JP3578147B2 - Atmospheric pressure ionization mass spectrometer - Google Patents

Description

[0001]
TECHNICAL FIELD OF THE INVENTION
According to the present invention, a sample solution is introduced into an atmospheric pressure ion source such as electrospray (ESI) to ionize, and ions generated by the ion source are guided to a mass spectrometer via an ion guide arranged in a vacuum chamber to perform mass spectrometry. Atmospheric pressure ionization mass spectrometer. In particular, the present invention relates to an atmospheric pressure ionization mass spectrometer capable of significantly reducing chemical noise and increasing the efficiency of collision induced dissociation (CID) in MS / MS.
[0002]
[Prior art]
Recently, research in the field of biotechnology has been remarkable, and its research subjects are diverse. In particular, proteins, peptides, DNAs, and the like play an extremely important role in living organisms, and have been studied by many researchers. Generally, only a trace amount of these biologically derived organic compounds is present in a complex matrix. In order to analyze a trace amount of a biologically relevant organic compound extracted from a living body, a liquid chromatograph direct mass spectrometer (LC / MS device) has come to play an important role. The LC / MS device is a device that separates a mixture by LC and performs qualitative and quantitative analysis with MS at high sensitivity. A typical ionization means used in LC / MS is electrospray ionization (ESI). ESI is an ionization procedure under atmospheric pressure, first disclosed by Fite in US Pat. No. 4,209,696. The analysis of trace organic compounds by coupling ESI with a chromatograph such as capillary electrophoresis (CE) is disclosed by Smith et al. In US Pat. No. 4,842,701. This ESI is known as a mild and highly sensitive ionization method, and has now been widely used for analyzing biologically relevant substances.
[0003]
ESI operates as follows. A voltage of several kV is applied between a metal capillary having an inner diameter of about 0.1 mm and a counter electrode arranged at a certain distance (about several tens of mm). When a sample solution is introduced into a metal capillary and a high voltage is applied, fine charged droplets are released into the atmosphere from the tip of the solution. The generated charged droplet flies in the air toward the counter electrode according to the electric field, and repeatedly collides with atmospheric molecules. As a result, the charged droplets are mechanically crushed, the evaporation of the solvent from the surface of the droplets is promoted, and the charged droplets are rapidly miniaturized. Finally, the ions in the charged droplets are released to the atmosphere. The ions fly in the atmosphere toward the counter electrode, pass through a thin tube or pore provided in the counter electrode, are led to a high vacuum mass spectrometer, and are subjected to mass analysis.
[0004]
In order to achieve high sensitivity and stabilization in ESI, it is important to produce a large number of uniform and fine charged droplets in the spraying stage as much as possible. Therefore, as a technique for improving the ESI method, a method has been proposed in which a metal thin tube is inserted into a thin tube having a large inner diameter, a sample solution flows through the thin metal tube, and a spray gas flows between two thin tubes. The promotion of vaporization from the surface of the droplet by the heat exchange between the droplet and the gas molecule and the mechanical crushing by the friction between the droplet and the gas molecule made it possible to spray a solution of about 1 ml / min stably. This technique was disclosed by Henion et al. In US Pat. No. 486,1988 and became known as Ion Spray. In ESI and Ion Spray, generation of fine droplets was achieved by spraying into the atmosphere, but sometimes giant droplets were generated in the sprayed droplets. When this giant droplet is introduced into the mass spectrometer, random noise is generated in the mass spectrum to deteriorate the S / N ratio, and consequently, the high sensitivity characteristic of MS is significantly impaired. Several techniques have been proposed to solve this problem. US Pat. No. 4,935,624 and US Pat. No. 5,122,670 disclose a technique for preventing the introduction of droplets by using a curtain gas. A curtain gas, such as dry nitrogen gas, is flown around the pores and the thin tube provided on the vacuum partition wall facing the ESI spray thin tube. It is intended to prevent the introduction of large droplets into a vacuum by the flow of curtain gas. On the other hand, ions can be taken into vacuum by an electric field. Further, US Pat. No. 5,235,186 discloses a method of heating a curtain gas to vaporize and dry fine droplets. However, since noise (chemical noise) derived from large droplets cannot be completely removed even by curtain gas, another method has been proposed in US Pat. No. 4,977,320. It is intended to heat a thin tube that takes in ions in a vacuum and to heat and evaporate the droplets while passing through the thin tube. This method was subsequently called a heated capillary (Heated Capillary) and has been adopted in many LC / MS devices.
