JP6593243B2 - Ion trap mass spectrometer - Google Patents

Description

  The present invention captures ions derived from sample components in an ion trap by the action of an electric field, dissociates the captured ions in one or more stages, and performs mass analysis of product ions generated by the dissociation. Relates to the device. The ion trap mass spectrometer referred to here is a mass spectrometer that detects product ions with an external detector while separating the product ions according to the mass-to-charge ratio by the ion trap itself, and releases the product ions from the ion trap. For example, a mass spectrometer that separates and detects a mass-to-charge ratio with an external mass separator such as a time-of-flight mass analyzer is also included.

  In order to identify polymer compounds and analyze their structures, in recent years, ions derived from the target compound are dissociated in one or more stages, and the product ions (fragment ions) generated thereby are mass-to-charge ratios. Mass spectrometry is widely used in which detection is performed separately according to the conditions. As such an apparatus for mass spectrometry, an ion trap time-of-flight mass spectrometer or the like is well known. As a technique for dissociating ions having a large molecular weight trapped in an ion trap in such a mass spectrometer, collision-induced dissociation (CID = Collision Induced Dissociation) in which dissociation is induced by colliding excited ions with a gas such as argon. The most common method is an infrared multi-photon dissociation (IRMPD) method in which a plurality of infrared photons are absorbed by ions to increase the internal energy of the ions and dissociate the ions. ing. Electron transfer dissociation (ETD) and electron capture dissociation (ECD) methods are widely used for dissociating ions derived from proteins and peptides.

  In the ETD method, negative molecular ions are irradiated as reactive ions in an ion trap, and collide with ions derived from sample components in the ion trap to cause an interaction. Due to this interaction, the electrons of the reaction ions move to the protons of ions derived from the sample components, and the protons change into hydrogen radicals. Radical species of ions generated by this reaction dissociate specifically in a bond. On the other hand, in the ECD method, electrons are irradiated into an ion trap, and electrons are added to protons of ions derived from sample components in the ion trap. As a result, the proton changes to a hydrogen radical, and the radical species of ions generated by this reaction dissociate specifically in a bond.

  Unlike the collisional dissociation methods such as the CID method, the ETD method and the ECD method are unpaired electron-induced dissociation methods, and thus the cleavage of the N-Cα bond of the peptide main chain occurs specifically. Therefore, c / z series fragment ions, which are difficult to generate with low energy CID, are actively generated. In addition, since the dissociation is carried out while retaining the modification site such as sugar chain, it is easy to identify the modified product and the modification site, which is useful for the structural analysis of the polymer compound.

  However, in the ETD method and the ECD method, monovalent ions derived from sample components are neutralized immediately after radical reaction, and theoretically, only dissociation of multivalent ions having two or more valences can be performed. Therefore, most of the generated ions are monovalent, and there is a difficulty in combination with a matrix-assisted laser desorption ionization (MALDI) ion source that is currently most widely used for ionization of biological samples. Further, the ETD method and the ECD method are generally effective only for positive ions, and it is difficult to dissociate negative ions. In addition, since the valence of ions decreases with each dissociation, the operation of performing multiple dissociations in order to generate immonium ions containing amino acid side chains has a valence equal to or greater than the number of amino acid residues. It can only be applied to ions that have it.

  In order to solve some problems in the conventional ion dissociation method, the present inventors have developed a hydrogen-attachment dissociation (hereinafter abbreviated as HAD method) and proposed it in Patent Document 1. In brief, the HAD method is a method of dissociating ions by attaching hydrogen radicals (= hydrogen atoms) to ions, and the dissociation mechanism itself is considered to be similar to the ECD method or ETD method. It can be said that this is an unpaired electron induced ion dissociation method. Although the dissociation mechanism is similar, the ECD method and ETD method dissociate the ions by giving electrons to the molecular ions, so that the valence of the molecular ions decreases with the dissociation. On the other hand, since hydrogen radicals are neutral and the valence of molecular ions does not decrease with dissociation in the HAD method, there is a great advantage that they can be applied to all ions including monovalent ions.

