costs and improving the stability of the obtained data.

It is a figure which shows the structure of nano LC-ESI-MS in the nano electro spray ionization method concerning this invention. It is a graph showing the change (gradient) of the organic solvent concentration in the separation solvent in nano LC-ESI-MS method. It is a figure which shows the distance between the column integrated needle in this application, and the capillary for gas supply. RNA synthesized according to the present invention nano LC-ESI-MS of the present invention equipped with an organic solvent-containing gas supply means, and old nano LC-ESI-MS supplying a mixture of an organic solvent and nitrogen gas by a solvent supply means It is a chromatogram of RNA-DNA hybrid. Nano LC-ESI-MS of the present invention provided with organic solvent-containing gas supply means, old nano LC-ESI-MS for supplying a mixture of organic solvent and nitrogen gas by solvent supply means, and organic solvent-containing gas supply means A chromatogram of the synthesized RNA or RNA-DNA hybrid by a common LC-ESI-MS without Without the cosolvent, the eluent and solute could not be ionized due to the formation of droplets at the column tip. It is a chromatogram of the synthesize | combined RNA or RNA-DNA hybrid by nano LC-ESI-MS of this invention equipped with the organic solvent containing gas supply means when the kind of organic solvent containing gas is changed. It is a chromatogram of human 5S-rRNA digest by nano LC-ESI-MS of this invention equipped with the organic solvent containing gas supply means. It is a figure which shows the base sequence (sequence number 1) of human 5S-rRNA. A blank in the base sequence is a site that is cleaved by RNase T1 digestion. The underlined portion in the base sequence is the base sequence corresponding to the peak detected in Example 3. In nano LC-ESI-MS of the present invention equipped with an organic solvent-containing gas supply means, the result of evaluating the relationship between the organic solvent-containing gas flow rate and the peak height of the chromatogram of the synthesized RNA or RNA-DNA hybrid Is a graph showing FIG. 10 is a graph showing the change in ionizing voltage with 0.1% formic acid-water in positive mode and a baseline. It is a chromatogram of the tryptic digest of human serum albumin by nano LC-ESI-MS of this invention equipped with the organic solvent containing gas supply means. It is a figure which shows the structure of general LC-ESI-MS.

The present invention will be specifically described below, but the present invention is not limited thereto.
Unless otherwise defined herein, scientific and technical terms used in connection with the present invention shall have the meanings that are commonly understood by those of ordinary skill in the art.

  The nanoelectrospray ionization method according to the present invention is a nano LC-ESI-MS method for analyzing ultra trace components, wherein a gas separated from the sample separated by LC is sprayed and ionized to the tip portion of the ESI needle. Supply.

  Usually, in the nano LC-ESI-MS method, it is possible to use an ESI needle integrated with a column on which LC is performed, in particular to eliminate post-column dead volume. This column-integrated needle is a silica gel column for reverse phase separation having a tapered structure packed with a silica gel packing material for reverse phase separation for liquid chromatography, and a column in which sample components separated in the packing material are tapered. By spraying immediately from the tip to prevent droplet formation, minute charged droplets can be obtained. The narrowed tip inner diameter of the column-integrated needle is preferably designed to be about 5 to 15 μm.

  The sample to be subjected to the nanoelectrospray ionization method according to the present invention is not particularly limited as long as it is generally applicable to the nano LC-ESI-MS method, and includes proteins, peptides, DNA, RNA, etc. Or mixtures thereof. The size of the protein, peptide, DNA or RNA is not particularly limited. In the present invention, in particular, a sample containing an enzyme-digested RNA oligomer, a peptide produced by enzymatic digestion of a protein, or a small RNA, which is difficult to analyze by conventional methods, is preferable.

  As used herein, a protein is a molecule comprising a polypeptide chain in which 20 types of natural L-α-amino acids are linked by a peptide bond (amide bond). The proteins may be naturally occurring or recombinantly produced by genetic engineering techniques. In addition, a molecule containing a polypeptide chain in which 2 to 20 naturally occurring L-α-amino acids are linked by a peptide bond may be particularly referred to as a peptide. The peptide may be one naturally occurring, one produced by chemical synthesis, one produced recombinantly by genetic engineering techniques, or one produced by enzymatic digestion of a protein. Enzymatic digestion of a protein means a reaction in which a protein is degraded by an enzyme such as a proteolytic enzyme to generate a peptide. The non-naturally occurring form of a protein or peptide means one obtained by artificially modifying a naturally occurring protein or peptide or one artificially produced by genetic engineering techniques or chemical synthesis.