[0005]
Chemical noise due to large droplets generated during spraying was greatly reduced by the use of curtain gas and a heated capillary. However, chemical noise was still present in the LC / MS instrument, which hindered the enhancement of the sensitivity of the LC / MS measurement. The reason why this chemical noise cannot be completely removed is considered as follows. When a gas is introduced into the vacuum from the atmosphere, the gas expands adiabatically and rapidly cools down to near zero degrees. The temperature of the gas is cooled down to about -263 ° C. (10 K) in a vacuum several mm away from the outlet of the thin tube having an inner diameter of 0.2 mm. When the cooled ions collide with polar molecules, such as water, which have also been cooled, the ions and molecules do not have internal energy enough to dissociate again, so that they form additional ions (cluster ions). This in turn causes the addition of water molecules in a snowball fashion, forming cluster ions. The cluster ions are distributed over a wide mass range and dissociate during mass analysis, so that random noise (chemical noise) is generated in the mass spectrum and an effective signal is suppressed.
[0006]
US Pat. No. 4,999,493 and US Pat. No. 5,015,845 show a technique for heating a first vacuum chamber (intermediate pressure chamber) for sampling gas and ions from the atmospheric pressure in order to prevent the generation of cluster ions. However, since the biomaterials targeted by ESI are easily thermally decomposed, heating at about 300 ° C. is the limit. With this level of heating, the cooling effect due to adiabatic expansion is superior, and the cooling temperature can be reduced from 10K to about 20K. That is, gas cooling cannot be eliminated. As a result, the chemical noise was reduced, but the chemical noise could not be eliminated. On the other hand, application to the field of biotechnology has led to the miniaturization of samples, which has led to a demand for higher sensitivity of LC / MS devices.
[0007]
A technique for heating the mass spectrometer not for LC / MS but for another purpose is disclosed in U.S. Pat. US Pat. No. 5,767,513 discloses a technique for heating an ion guide (octapole) disposed between an ion source and a mass spectrometer by Dressler. This relates apparatus used to study ion-molecule reactions between the ions and the Na and D ₂ generated in the electron ionization is ionized under a high vacuum (EI). The ions generated by the EI are guided to an octapole to which a high frequency is applied, where an ion molecule reaction is caused. Octapole acts as a reaction cell. The reaction cell and the octapole rod itself were heated to 1,000 K to prevent side reactions and contamination in the reaction cell. In US Pat. No. 5,767,513, ions and gases are introduced and generated in a housing kept in a high vacuum, so that adiabatic expansion and cooling do not occur at all. There is no description regarding introduction of ions generated by atmospheric pressure ionization into vacuum, adiabatic expansion, cooling, and the like. There is no description about improving the MS / MS efficiency of biological macromolecules or removing chemical noise.
[0008]
Heating the ion trap itself is shown by Parermo in US Pat. No. 5,796,100. This relates to electron ionization (EI) in which a sample gas is introduced into an ion trap, and electrons are bombarded and ionized. It is intended to reduce the introduction of the neutral sample molecules before ionization adsorbed on the electrode surface in the ion trap and thermal decomposition. U.S. Pat. No. 5,994,697 indicated that Kato showed heating of an LC / MS ion trap. Here, in order to eliminate water molecules adsorbed in the ion trap or on the electrode surface, a method of heating the ion trap itself or heating the buffer gas and sending it into the electrode is shown. In US Pat. No. 5,796,100 and US Pat. No. 5,994,697, there is no description about introduction of ions generated by atmospheric pressure ionization into a vacuum, problems associated with adiabatic expansion and cooling, and means for solving the problems. There is no description about the improvement of MS / MS efficiency of a biopolymer.