  However, the HAD method has a low ion dissociation efficiency as in the ECD method and the ETD method, so that the detection sensitivity of product ions is low. Further, the HAD method has a low sequence coverage (ratio of the number of dissociated peptide bonds to the total number of peptide bonds in the main chain) indicating the coverage of dissociated sites. If the sequence coverage is low, there is little information obtained from the product ion spectrum of the peptide, so that there is a problem that the estimation accuracy of the amino acid sequence is likely to be lowered or impossible to estimate. Also, in the HAD method, hydrogen moves within the molecular ion, and the mass of the dissociated ion changes by ± several Da by the amount of the transferred hydrogen (this is called hydrogen transfer). As a result, the peak of the dissociated ion is dispersed and intensified. Is also a problem. However, hydrogen transfer is thought to occur when it takes time to dissociate after the hydrogen is attached. If the time until dissociation is shortened by improving the ion dissociation efficiency, hydrogen transfer will occur at the same time. Is also considered to be suppressed.

  In order to improve the ion dissociation efficiency, in the ECD method and the ETD method, a method of giving energy from the outside auxiliary (generically referred to as a supplemental activation method) has been attempted. As a supplemental activation method, the same operation as the CID method is performed with a low energy that does not dissociate ions (Collisional Activation method (see Non-Patent Document 4 etc.)), similar to the IRMPD method. IR activation (IR Activation) method (see Non-Patent Documents 5 and 6, etc.) in which the operation is performed with a low energy that does not dissociate the ions, and the ions constituting the ion trap are heated and the ions are passed through the gas. Thermal activation (Thermal Activation) method (see Non-Patent Documents 1 to 3) that increases the energy of ions by heat bathing has been attempted, and effects such as improved ion dissociation efficiency and improved sequence coverage have been reported. ing.

  On the other hand, the HAD method is a recently established method, and the supplemental activation method has hardly been studied. Patent Document 1 shows that the collaborative activation method is effective as one method for improving the ion dissociation efficiency of the HAD method, but there is no other application example of the supplemental activation method. . Although application of the collaborative activation method to the HAD method has been confirmed to be effective, the value or frequency of the voltage applied to the electrode of the ion trap to excite ions, the reaction time, the background gas Appropriate conditions such as pressure vary depending on the type of sample, and adjustment of such conditions is quite troublesome. In addition, if the conditions are adjusted incorrectly, dissociation occurs not by the HAD method but by the CID method, and post-translational modifications such as sugar chains are eliminated, making it difficult to identify peptides that have undergone post-translational modifications. There is also.

International Publication No. 2015/133259

Butcher (David J. Butcher) and three others, "Thermal Dissociation of Gaseous Bradykinin Ions", Journal of Physical Chemistry A, 1999, Vol. 103 , Pp.8664-8671 Sharon J. Pitteri and two others, “Electron-Transfer Ion / Ion Reactions of Dublin Prototype Peptides: Effect of Elevated Bath Gas Temperature (Electron- Transfer Ion / Ion Reactions of Doubly Protonated Peptides: Effect of Elevated Bath Gas Temperature), Anal. Chem. 2005, Vol. 77, pp. 5562-5669 Blue Car (Kathrin Breuker) and 4 others, “Detailed Unfolding and Folding of Gaseous Ubiquitin Ions Characterized by Electron Capture Dissociation) ”, Journal of American Chemical Society, 2002, Vol. 124, pp. 6407-6420 Danielle L. Swaney and five others, “Supplemental Activation Method for High-Efficiency Electron-Transfer Dissociation of Doubly Prototype Peptide Precursors (Supplemental Activation Method for High-Efficiency Electron-Transfer Dissociation of Doubly Protonated Peptide Precursors), Anal. Chem. 2007, Vol. 79, pp.477-485 Aaron R. Ledvina and six others, “Infrared Photo-Activation Reduces Peptide Folding and Hydrogen Atom Migration” Following IT Tandem Mass Spectrometry ( Infrared Photo-Activation Reduces Peptide Folding and Hydrogen Atom Migration Following ETD Tandem Mass Spectrometry), Angelwandte Chemie International Edition, 2009, Vol. 48, Issue 45, pp.8526-8528 Aaron R. Ledvina and 7 others, “Activated-Ion Electron Transfer Dissolution Improves The Ability of Electron Transfer Dissolution To Identity Peptides・ Activated-Ion Electron Transfer Dissociation Improves the Ability of Electron Transfer Dissociation to Identify Peptides in a Complex Mixture ”, Anal. Chem. 2010, Vol. 82, pp.10068? 10074

  The present invention has been made to solve the above-mentioned problems, and an object of the present invention is to provide an ion trap mass spectrometer capable of improving ion dissociation efficiency and sequence coverage by the HAD method. It is.