  In one aspect, the proteins or peptides to be analyzed according to the present invention may be naturally occurring and non-naturally occurring proteins, and various polypeptides and synthetic polypeptides produced by these enzyme digestions. The length of the peptide generated by enzymatic digestion of the protein is not particularly limited.

  DNA (deoxyribonucleic acid) has D-2-deoxyribose as a sugar component, and adenine (A), guanine (G), cytosine (C) or thymine (T) as a base is linked to the 1 'carbon of the sugar And, with deoxyribonucleotides in which the phosphate is linked to the 5 'carbon of the sugar as a basic unit, the phosphate forms a phosphodiester bond between the 3' and 5 'carbons of the sugar between each nucleotide, The deoxyribonucleotide is a polymerized molecule. RNA (ribonucleic acid) has D-ribose as a sugar component, and adenine (A), guanine (G), cytosine (C) or uracil (U) as a base is linked to the 1 'carbon of the sugar, and The ribonucleotide is a phosphate diester bond formed between the 3 'and 5' carbons of the sugar between each nucleotide, with the ribonucleotide in which the phosphate is linked to the 5 'carbon of the sugar as a basic unit Is a polymerized molecule. DNA or RNA may be naturally occurring ones, those obtained by degrading naturally occurring ones by enzyme treatment or chemical treatment, those produced by chemical synthesis, or those produced by genetic engineering techniques. May be The non-naturally occurring form of DNA or RNA means synthetic DNA or RNA and artificially modified such as Locked NA, S-modified RNA and the like.

  In one embodiment, the RNA to be analyzed according to the present invention is a functional non-translated RNA (ncRNA) of about 20 to 30 nucleotides contained in a biological sample such as a microRNA (miRNA) or siRNA having a base length of about 30 bases Base length generated by enzymatic digestion of large RNA such as natural RNA such as tRNA with about 70 bases, non-natural RNA synthesized for the purpose of drug development etc., and ribosomal RNA (rRNA) with a base length of 6000 bases It may be an oligonucleotide (enzyme-digested oligomer) of about 2 to 50 bases or the like. The DNA or RNA to be analyzed according to the present invention may be natural and non-natural DNA, DNA-RNA hybrids, and various oligonucleotides produced by these enzyme digestions. As used herein, small RNA refers to RNA of 2 to 100 nucleotides, preferably 2 to 70 nucleotides, preferably 2 to 50 nucleotides, 2 to 30 nucleotides, 2 to 20 nucleotides, or 2 to 15 nucleotides.

The sample to be subjected to the nanoelectrospray ionization method according to the present invention can be used by dissolving it in a buffer solution, a salt solvent or the like in the analysis.
FIG. 12 is a schematic view showing the configuration of a conventional general nano LC-ESI-MS.

In FIG. 12, the nano LC-ESI-MS includes a sample separation unit 12 and a mass analysis unit 14.
The sample separation 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 the respective components are appropriately connected by a flow path There is. The mass analysis means 14 has a sample introduction orifice 30 and a mass spectrometer 32 connected thereto, and the orifice 30 is disposed so as to face the pointing direction of the tip of the column integrated needle 28.

  In the sample separation means 10, an analysis sample appropriately dissolved in water or the like is introduced from the injector 22 and travels on the separation solvent fed at a set flow rate from the separation solvent delivery pump 20 and reaches the gradient generator 24. The gradient generator 24 adjusts the composition and concentration of the separation solvent according to the elution characteristics of the sample components, and controls the separation of the sample components in the subsequent column integrated needle 28 more precisely. The column-integrated needle 28 is packed with a packing material for liquid chromatography, and the sample introduced together with the separation solvent is the difference in affinity for the separation solvent prepared by the gradient generator 24 and the packing material. The components are separated accordingly. Further, to the column-integrated needle 28, a voltage of about −1 to −2 kV is applied when performing mass analysis in the negative mode with the spray electrode 26, and a voltage of about +1 to +2 kV is applied when performing mass analysis in the positive mode. The sample components separated in the column are sprayed from the tip of a needle integrated with the column to form minute charged droplets in which a large number of separation solvent molecules are associated with ions. The sample components formed into droplets are ionized by the ions inside the droplets and introduced into the mass analysis means 14 from the sample introduction orifice 30 in the direction indicated by the needle tip.