[0009]
When a peptide, protein, DNA, or the like is ionized by ESI by ESI, these molecules give a multiply-charged ion having a plurality of charges to one ion. The CPU can determine the molecular weight of the sample from the plurality of multiply charged ions. However, it is not possible to identify these bio-related molecules only by information on molecular weight. Identification of proteins, DNA, and the like requires information on the sequences of amino acids and nucleic acids that constitute these bio-related molecules. However, it is not necessary to know the entire sequence of DNA or protein, but only the sequence of a part of the structure. A library can be searched from this partial sequence and information on the molecular weight to identify proteins and DNA. MS / MS is a mass spectrometer that gives this structural information.
[0010]
As the MS / MS, a tandem MS in which a plurality of MSs are connected in series, an ion trap mass spectrometer, and the like are used. The ions generated by ESI are directed to the first MS, isolated into one precursor ion, and the other ions are excluded. Next, the isolated precursor ions are guided to a collision cell provided between the first MS1 and the second MS2, and dissociation (CID) is caused by collision between the target gas and the ions. The generated product ions are led to a second MS, subjected to mass spectrometry, and a mass spectrum is obtained.
[0011]
An ion trap mass spectrometer is an apparatus that can achieve MS / MS with one MS. Ionization, introduction and accumulation of ions into an ion trap, MS / MS, and acquisition of a mass spectrum of product ions are performed in a time sharing manner. MS / MS is composed of a plurality of steps, and isolation of precursor ions and resonance dissociation of precursor ions are also performed in a time-division manner.
[0012]
In MS / MS, ions are excited by resonance, and the kinetic energy is gradually converted into the internal energy of the ions by the collision of the ions with buffer gas molecules having a pressure of about 0.1 Pa many times. Is a method of cleavage. When ions and gas molecules are cooled down to 10 K, long-time excitation is required before ions are dissociated. Also, what biopolymers such as DNA and proteins, internal energy is converted by one collision with low mass molecules buffer gas such consists of several parts per million of the energy of the collision hundreds of thousands parts per , Excitation is not performed sufficiently. Therefore, it has been considered that collision-induced dissociation (CID) of these biopolymers is difficult.
[0013]
A method to solve this difficulty has been proposed. An ion (IR) laser is irradiated to the ions trapped in the ion trap to heat the ions themselves, and the ions are resonantly excited to dissociate the ions. This technique is called infrared multiphoton dissociation (IRMPD). Cooks et al. Compare photodissociation of small molecules with CID (Cooks, Int. J. Mass Spectrom. Ion Processes, 75 (1987) 345-352).
[0014]
McLafferty et al. Reported the use of IRMPD in an ICR mass spectrometer (FT-MS) to cleave polyvalent ions of a protein having a molecular weight of 29 kDa (McLafferty, Anal. Chem., 66 (1994) 2809-2815). .
[0015]
In addition, a method has been proposed in which ions trapped in an ion trap mass spectrometer or an ICR mass spectrometer are heated from the surroundings without using a laser to thermally decompose the ions. This is known as Dissociation BIRD. Williams et al. Report that BIRD can cleave polyvalent ions of a 8.6 kDa protein to obtain structural information (Williams, Anal. Chem., 69 (1997) 1119-1126).
[0016]
[Problems to be solved by the invention]
When ions generated by an atmospheric pressure ion source are introduced into a vacuum chamber for mass analysis, the ions are rapidly cooled by adiabatic expansion. This rapid cooling generates cluster ions in the ion guide and ion trap. In order to excite ions that have once been cooled and become cluster ions, it is necessary to dissociate the added polar molecules. After this, the ions must be excitedly dissociated. Therefore, the cluster ion require injection of more energy, the efficiency of CI D in the ion trap is significantly reduced. Conversely, excessive excitation can sometimes lead to ion decomposition.
[0017]
Further, since the temperature of the ion introduced into the ion trap mass spectrometer and the ion introduced at the beginning of the accumulation period and the ion introduced at the end of the accumulation period are uneven, the efficiency of the CID varies.
In view of the above, an object of the present invention is to provide an atmospheric pressure ionization mass spectrometer capable of improving the CID efficiency by shortening the ion excitation time.
[0018]
[Means for Solving the Problems]
The present invention solves such a problem, and heats ions by heating an ion guide space where cooling by adiabatic expansion occurs. Also, the ion trap space is heated.