  The present inventor has repeatedly conducted experiments in which the thermal activation method is applied to the HAD method, paying attention to the thermal activation method among supplemental activation methods that have been attempted by the ETD method and the ECD method. As a result, when the thermal activation method is applied to the HAD method, the post-translational modification of the peptide subjected to the post-translational modification can be eliminated by appropriately controlling the temperature of the heat source for exposing the ions to heat. It was confirmed that the ion dissociation efficiency can be sufficiently improved, and when the thermal activation method is not applied, it is possible to improve the sequence coverage by cleaving a bond that hardly causes dissociation. The present invention has been made based on such experimental findings.

That is, the present invention made to solve the above problems is an ion trap mass spectrometer that performs mass analysis of product ions generated by dissociating ions derived from sample components inside an ion trap,
a) an ion trap that traps ions derived from sample components by the action of an electric field;
b) a hydrogen radical irradiation part that dissociates the ions trapped in the ion trap by irradiating the radicals with hydrogen radicals;
c) a heat application unit that promotes dissociation of ions by the hydrogen radicals by applying heat to the ions trapped in the ion trap;
It is characterized by having.

  The ion trap has either a three-dimensional quadrupole configuration including one ring electrode and a pair (two) of end cap electrodes, or a linear configuration including a plurality of rod electrodes.

  In the ion trap mass spectrometer according to the present invention, the ions trapped in the ion trap by the action of an electric field are irradiated with hydrogen radicals of a predetermined flow rate from the hydrogen radical irradiation unit (that is, dissociation by the HAD method). Although it is generated, the heat application unit applies heat to the trapped ions during or after the irradiation of the hydrogen radical. Thereby, the internal energy of the trapped ions is increased, the bonds in the molecules are easily broken, and the ion dissociation efficiency is improved. In addition, even if the site does not exceed the binding energy by only being irradiated with hydrogen radicals, it is activated by heat and the internal energy of the ions exceeds the binding energy, so the number of dissociated sites increases and sequence coverage is increased. improves. Further, when the heat-applied ions reach temperature equilibrium, no further energy is given to the ions, so that the energy given to the ions by the heat can be adjusted only by the temperature. Therefore, it becomes easy to adjust the dissociation conditions so that post-translational modifications and the like are not desorbed while improving dissociation efficiency and sequence coverage as much as possible.

  The ion dissociation operation is performed inside the ion trap. The mass separation of the product ions generated by the dissociation may use the function of the ion trap itself, or the product ions are discharged from the ion trap. Later, a mass separator (eg, a time-of-flight mass separator) provided outside the ion trap may be used.

  The ion trap mass spectrometer according to the present invention preferably further includes an ion source based on a matrix-assisted laser desorption ionization (MALDI) method, and monovalent ions generated by the ion source are included in the ion trap. It is preferable that dissociation be performed.

  Unlike the ECD method and the ETD method, the HAD method does not decrease the valence of ions during dissociation, so that monovalent ions can be dissociated well. In the MALDI method, monovalent ions are mainly generated. However, according to the above configuration, the monovalent molecular ions generated by the ion source by the MALDI method, introduced into the ion trap, and trapped are accurately dissociated. The mass information of various product ions obtained thereby can be collected.

In the ion trap mass spectrometer according to the present invention, the method for applying heat to the ions trapped in the ion trap is not particularly limited. As one embodiment of such an ion trap mass spectrometer,
The heat applying unit includes a ceramic heater, which is an insulator, disposed between a plurality of electrodes constituting the ion trap, a heating power source unit that supplies heating power to the ceramic heater, and a heat medium in the ion trap. And a gas introduction part for introducing a buffer gas.

For example, in the case of a three-dimensional quadrupole ion trap, the ceramic heater may be arranged between the ring electrode and the end cap electrode so as to be in contact with each other.
In such a configuration, the electrode itself is heated by the ceramic heater, and heat is applied to the ions by the buffer gas having heat contacting the ions, and the ions are heated by the radiant heat from each heated electrode. Since most of the surroundings of the trapped ions are surrounded by electrodes, heat can be efficiently applied to the ions, and stable improvement in ion dissociation efficiency and improvement in sequence coverage can be expected.