  FIG. 1 is a schematic view showing a nanoelectrospray ionization device of the present invention. The same reference numerals are given to members having the same configurations as those of the conventional nano electrospray ionization device described above, and in the following description, portions different from the conventional nano electrospray ionization device described above will be described in detail.

  The nano LC-ESI-MS of the present invention shown in FIG. 1 includes an organic solvent-containing gas supply means 36 in addition to the sample separation means 12 and the mass analysis means 14. The organic solvent-containing gas supply means 36 has a gas supply unit 38, an organic solvent reservoir 40, and a capillary 42 for gas supply. The gas supply unit 38 and the organic solvent reservoir 40, and the organic solvent reservoir 40 and the gas supply capillary 42 are connected by a flow passage 46 and a flow passage 48, respectively. Here, the organic solvent reservoir 40 has a hollow cylindrical shape, and the end of the flow path 46 for connection with the gas supply unit 38 and the end of the flow path 48 for connection with the gas supply capillary 42 are both organic solvents It is arranged not to contact the organic solvent in the reservoir 40.

  The direction in which the tip of the gas supply capillary 42 points is directed to the tip of the column-integrated needle 28 of the sample analysis means 12. In order to set the tip of the gas supply capillary 42 so that the tip of the gas supply capillary 42 faces the tip of the column integrated needle 28 as described above, the gas supply capillary 42 is installed with the column integrated needle 28 although not particularly shown. It is possible to employ a device configuration that is held by a three-dimensional positioning holder installed on the same base as the base being mounted. The holder holds the central portion of the gas supply capillary 42 and is rotatable in the vertical and horizontal directions, and the tip of the gas supply capillary 42 can be positioned in various directions. As a result, even if the tip position of the column integrated needle 28 is changed variously, the tip of the gas supply capillary 42 can be moved following it.

In order to stir the organic solvent in the organic solvent reservoir 40, it may further have a stirrer 44.
As the gas supply unit 38, a compressor or a gas cylinder can be used.

The flow path 46 and the flow path 48 are not limited in material and shape as long as they are tubular bodies that can supply the organic solvent-containing gas, but for example, resin piping can also be used.
In the configuration of the present invention, the steps leading to ionization after introduction and separation of the sample in the sample separation means 12 and mass analysis in the mass analysis means 14 are the configurations of the general nano LC-ESI-MS described above (FIG. 12) Is the same as

  However, in the configuration of the present invention, the gas supplied by the gas supply unit 38 at the set pressure in the organic solvent-containing gas supply means 36 simultaneously with the capillary 42 for gas supply after passing through the organic solvent reservoir 40 simultaneously with the above steps. By being introduced, the organic solvent-containing gas is supplied from the end of the gas supply capillary 42 to the end of the column-integrated needle 28 of the sample separation means 12.

  Thus, one embodiment of the present invention is a nanoelectrospray ionization device for carrying out the nanoelectrospray ionization method of the present invention, which is filled with a filler for liquid chromatography and is provided at a sample introduction orifice of a mass spectrometer. A column integrated needle 28 disposed in contact or close proximity, a spray electrode 26 connected to the column integrated needle, and an organic solvent containing gas supply means 36, wherein the organic solvent containing gas supply means 36 , A gas supply unit 38, an organic solvent reservoir 40, and a capillary 42 for gas supply.

  In the present application, the separation solvent is a mobile phase generally used in liquid chromatography (LC), and is a water or a mixed system of a salt solution and an organic solvent (eg, methanol solution, acetonitrile solution, formic acid aqueous solution, triethylamine acetate aqueous solution etc.) Is preferably used.