[0019]
For this purpose, an ion guide is provided which has an atmospheric pressure ion source for generating ions under atmospheric pressure by introducing a sample solution into a fine narrow tube, and the ions generated by the ion source are placed in a vacuum chamber and a high frequency is applied. In the atmospheric pressure ionization mass spectrometer which leads the mass spectrometer to the mass spectrometer through mass spectrometry and gives the mass spectrum, the ion guide and the mass spectrometer are heated at the same time.
[0020]
Further, an atmospheric pressure ion source for introducing ions of the sample solution into a fine capillary to generate ions at atmospheric pressure is provided, and the ions generated by the ion source are supplied to a vacuum chamber through a capillary or a pore provided in a vacuum partition. In the atmospheric pressure ionization mass spectrometer which introduces the gas into the mass spectrometer through the ion guide to which a high frequency is applied and gives a mass spectrum, the ions between the mass spectrometer move and trap from the capillary or the pore. Be sure to heat the space.
[0021]
BEST MODE FOR CARRYING OUT THE INVENTION
FIG. 1 shows an apparatus according to an embodiment of the present invention. The sample component solution separated by the liquid chromatograph LC1 is sent to the ESI probe 3 of the ESI ion source 4 via the capillary tube 2. The ESI probe 3 is arranged on an XYZ three-axis positioning device 9. The sample solution sent from the LC 1 is sent to the nozzle of the ESI probe 3 and is sprayed from the nozzle tip into the atmosphere as a spray ion stream (charged droplet) 6. Ions are released from the charged liquid droplets into the atmosphere, and are taken into the vacuum chamber 12 from the thin tubes 8 (including fine pores) provided on the vacuum partition 11 and heated by the heater 40. The mass spectrometer comprises several chambers 12, 15 and 19 having different pressures, and each chamber is evacuated by independent vacuum pumps 20, 21 and 22. The ions are introduced into a mass spectrometer 17 via a high-frequency ion guide 16 and subjected to mass analysis. When the high-frequency voltage supplied from the mass sweep power supply 23 is swept, the ions are separated for each mass, and the ion current is detected by the detector 18. The signal of the ion current corresponding to each mass is sent to the control data processor 24 and collected as a mass spectrum.
[0022]
Radio frequency ion guide 16 includes a plurality (4,6,8, ... present) cylindrical electrodes are disposed parallel on the same circumference. Every other electrode is commonly wired, and a high frequency is applied between the two electrode groups. A cylindrical shield tube 43 covers the high-frequency ion guide 16 for shielding the high-frequency ion guide. The shield cylinder 43 plays a role of keeping the gas 44 introduced into the shield at a constant pressure (about 0.1 Pa) together with the high-frequency shield. An inert gas such as argon (Ar) or nitrogen gas (N ₂ ) gas is introduced into the shield. A heater 41 is disposed around the shield tube 43 as a heating means so that the shield tube and the high-frequency ion guide 16 can be heated. Further, the introduced gas itself may be heated by the heater 45. The electrodes of the ion trap mass spectrometer 17 are heated by a heater 25 as heating means. The number of heaters 25 may be increased for heating the entire electrode. Electric current is sent from the power sources 42 and 26 controlled by the control data processing device 24 to the heaters 41 and 25. Thus, the high-frequency ion guide 16 and the introduced gas disposed in the space inside the shield cylinder 43 are heated from 200 ° C. to 300 ° C. The ions and gas molecules cooled down to several tens of K in the vacuum chamber 12 through the pores at the tip of the skimmer 13 are introduced into the high-frequency ion guide 16 from the opening of the shield cylinder 43. The introduced ions vibrate due to the high-frequency electric field in the high-frequency ion guide and move toward the mass spectrometer while gradually converging on the central axis. Ions moving while oscillating a high-frequency ion guide 16 medium frequently collide with the heated hot gas molecules. Thereby, heat exchange between the gas molecules and the ions is performed, and the ions are rapidly heated. The ions heated in the high-frequency ion guide 16 are sent to a high vacuum chamber 19 where the ion trap mass spectrometer is placed, and introduced into the ion trap mass spectrometer 17. The ion trap mass spectrometer 17 is heated to 200 to 300 ° C. by a heater 25 as heating means. That is, the space for trapping ions is heated. Therefore, the introduced ions are introduced and accumulated in the ion trap mass spectrometer 17 while maintaining the temperature. Thereby, the ions can be subjected to the MS / MS operation while being kept at a high temperature. The high-temperature ions rapidly increase in internal energy due to resonance excitation and collision with high-temperature buffer gas molecules in the ion trap electrode. As the ion's internal energy increases above the binding energy, the ion cleaves to form product ions. Next, the control data processing device 24 controls the power supply 23 to sweep the high frequency voltage applied to the ring electrode of the ion trap, thereby acquiring a mass spectrum of the generated product ions.