As another embodiment of the ion trap mass spectrometer according to the present invention,
The heat application unit may include a gas heating unit that heats a buffer gas outside the ion trap, and a gas introduction unit that introduces a buffer gas heated by the gas heating unit into the ion trap. Good. Here, the gas heating unit may include a heater that heats a gas introduction tube for introducing a buffer gas into the ion trap, and a heating power supply unit that supplies heating power to the heater.
In such a configuration, heat can be applied to the ions when the buffer gas having heat exclusively contacts the ions.

  In the ion trap mass spectrometer, the buffer gas may also serve as a cooling gas used to cool the ions trapped in the ion trap. Thereby, the component added in order to give heat to ion can be suppressed to the minimum.

In the ion trap mass spectrometer according to the present invention, the change in ion dissociation efficiency with respect to the change in temperature when the heat is applied to the ions from the heat applying unit is not uniform within the compound molecule. The dissociation efficiency may be reduced. This can be presumed to be because the three-dimensional structure called folding formed by intramolecular hydrogen bonding, which affects the ease and difficulty of dissociation, changes due to heating.
Therefore, by utilizing this phenomenon, in the ion trap mass spectrometer according to the present invention, a plurality of product ions derived from one sample component each obtained when the temperature at the time of applying heat to the ions from the heat applying unit is changed. It is preferable to further include a data processing unit that estimates the structure information of the sample component using the intensity information.

According to the ion trap mass spectrometer of the present invention, the ion dissociation efficiency can be improved and the sequence coverage can be improved as compared with the conventional HAD method. Further, by appropriately adjusting the temperature of the heat source of heat applied to the ions, sequence coverage can be improved without desorbing post-translational modifications such as sugar chains. As a result, product ion information obtained by MS ⁿ analysis increases, so that the accuracy of identification and structural analysis of target samples such as glycopeptides is improved.

BRIEF DESCRIPTION OF THE DRAWINGS The schematic block diagram of the ion trap mass spectrometer which is 1st Example of this invention. The figure which shows transition of the measured product ion spectrum with respect to the change of the preset temperature of a ceramic heater in the ion trap mass spectrometer of 1st Example. The graph which put together the transition of the peak intensity | strength of each product ion with respect to the change of the setting temperature of a ceramic heater in the ion trap mass spectrometer of 1st Example. The figure which shows the product ion spectrum obtained when the ceramic heater temperature was 150 degreeC in the ion trap mass spectrometer of 1st Example. The schematic block diagram of the ion trap mass spectrometer which is another Example (2nd Example) of this invention. The graph which shows the result of having measured the relationship between the intensity | strength of each product ion derived from a renin, and the setting temperature of a ceramic heater in the ion trap mass spectrometer of 1st Example. The schematic block diagram of the ion trap mass spectrometer which is the other Example (3rd Example) of this invention.

An ion trap mass spectrometer according to a first embodiment of the present invention will be described with reference to the accompanying drawings.
FIG. 1 is a schematic configuration diagram of an ion trap mass spectrometer of the present embodiment. The mass spectrometer according to the present embodiment includes an ion source 1 that ionizes components in a target sample and an ion generated by the ion source 1 in a vacuum chamber (not shown) maintained in a vacuum atmosphere by the action of a high-frequency electric field. An ion trap 2 to be captured, a time-of-flight mass separation unit 3 that separates ions ejected from the ion trap 2 according to a mass-to-charge ratio, and an ion detector 4 that detects the separated ions are provided. The ion trap mass spectrometer of the present embodiment further includes a hydrogen radical irradiation unit 5 for introducing hydrogen radicals into the ion trap 2 in order to dissociate ions trapped in the ion trap 2, and the ion trap 2 A gas supply unit 6 for supplying a predetermined gas, a trap voltage generation unit 7, a control unit 8, a data processing unit 9, and a heater power supply unit 10.

  Here, the ion source 1 is a MALDI ion source, and the sample is irradiated with laser light to ionize components in the sample. The ion trap 2 is a three-dimensional quadrupole ion trap that includes an annular ring electrode 21 and a pair of end cap electrodes 22 and 24 arranged to face each other with the ring electrode 21 interposed therebetween. In response to an instruction from the control unit 8, the trap voltage generation unit 7 applies either a high-frequency voltage or a DC voltage to the electrodes 21, 22, 24 at a predetermined timing, or a voltage obtained by combining them. The time-of-flight mass separator 3 is a linear type in this example, but may be a reflectron type, a multi-turn type, or the like, and is not a time-of-flight type mass separator, for example, using the ion separation function of the ion trap 2 itself Thus, a mass separator or an orbitrap may be used.