  In the general nano LC-ESI-MS method, in order to separate trace components contained in the sample more precisely depending on the affinity (solubility) with the organic solvent in the separation solvent, it is usually carried out in the gradient generator 24 The composition ratio of the separation solvent is adjusted to a gradient so that the solvent concentration gradually increases from low concentration. An ideal gradient line (concentration gradient of organic solvent in separation solvent) in such adjustment is as illustrated in the upper part of FIG.

  However, in the actual measurement, at the stage where the concentration of the organic solvent in the separation solvent is low in the gradient, that is, in the initial stage of the gradient analysis, the organic solvent is as low as a few percent or less, It is expected to be much larger than the organic solvent alone. Therefore, the separation solvent at the initial stage often remains at the tip of the column-integrated needle 28 by surface tension to form droplets. Since the tip of the column-integrated needle used for nanoanalysis is extremely small, when the droplet is generated, as shown in the lower part of FIG. 2, the state where spraying of sample components is interrupted intermittently (state A) or completely It becomes (State B), and it is impossible to continue normal analysis.

  Therefore, as represented by the configuration of the present invention and the configuration of the above embodiment shown in FIG. 1, in the method according to the present invention, the needle tip which is the spray port simultaneously with the spraying of the sample component from the column integrated needle By supplying an organic solvent-containing gas to the unit, the separated sample components are mixed with the organic solvent contained in the gas, and are prevented from stopping at the jet nozzle, thereby eliminating the formation of the droplets that impede the spraying. It is

  In the nano LC-ESI-MS method including the nanoelectrospray ionization of the present invention illustrated in FIG. 1, the sample to be introduced is a solution of a solution dissolved in an appropriate solvent such as water, ethanol, acetone etc. as needed. It is preferable to be in the state. The flow rate of the separation solvent and the sample in the column-integrated needle 28 is preferably 50 to 500 nl / min, although it depends on the components of the sample and the type of the separation solvent.

  In the present application, the organic solvent-containing gas is a concept including a gas containing a vaporized organic solvent, a gas containing a mist-like organic solvent, or a gas containing both of them. Particularly preferably, the organic solvent-containing gas is a gas in which the organic solvent is saturated. The organic solvent-containing gas is generated at the top of the organic solvent reservoir 40 by the vaporization of the organic solvent in the reservoir. Stirring the organic solvent in the organic solvent reservoir 40 with, for example, the stirrer 44 is effective from the viewpoint of bringing the interior of the reservoir close to vapor-liquid equilibrium. It is understood that the gas in which the organic solvent is saturated is generated in the space above the storage when the inside of the organic solvent storage 40 is in vapor-liquid equilibrium.

  In the method according to the present invention, the gas supply capillary 42 is not particularly limited in material and shape as long as it is a tubular body capable of supplying an organic solvent-containing gas, but the inner diameter is 0.2 to 1.0 mm, preferably It may be 0.2 to 0.75 mm or 0.4 to 0.6 mm. The tip of the gas supply capillary 42 may be cut in a direction perpendicular to the direction in which the capillary is indicated, and may have a tapered structure (i.e., a needle-like structure).

The flow rate of the organic solvent-containing gas supplied from the gas supply capillary 42 to the tip of the column-integrated needle 28 is not particularly limited, and is, for example, 10 to 1,500 ml / min, preferably 30 to 1,400 ml / min, 300 to 1,400 ml / min. It may be a minute. The flow rate of the organic solvent-containing gas is determined by the combination of the pressure of the gas supplied from the gas supply unit 38 and the inner diameter of the gas supply capillary 42. For example, after setting the pressure of the gas supplied from the gas supply unit 38 to 1.0 kg / cm ² and adopting the inside diameter of the above range for the gas supply capillary 42, the flow rate of the organic solvent-containing gas The above-mentioned range can be achieved.