[0023]
The heating temperature of the high-frequency ion guide 16 and the heating temperature of the ion trap mass spectrometer 17 can be set independently by the control data processor 24. Cooling by adiabatic expansion is performed, and the set temperature of the high-frequency ion guide having a short stay time (several tens of microseconds) is set higher than the temperature of an ion trap mass spectrometer having an ion stay time of several hundred milliseconds. I do. In peptide analysis, good results were obtained when the temperature of the high-frequency ion guide was 300 ° C and the temperature of the ion trap mass spectrometer was 200 ° C.
[0024]
FIG. 3 shows an example of a mass spectrum obtained by an LC / MS apparatus in which a high-frequency ion guide having no heating means and an ion trap mass spectrometer are combined. a, b, and c are mass peaks derived from samples and impurities. On the other hand, mass peaks appearing over the entire mass region have no known assignment, and must be excluded from information as chemical noise. This chemical noise becomes remarkable when the sensitivity of the apparatus is increased when the amount of the sample is very small. As shown in FIG. 3 (2), the c ion corresponding to the pseudo molecular ion of the sample is isolated, and MS / MS is performed. Excitation of c-ions causes collision-induced dissociation (CID) in the ion trap. However, when the ions have a mass exceeding 1000 and the ions are cooled, the CID is not performed efficiently, and the product ions have minute peaks, making it difficult to distinguish them from noise. As shown in FIG. 3 (3), the excited pseudo-molecular ions remain in the mass spectrum with almost no change in intensity.
[0025]
When this sample is heated with a high-frequency ion guide and an ion trap mass spectrometer as in this method, a mass spectrum with little chemical noise is obtained as shown in FIG. By improving the S / N ratio, ions of the signals d and e buried in the chemical noise can be clearly identified. In the MS / MS measurement, first, a precursor ion c is isolated. The precursor ions are sufficiently heated by heating the high-frequency ion guide and the ion trap mass spectrum. By exciting the heated precursor ions, molecules having a mass exceeding 1000 can be cleaved, and a product ion mass spectrum as shown in (3) of FIG. 2 can be obtained. The amount of the precursor ion c is greatly reduced by cleavage. On the other hand, the product ions p1 to p4 clearly appear.
[0026]
The ion trap mass spectrometer temporarily stores ions in the ion trap and then performs MS / MS measurement and mass sweep. In order to avoid the influence of space charge in the ion trap during the accumulation process, the accumulation time is shortened when the amount of ions is large, and the accumulation time is lengthened when the ion current is small. That is, the ion introduction accumulation time is constantly changing. According to this method, even if the ion introduction accumulation time changes, the ion temperature does not change, so that product ions can be obtained with good reproducibility by MS / MS.
[0027]
Here, ESI is mainly described, but the present invention relates to atmospheric pressure chemical ionization (APCI) and ion spray (Ion Spray), sonic spray (Sonic Spray), nano spray (Nano Spray), etc., which are improvements of ESI. Applicable to coupling with ionization technology.
[0028]
According to this method, cooling of ions and gas from the ion source to the ion trap mass spectrometer can be prevented. As a result, generation of cluster ions can be prevented, and chemical noise can be further reduced.
[0029]
The present invention is not limited to an ion trap mass spectrometer, but includes a quadrupole mass spectrometer (QMS), a triple stage quadrupole mass spectrometer (TSQ), an ICR mass using an atmospheric pressure ion source and a high frequency ion guide. It can also be applied to analyzers.