The hydrogen radical irradiation unit 5 includes a hydrogen radical supply source 51 that stores hydrogen radicals or generates hydrogen radicals, a valve 52 that can adjust the flow rate, a nozzle 53 that ejects hydrogen radicals, and an ejection flow from the nozzle 53. And a skimmer 54 that has an opening on the central axis and separates a gas such as diffusing hydrogen molecules to extract a small-diameter hydrogen radical flow.
The gas supply unit 6 includes a gas supply source 61 that stores helium, argon, or the like used as a buffer gas or a cooling gas, a valve 62 that can adjust the flow rate, and a gas introduction pipe 63.

An analysis operation in the ion trap mass spectrometer of the present embodiment will be schematically described.
Various ions (mainly monovalent ions) generated from a sample such as a peptide mixture in the ion source 1 are ejected from the ion source 1 in the form of packets and pass through an ion introduction hole 23 formed in the inlet end cap electrode 22. It is introduced inside the ion trap 2. The peptide-derived ions introduced into the ion trap 2 are captured by a high-frequency electric field formed in the ion trap 2 by a voltage applied to the ring electrode 21 from the trap voltage generator 7. After that, a predetermined voltage is applied from the trap voltage generator 7 to the ring electrode 21 and the like, thereby exciting ions included in a mass-to-charge ratio range other than ions having a specific mass-to-charge ratio. 2 is excluded. Thereby, precursor ions having a specific mass-to-charge ratio are selectively captured in the ion trap 2.

  Subsequently, the valve 62 is opened in the gas supply unit 6, and an inert gas such as He is introduced into the ion trap 2 as a cooling gas, whereby the precursor ions are cooled. As a result, the precursor ions are converged near the center of the ion trap 2. In this state, the valve 52 of the hydrogen radical irradiation unit 5 is opened, and a gas containing hydrogen radicals (hydrogen atoms) is ejected from the nozzle 53. A gas such as hydrogen gas (hydrogen molecules) is removed by the skimmer 54 positioned in front of the jet flow, and the hydrogen radicals that have passed through the opening of the skimmer 54 are formed into a thin beam and drilled in the ring electrode 21. It passes through the radical particle inlet 26 which is made. This hydrogen radical is introduced into the ion trap 2 and irradiated to the precursor ions trapped in the ion trap 2.

  At this time, the opening degree of the valve 52 and the like are adjusted in advance so that the flow rate of hydrogen radicals irradiated to the ions is equal to or higher than a predetermined flow rate. The irradiation time of hydrogen radicals is also set appropriately in advance. As a result, the precursor ion undergoes unpaired electron-induced dissociation, and a peptide-derived product ion is generated. The produced various product ions are trapped in the ion trap 2 and cooled. Thereafter, a high DC voltage is applied from the trap voltage generator 7 to the end cap electrodes 22 and 24 at a predetermined timing. As a result, ions trapped in the ion trap 2 receive acceleration energy, and the ion injection holes 25 It is injected all at once.

  In this way, ions having a constant acceleration energy are introduced into the flight space of the time-of-flight mass separation unit 3 and separated according to the mass-to-charge ratio while flying in the flight space. The ion detector 4 sequentially detects the separated ions, and the data processing unit 9 receiving the detection signal creates a time-of-flight spectrum with the time point of emission of ions from the ion trap 2 being time zero, for example. And the mass spectrum by a fragment ion is created by converting the flight time into the mass-to-charge ratio using the mass calibration information obtained in advance. The data processing unit 9 identifies components (peptides) in the sample by performing predetermined data processing based on information (mass information) obtained from the mass spectrum.

  In the ion trap mass spectrometer of this embodiment, product ions are generated by dissociating ions by directly irradiating the ions trapped in the ion trap 2 with hydrogen radicals. Although ions are basically dissociated by irradiation with hydrogen radicals as disclosed in Patent Document 1, this ion trap mass spectrometer increases ion dissociation efficiency and increases ion dissociation sequence coverage. Has a characteristic structure as described below in order to reduce the binding site specificity.