  The organic solvent-containing gas supplied from the gas supply capillary 42 is not particularly limited as long as it is a gas containing an organic solvent. Preferably, the organic solvent is an organic solvent having solubility in water. The organic solvent having solubility in water is not particularly limited, but may optionally be mixed with an organic solvent having a solubility in water of 1% or more, preferably 5% or 10% or more, more preferably water. Organic solvents. Specific examples of such organic solvents include alcohols such as methanol, ethanol or isopropyl alcohol (IPA), acetonitrile, acetone, methyl acetate, ethyl acetate, dichloromethane and the like. Preferred organic solvents are methanol, acetonitrile, acetone, ethyl acetate. A further preferred organic solvent is methanol. Further, as the gas, any gas may be used as long as it is air or an inert gas, and suitable gases include air or nitrogen.

The temperature of the organic solvent-containing gas supplied from the gas supply capillary 42 is not particularly limited, but may preferably be room temperature, for example, 20 to 30 ° C., preferably 25 ° C.
In the method according to the present invention, the column-integrated needle 28 is disposed close to the sample introduction orifice 30 of the mass spectrometer 32 to the extent that ionized sample components can be introduced, and its tip is directed to the orifice. The gas supply capillary 42 is disposed in contact with or in close proximity to the column integrated needle 28, and the tip thereof is directed to the column integrated needle. The distance between the tip of the column-integrated needle 28 and the tip of the gas supply capillary 42 (the distance represented by FIG. 3A) is preferably about 0 to 2000 μm. 0 μm means that the tip of the gas supply needle 42 is in contact with the column integrated needle 28. 2000 μm is a measure of the distance that the organic solvent-containing gas supplied from the tip of the gas supply capillary 42 can reach the column-integrated needle 28, and a more appropriate distance allows the organic solvent-containing gas to move to the needle It can adjust suitably in the range.

  Furthermore, in electrospray ionization using a column-integrated needle (ESI needle), droplets that interfere with the spray of the sample component from the needle tip tend to be generated from 1 to 2 mm behind the spray nozzle . Therefore, in the present invention, the tip of the gas supply capillary 42 is preferably disposed about 1 to 2 mm behind the tip of the column-integrated needle 28 (the distance shown in FIG. 3B). By arranging the column-integrated needle 28 and the gas supply capillary 42 in this manner, it is possible to more reliably eliminate the formation of droplets and continue normal ion formation.

  From the point of determining the arrangement of these needles, the nanoelectrospray ionization method according to the present invention uses a nanoelectrospray ionization device provided with positioning means for adjusting the arrangement of the column integrated needle 28 and the capillary 42 for gas supply. Is preferred. That is, in the nanoelectrospray ionization apparatus according to the present invention, the tip of the column integrated needle 28 is positioned in contact with or close to the sample introduction orifice 30 of the mass spectrometer 32, and the tip of the capillary 42 for gas supply is It has a structure that can be placed in contact with or close to the tip of the column-integrated needle.

  The method and apparatus of the present invention are not limited to the embodiments described above, and various modifications can be made without departing from the spirit of the present invention. For example, a double structure tube in which a gas supply capillary is disposed to cover the outer periphery of the column-integrated needle 28 can also be used.

Hereinafter, the nanoelectrospray ionization method and apparatus according to the present invention will be specifically described by way of examples, but the present invention is not limited thereto.
Example 1 Comparison with Conventional Methods RNA or RNA-DNA hybrid (50 fmol each) synthesized using nano LC-ESI-MS (q-ex: quadrupole orbitrap mass spectrometer) configured as shown in FIG. ) Was used as a sample to evaluate nanoelectrospray ionization according to the present invention. The nucleic acids used as the synthesized RNA or RNA-DNA hybrid are as follows.
5'-CACCA-3 '
5'-UUUCGU (dC) (dA) -3 '
5'-CUCAGU (dT) (dT) -3 '
5'-AAUUCGA (dT) (dT) -3 '
The conditions in the sample separation means are as follows. As a column, Develosil C30 3 μm 0.15 mm ID × 65 mm L (Nomura Chemical Co., Ltd.) was used. The flow rate of the eluent was 100 nL / min, and the sample was separated by the gradient elution method. Solution A: triethylamine, pH 7.0: methanol = 9: 1, solution B: 10 mM triethylamine-acetic acid, pH 7.0: acetonitrile = 6: 4, 0% solution B → 40% solution B / 30 minutes, 40% solution B → 70% solution B / 1 minute, kept at 70% solution B for 10 minutes, eluted with a 70% solution B → 0% solution B / 1 minute.