[0030]
In the present invention, it is important to heat ions and gas in a form that compensates for cooling due to adiabatic expansion. Therefore, it is necessary to keep the heated state from the outlet of the thin tube 8 where the adiabatic expansion occurs to the mass spectrometer 17. The heating means may be a heater as shown in FIG. 1, or may be an infrared lamp or an infrared ray irradiation by a laser. In short, it is important that the ions are heated until they are detected by the detector 18.
[0031]
【The invention's effect】
According to the present invention, product ions can be efficiently generated even from biopolymers such as peptides, proteins, and DNA. Further, a highly reproducible product ion current value, that is, a product ion mass spectrum can be obtained regardless of the ion introduction accumulation time.
In addition, the suppression of chemical ions has made it possible to increase the sensitivity of the LC / MS device.
[Brief description of the drawings]
FIG. 1 is a configuration diagram of an atmospheric pressure ionization mass spectrometer according to one embodiment of the present invention.
FIG. 2 is a mass spectrum diagram without chemical noise.
FIG. 3 is a product mass spectrum diagram.
[Explanation of symbols]
DESCRIPTION OF SYMBOLS 1 ... LC, 2 ... Capillary tube, 3 ... ESI probe, 4 ... ESI ion source, 5 ... High voltage power supply, 6 ... Spray ion flow, 7 ... Ion source space, 8 ... Narrow tube, 9 ... XYZ 3-axis positioning mechanism, 11 , 14: vacuum partition, 12, 15: vacuum chamber, 13: skimmer, 16: high frequency ion guide, 17: mass spectrometer, 18: detector, 19: high vacuum chamber, 20, 21, 22, ... vacuum pump, 23 ... mass sweep power supply, 24 ... control data processing device, 25, 40, 41, 45 ... heater, 26, 42 ... heater power supply, 43 ... shield cylinder, 44 ... gas reservoir.

Claims (8)

It has an atmospheric pressure ion source that generates ions under atmospheric pressure by introducing a sample solution into a fine capillary, has three chambers evacuated by a pump, and a skimmer with pores in one of the chambers. A mass spectrometer is arranged in another chamber, and an ion guide to which a high frequency is applied is arranged in a chamber between them, and ions generated in the ion source are sequentially sent from the skimmer to the ion guide. guidance to the mass spectrometer through, a atmospheric pressure ionization mass spectrometer to provide mass spectra,
A shield tube covering the ion guide, disposed around the shield tube, a heating unit for heating the shield tube, and a heating unit for heating the mass spectrometer,
Wherein when introduced ions passing through the ion guide from the pores, atmospheric pressure ionization mass spectrometer, characterized in that it is heated. 2. The atmospheric pressure ionization mass spectrometer according to claim 1, wherein the ion guide and the mass spectrometer are independently heated and controlled. 3. The atmospheric pressure ionization mass spectrometer according to claim 1, wherein a heating temperature of the ion guide is set higher than a heating temperature of the mass spectrometer. The atmospheric pressure ionization mass spectrometer according to any one of claims 1 to 3, wherein the ion source is an electrospray (ESI) ion source. The atmospheric pressure ionization mass spectrometer according to any one of claims 1 to 3, wherein the ion source is an ion spray ion source. The atmospheric pressure ionization mass spectrometer according to any one of claims 1 to 3, wherein the ion source is a nanospray ion source. 4. An atmospheric pressure ionization mass spectrometer according to claim 1, wherein said ion source is an atmospheric pressure chemical ionization (APCI) ion source. It has an atmospheric pressure ion source that generates ions under atmospheric pressure by introducing a sample solution into a fine capillary, has three chambers evacuated by a pump, and a skimmer with pores in one of the chambers. A mass spectrometer is arranged in another chamber, and an ion guide to which a high frequency is applied is arranged in a chamber between them, and ions generated in the ion source are sequentially sent from the skimmer to the ion guide. guidance to the mass spectrometer through, a atmospheric pressure ionization mass spectrometer to provide mass spectra,
A shield tube for covering the ion guide, a heating unit disposed around the shield tube and heating the shield tube, a gas introduction unit for introducing an inert gas into the shield tube, and the inert gas Heating means for heating,
An atmospheric pressure ionization mass spectrometer, wherein the ions introduced from the pores are heated when passing through the ion guide .

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