  That is, in this ion trap mass spectrometer, in order to maintain the electrical insulation between the ring electrode 21 of the ion trap 2 and the end cap electrodes 22, 24, the relative positions of the electrodes 21, 22, 24 are maintained. The insulator (alumina ceramic) member is replaced with a ceramic heater 28. The ceramic heater 28 is connected to the heater power supply unit 10. When the heater power supply unit 10 supplies heating power to the ceramic heater 28 under the control of the control unit 8, the ceramic heater 28 generates heat. The electrodes 21, 22, 24 are also heated by heat conduction from the ceramic heater 28. A thermocouple (not shown) is embedded in the ceramic heater 28, the heating power supplied based on the monitor temperature of the ceramic heater 28 by the thermocouple is adjusted, and the amount of heat generated by the ceramic heater 28 is feedback controlled. Thereby, the ceramic heater 28 is accurately adjusted to the target temperature.

  In the state where the electrodes 21, 22, 24 of the ion trap 2 are heated by the ceramic heater 28, the product ions are discharged from the ion trap 2 at the time when hydrogen radicals are introduced into the ion trap 2 as described above, and the mass is increased. Until the time of analysis, He gas, which is a buffer gas, is intermittently introduced from the gas supply unit 6 into the ion trap 2. The heat of each electrode 21, 22, 24 of the ion trap 2 propagates to the ions via the buffer gas, and the ions are activated by this heat, that is, the energy by the heat is applied to improve the ion dissociation efficiency. In addition, bonds that are difficult to be broken (ie, binding sites with higher binding energy) are more likely to be broken without application of heat, and more types of product ions are likely to be generated, improving sequence coverage. .

An experiment for confirming the effect of the ion trap mass spectrometer of the present embodiment will be described. In this experiment, the set temperature of the ceramic heater 28 is set in a plurality of stages between 60 ° C. and 170 ° C., and a monovalent protonated ion ([M + H] ⁺ ) of mucin 5AC which is an O-linked glycopeptide The product ion spectrum obtained by dissociating was obtained. FIG. 2 is a diagram showing the transition of the actually measured product ion spectrum with respect to the change in the set temperature of the ceramic heater 28.

  In FIG. 2, product ions (c8, c9, c11 ions) whose peak intensity changes particularly with respect to temperature change are surrounded by ellipses. In these product ions, it can be seen that the peak intensity clearly increases as the set temperature of the ceramic heater 28 rises between 60-150 ° C. For example, a peak of c8 ions is hardly observed when the temperature of the ceramic heater 28 is 60 ° C., whereas a peak is observed with a sufficient intensity when the temperature of the ceramic heater 28 is 150 ° C.

  FIG. 3 is a graph summarizing the transition of the peak intensity value of each product ion with respect to the change in the set temperature of the ceramic heater 28. From this figure, it can be seen that the peak intensity is increased by a maximum of about 15 times for the c9 ion in particular, and the ion dissociation efficiency is greatly improved. In addition, the dissociated sites were only 6 sites at 60 ° C, but increased to 10 sites at 150 ° C and 170 ° C, and it was confirmed that the sequence coverage was improved from 33% to 47%. This is presumed that because the set temperature increases and the thermal energy applied to the ions increases, the binding sites that were not cleaved when the set temperature was low and that had a relatively high binding energy were also cleaved. it can.

  FIG. 4 is an enlarged view of the product ion spectrum obtained when the ceramic heater temperature is 150 ° C. with respect to Mucin 5AC. In the CID method, which is an ion dissociation method that is not an unpaired electron induction type, N-acetylgalactosamine bound to threonine (abbreviated symbol T), the third and thirteenth amino acids from the N-terminal side of the amino acid sequence of Mucin 5AC ( It is known that product ions may be generated from which GalNAc) is desorbed. In addition, even when the dissociation promotion method by excitation of ions by a resonance electric field (the above-described collaborative activation method) is applied to the HAD method, the voltage applied to the electrode of the ion trap, the reaction time, the background gas pressure, etc. are appropriately set. The present inventors have experimentally confirmed that product ions from which N-acetylgalactosamine is eliminated can be produced without adjustment.

  On the other hand, in the product ion spectrum shown in FIG. 4, the product ion from which N-acetylgalactosamine was desorbed was not detected, and simply by setting the temperature appropriately, without desorbing N-acetylgalactosamine. It can be confirmed that ion dissociation by cleaving the peptide bond can be realized. As described above, the ion trap mass spectrometer of the present example has the effect that the ion dissociation efficiency and sequence coverage of the HAD method can be improved without detaching the post-translational modification of the peptide by simple control. This can be confirmed from the experimental results.