The mass spectrometer was used in negative mode and the ionization voltage was -1300V or -1400V.
Further, as the organic solvent-containing gas, air containing vaporized methanol was used. The inner diameter of the gas supply capillary was 0.50 mm, and the pressure of the compressor was 1.0 kg / cm ² . The temperature of the organic solvent-containing gas supply means was 25.degree.

Also, for comparison, a conventional general LC-ESI-MS (FIG. 12, sometimes referred to as “no auxiliary device” in the present specification or drawings) not provided with an organic solvent-containing gas supply means The same analysis was performed using. Then, instead of the organic solvent-containing gas supply means of the present application, LC-ESI-MS disclosed in JP 2010-281777 A, in which a mixture of an organic solvent and nitrogen gas is supplied by the solvent supply means. In the drawings, analysis was performed using “old type” or “old type auxiliary device”), and comparison with the method of the present invention was performed. The mixture of organic solvent and nitrogen gas was a mixture of acetonitrile and nitrogen gas, and the nitrogen gas cylinder pressure was 1.0 kg / cm ² , temperature 25 ° C., tip gas flow rate 20 mL / min, acetonitrile flow rate: 300 nL / min .

The chromatograms obtained are shown in FIGS. 4 and 5.
In the LC-ESI-MS without auxiliary equipment (FIG. 5 lower), in the electrospray ionization step, droplets are generated at the tip of the needle that sprays the sample solution, and soon after the start of the test it becomes impossible to spray and obtain analytical data. could not.

  In the nano LC-ESI-MS (FIG. 4 upper stage and FIG. 5 upper stage) of the present invention and the old nano LC-ESI-MS (FIG. 4 lower stage and FIG. 5 middle stage), the peak of the target substance was detected. In the nano LC-ESI-MS of the present invention, each component in the sample appeared in the form of a single peak. However, in the old nano LC-ESI-MS, the peak tip had a cracked shape. In addition, the sensitivity was improved particularly for the elution of the organic solvent 20% or less in the eluent of LC. This indicates that the nano LC-ESI-MS of the present invention equipped with an organic solvent-containing gas supply means is capable of highly accurate analysis of sample components.

Example 2 Examination of the Type of Organic Solvent As the organic solvent-containing gas, the air according to the present invention was used in the same manner as in Example 1 except that the air containing vaporized methanol, acetone, methyl acetate, ethyl acetate, dichloromethane or hexane was used. Analysis was performed by LC-ESI-MS.

The obtained chromatogram is shown in FIG.
When air containing vaporized hexane was used as the organic solvent-containing gas, analytical data could not be obtained (data not shown). It is inferred that hexane did not support spraying and ion formation of the sample component from the column integrated needle, since hexane has 0% solubility in water.

  When air containing a gas of methanol, acetone, methyl acetate, ethyl acetate or dichloromethane is used as the organic solvent-containing gas, the spray of sample components from the column-integrated needle and the formation of ions progress stably, resulting in high accuracy. Analytical data of were obtained. In particular, when air containing vaporized methanol is used as the organic solvent-containing gas, it is clear that the peak of the sample component is high, and more sensitive analysis is possible.

Example 3: Analysis of human 5S-rRNA The nano LC-ESI-MS of the present invention was carried out in the same manner as in Example 1 except that RNase T1 digested product (50 fmol) of human 5S-rRNA (SEQ ID NO: 1) was used as a sample. The analysis was done in In addition, air containing vaporized methanol, acetonitrile, or acetone was used as the organic solvent-containing gas.

  The obtained chromatogram is shown in FIG. The relationship between the nucleotide sequence of human 5s-rRNA and the nucleotide sequence corresponding to the peak detected in Example 3 is shown in FIG. In the base sequence of FIG. 8, the underlined part shows the base sequence corresponding to the peak detected in Example 3.

  Also when a human 5s-rRNA digest was used as a sample, spraying of sample components from the column-integrated needle and ion formation progressed stably, and high-accuracy analytical data was obtained. In addition, there was no significant difference in the chromatograph when using any of the vaporized methanol, acetonitrile, or air containing acetone as the organic solvent-containing gas. It became clear that highly sensitive analysis was possible using any of the organic solvent-containing gases.

Example 4: Investigation of organic solvent-containing gas flow rate The same synthesis as in Example 1 was carried out except that tubes with an inner diameter of 0.13 mm, 0.25 mm, 0.50 mm, and 0.75 mm were used as capillaries for gas supply. Analysis was performed using RNA or RNA-DNA hybrid as a sample. Since the pressure of the compressor is fixed at 1 kg / cm ² , the flow rate of the organic solvent-containing gas can be changed by changing the inner diameter of the gas supply capillary. The relationship between the inner diameter of the gas supply capillary and the gas flow rate was as follows.

Each component of the sample was evaluated by the relative value of peak height in the chromatograph. The results are shown in FIG.
When the inner diameter of the gas supply capillary was 0.13 mm, no atomization and ion formation occurred, and analytical data could not be obtained.

  On the other hand, when the inside diameter of the gas supply capillary was 0.25 mm, 0.50 mm, and 0.75 mm, the spray and the ion formation of the sample component from the column-integrated needle progressed stably. In particular, when the inner diameter of the gas supply capillary is 0.50 mm, the stability of the spray and ionization formation is high, and when installing the gas supply capillary toward the tip of the column integrated needle, it is good. Because the range of positions where chromatograms can be obtained is wide, there is also an advantage that installation is easy.

Example 5: Ionization in Positive Mode Using nano LC-ESI-MS of the configuration shown in FIG. 1, the change in ionization voltage with 0.1% formic acid-water, which is the initial condition of nano LC-MS proteomics analysis Baseline was assessed.

Specifically, tryptic digests of proteins (50 fmol) were analyzed by nano LC to investigate the ease of spraying with 0.1% formic acid-water, the most difficult to ionize initial solvent.
The conditions in the mass spectrometric means are as follows. The column used mightySiLC18 3 micrometers and column size 0.1 mmIDx120 mmL (Kanto Chemical Co., Ltd.). The sample was eluted by flowing 0.1% formic acid-water at a flow rate of 100 nL / min.

The mass spectrometer was used in positive mode and the ionization voltage was lowered by 0.1 kV from +1.6 kV every minute.
As the organic solvent-containing gas, air containing vaporized methanol was used. The inner diameter of the gas supply capillary was 0.50 mm, and the pressure of the compressor was 1.0 kg / cm ² . The temperature of the organic solvent-containing gas supply means was 25.degree.

  Also, for comparison, a conventional general LC-ESI-MS (FIG. 12, sometimes referred to as “no auxiliary device” in the present specification or drawings) not having an organic solvent-containing gas supply means is used. The same analysis was performed.

A graph of the obtained baseline is shown in FIG.
As can be read from the baseline (indicated as upper row in FIG. 10, “without sheath”) without an auxiliary device, the spray stops at an ionization voltage of +1.3 kV when the organic solvent-containing gas supply means is not provided. Droplets were generated at the tip and were not ionized.

  In the case of using the nano LC-ESI-MS of the present invention, it was ionized to +0.9 kV as can be read from the lower baseline in FIG. This indicates that the nano LC-ESI-MS of the present invention is effective for deterioration of the column tip due to long-term use, etc., and is good for continuous operation load.

Example 6 The analysis was performed by the nano LC-ESI-MS of the present invention of the present invention, using a tryptic digest of human serum albumin as an analysis sample of an enzymatic digest of a protein .

  The conditions in the mass spectrometric means are as follows. The column used mightySiLC18 3 micrometers and column size 0.1 mmIDx120 mmL (Kanto Chemical Co., Ltd.). The flow rate of the eluent was 100 nL / min, and the sample was separated by the gradient elution method. Solution A was eluted with 0.1% formic acid-water, solution B was 0.1% formic acid-acetonitrile, and the solution B was eluted with a gradient of 0% → B solution 35% / 18 minutes.