Next, an ion trap mass spectrometer of another embodiment (second embodiment) of the present invention will be described with reference to FIG. In FIG. 5, the same components as those in the first embodiment are denoted by the same reference numerals.
In the ion trap mass spectrometer of the first embodiment, the ceramic heater 28 is disposed between the ring electrode 21 and the end cap electrodes 22 and 24. In the second embodiment, instead, the gas supply unit 6 , A gas introduction pipe heater 64 is added around a gas introduction pipe 63 that supplies gas from the gas supply source 61 into the ion trap 2. Heating power is supplied to the gas introduction pipe heater 64 from the heater power source 10 to heat the gas introduction pipe 63 in advance, and the gas is supplied at the same timing as when the buffer gas is introduced into the ion trap 2 in the above embodiment. He gas that is a buffer gas is introduced into the ion trap 2 from the unit 6. At this time, the buffer gas is heated by the gas introduction pipe 63 near the heater 64 and enters the ion trap 2 at a high temperature. For this reason, when a high-temperature buffer gas collides with ions, the heat of the buffer gas propagates to the ions, and ion dissociation by irradiation with hydrogen radicals is promoted as in the above-described embodiment.

  Further, any configuration other than the above embodiment may be used as long as heat can be given to the ions trapped in the ion trap 2 with a certain degree of stability.

  In the first and second embodiments, the ion dissociation efficiency and the sequence coverage are improved by heat bathing the ions more directly through the gas or with the gas. In addition, the thermal activation method can be used more actively. That is, as explained in FIG. 2, FIG. 3 and the like, normally, when the thermal activation method is used in combination with the HAD method, the heating temperature (the set temperature of the ceramic heater 28 in the configuration of FIG. 1) can be raised within a predetermined temperature range. The higher the value, the higher the ion dissociation efficiency. However, according to the study by the present inventors, in some compounds, the detection sensitivity of some product ions decreases as the heating temperature is increased, that is, the ion dissociation efficiency associated with the product ions increases the heating temperature. It turned out that it fell so much.

  FIG. 6 shows an actual measurement of the relationship between the intensity of a plurality of types of product ions derived from Renin, which is a kind of peptide, and the set temperature of the ceramic heater 28, using the ion trap mass spectrometer of the embodiment shown in FIG. It is a graph which shows the result. As shown in FIG. 6B, the strength of many product ions increases with increasing temperature, that is, the ion dissociation efficiency is improved. On the other hand, as shown in FIG. 6A, the strength of the two types of product ions c12 and c13 decreases with increasing temperature, and the C-terminal bond of c12 and c13 ions increases in temperature. It shows that it is harder to cut.

  Thus, although the mechanism by which the behavior of ion dissociation efficiency differs is not elucidated, it can be estimated from the above experimental results that the mechanism is as follows. That is, at room temperature (or a relatively low temperature range), a three-dimensional structure (folding) due to intramolecular hydrogen bonding occurs in the region of amino acid residues D to V (DRVYIHPFHLLV) on the N-terminal side (left side) of the amino acid sequence. In this region, it is difficult to dissociate (bonds are not easily broken). On the other hand, at this time, the bond on the C-terminal side of the c12 ion and the c13 ion is outside the three-dimensional structure due to the intramolecular hydrogen bond and is relatively easily dissociated. When this ion is heated from this state, the steric structure due to the intramolecular hydrogen bond is relaxed, and dissociation is likely to occur even in the region of amino acid residues D to V. The bonds of c8 ion to c11 ion are more easily dissociated than 13 ions, and it can be considered that this property appears. In other words, there are places that are easily dissociated and places that are difficult to dissociate due to the three-dimensional structure formed by intramolecular hydrogen bonding. It can be inferred that the difference in the behavior of ion dissociation efficiency due to temperature as described above occurs.

  FIG. 7 shows a schematic configuration of the ion trap mass spectrometer of the third embodiment incorporating data processing for estimating the structure of a compound based on such estimation. In this ion trap mass spectrometer, the control unit 8 controls the heater power supply unit 10 so that the set temperature of the ceramic heater 28 changes with a predetermined step width within a predetermined temperature range. Further, every time the set temperature changes, the control unit 8 performs mass analysis of product ions using precursor ions as molecular ions derived from target components in the same sample, the ion source 1, the ion trap 2, and the flight time. The mold mass separation unit 3 and the ion detector 4 are controlled. As a result, product ion spectra at different set temperatures are obtained in the data processing unit 9.

  The product ion intensity extraction unit 91 extracts the peak intensities at a plurality of mass-to-charge ratios determined in advance (or automatically extracted) from the plurality of product ion spectra as the product ion intensity. From the result of the product ion intensity extraction unit 91, the change in the product ion intensity with respect to the change in the set temperature of the ceramic heater 28 is determined for each product ion. Therefore, the structure analysis unit 92 includes a binding site corresponding to a product ion whose strength increases with an increase in set temperature, and a binding site corresponding to a product ion whose strength decreases as the set temperature increases. The chemical structure of the target component is displayed on the screen of the display unit 11 so that it can be identified and visually identified. Alternatively, the display mode of the structural information can be modified as appropriate, such as graphically depicting how the three-dimensional structure due to intramolecular hydrogen bonding changes with temperature changes.

  It should be noted that the above embodiment is merely an example of the present invention, and it should be understood that modifications, additions, and modifications as appropriate within the spirit of the present invention are included in the scope of the claims of the present application. For example, in the ion trap mass spectrometer of the above embodiment, the ion trap has a three-dimensional quadrupole configuration, but may be a linear ion trap.

DESCRIPTION OF SYMBOLS 1 ... Ion source 2 ... Ion trap 21 ... Ring electrode 22, 24 ... End cap electrode 23 ... Ion introduction hole 25 ... Ion injection hole 26 ... Radical particle introduction port 28 ... Ceramic heater 3 ... Time-of-flight mass separation part 4 ... Ion Detector 5 ... Hydrogen radical irradiation part 51 ... Hydrogen radical supply source 52 ... Valve 53 ... Nozzle 54 ... Skimmer 6 ... Gas supply part 61 ... Gas supply source 62 ... Valve 63 ... Gas introduction pipe 64 ... Gas introduction pipe heater 7 ... Trap Voltage generation unit 8 ... control unit 9 ... data processing unit 91 ... product ion intensity extraction unit 92 ... structure analysis unit 10 ... heater power supply unit 11 ... display unit

Claims (7)

An ion trap mass spectrometer for mass-analyzing product ions generated by dissociating ions derived from sample components inside an ion trap,
a) an ion trap that traps ions derived from sample components by the action of an electric field;
b) a hydrogen radical irradiation part that dissociates the ions trapped in the ion trap by irradiating the radicals with hydrogen radicals;
c) a heat application unit that promotes dissociation of ions by the hydrogen radicals by applying heat to the ions trapped in the ion trap;
An ion trap mass spectrometer comprising: The ion trap mass spectrometer according to claim 1,
An ion trap mass spectrometer, further comprising an ion source based on a matrix-assisted laser desorption ionization method, wherein monovalent ions generated by the ion source are dissociated in the ion trap. The ion trap mass spectrometer according to claim 1 or 2,
The heat applying unit includes a ceramic heater, which is an insulator, disposed between a plurality of electrodes constituting the ion trap, a heating power source unit that supplies heating power to the ceramic heater, and a heat medium in the ion trap. A gas introduction part for introducing a buffer gas as
An ion trap mass spectrometer. The ion trap mass spectrometer according to claim 1 or 2,
The heat applying unit includes a gas heating unit that heats a buffer gas outside the ion trap;
A gas introduction part for introducing a buffer gas heated by the gas heating part into the ion trap;
An ion trap mass spectrometer. The ion trap mass spectrometer according to claim 4,
The gas heating unit includes a heater that heats a gas introduction pipe for introducing the buffer gas into the ion trap, a heating power supply unit that supplies heating power to the heater,
An ion trap mass spectrometer. The ion trap mass spectrometer according to claim 3 or 4,
The ion trap mass spectrometer is characterized in that the buffer gas also serves as a cooling gas used for cooling ions trapped in the ion trap. The ion trap mass spectrometer according to claim 1,
Data processing for estimating structure information of a sample component using intensity information of a plurality of product ions derived from one sample component obtained when the temperature at the time of applying heat to the ions from the heat applying unit is changed The ion trap mass spectrometer further comprising a unit.

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