The mass spectrometer was used in positive mode and the ionization voltage was +1.2 kV, +1.3 kV, +1.4 kV, +1.5 kV or +1.6 kV.
Further, as the organic solvent-containing gas, air containing vaporized methanol was used. The inner diameter of the gas supply capillary was 0.50 mm, and the pressure of the compressor was 1.0 kg / cm ² . The temperature of the organic solvent-containing gas supply means was 25.degree.

  Also, for comparison, a conventional general LC-ESI-MS (FIG. 12, sometimes referred to as “no auxiliary device” in the present specification or drawings) not having an organic solvent-containing gas supply means is used. The same analysis was performed.

  The obtained chromatogram is shown in FIG. The left side of FIG. 11 is a chromatogram without the auxiliary device, and the right side is a chromatogram in which the ionization voltage was changed in the range of +1.2 kV to +1.6 kV, respectively, using the nano LC-ESI-MS of the present invention.

  In the absence of auxiliary equipment, the spray became unstable at an ionization voltage of +1.3 kV, and when the initial concentration of organic solvent was low, droplets were generated at the tip of the leaf and column and no ions were formed. In addition, when the ionization voltage was +1.2 kV, it did not ionize until the end.

  In the case of using the nano LC-ESI-MS of the present invention, it was ionized at almost the same intensity up to +1.2 kV, and a chromatogram was stably obtained. This indicates that the nano LC-ESI-MS of the present invention shows that the spray is stable even in actual analysis.

12 sample separation means 14 mass analysis means 18 separation solvent 20 separation solvent delivery pump 22 injector 24 gradient generator 26 spray electrode 28 column integrated needle 30 sample introduction orifice 32 mass spectrometer 36 organic solvent containing gas supply means 38 gas supply unit 40 Organic solvent reservoir 42 Capillary 44 for gas supply Stirrer 46 Channel 48 Channel

Claims (7)

A separation solvent containing a sample is introduced into a column-integrated needle packed with a packing material for liquid chromatography, and a voltage is applied to the tip of the needle to separate the separated sample components from the tip of the needle to a mass spectrometer Spraying and ionizing toward the sample introduction orifice of
Introducing an organic solvent-containing gas into the gas supply capillary and supplying the gas from the capillary tip to the column-integrated needle tip;
A nanoelectrospray ionization method comprising performing the two steps simultaneously.   The sample is a natural RNA having a base length of about 2 to 100 bases, a non-natural RNA, an enzyme-digested RNA oligomer, a natural or non-natural DNA, a DNA-RNA hybrid, an enzyme-digested DNA oligomer, a natural The method according to claim 1, characterized in that it comprises a type and non-natural type protein, a peptide produced by enzymatic digestion of a protein or a synthetic polypeptide.   The method according to claim 1 or 2, wherein the gas is nitrogen gas or air containing a vaporized organic solvent, and the organic solvent is an organic solvent having solubility in water.   The method according to claim 3, wherein the organic solvent having water solubility is alcohol, acetonitrile, acetone, methyl acetate, ethyl acetate, dichloromethane.   The method according to any one of claims 1 to 4, wherein the inner diameter of the gas supply capillary is 0.2 to 1.0 mm.   The method according to any one of claims 1 to 5, wherein the flow rate of the gas is 10 to 1500 mL / min. A nanoelectrospray ionization device for carrying out the method according to claims 1-6, wherein
A column-integrated needle packed with a packing material for liquid chromatography and placed in contact with or in close proximity to a sample introduction orifice of a mass spectrometer;
A spray electrode connected to the column-integrated needle,
An organic solvent-containing gas supply means;
Equipped with
The organic solvent-containing gas supply means is
A gas supply unit,
An organic solvent reservoir,
And a gas supply capillary,
The gas supply unit is connected to the organic solvent reservoir via a flow path to supply a gas to the organic solvent reservoir.
The organic solvent reservoir is connected to the gas supply capillary via a flow path, and supplies an organic solvent-containing gas generated by supplying the gas to the organic solvent reservoir to the gas supply capillary. And
The gas supply capillary is disposed such that its tip is in contact with or close to the tip of the column-integrated needle.
A nanoelectrospray ionization device characterized in that.