JP4701720B2 - MALDI ion trap mass spectrometer and analysis method - Google Patents

Description

  In the present invention, MALDI (Matrix-Assisted Laser Desorption / Ionization) is used as an ion source, and ions generated by the ion source are once accumulated in an ion trap storage device such as a three-dimensional ion trap device, and then mass spectrometry is performed. The present invention relates to a MALDI ion trap mass spectrometer and an analysis method using the same.

  The outline of mass spectrometry using a MALDI ion trap mass spectrometer is as follows. A sample placed on a MALDI sample tray is irradiated with a laser beam to ionize the sample. The ionized sample is converged by an ion lens and sent to an ion trap. In the ion trap, an appropriate voltage is applied to the electrode at that time to trap incoming ions in the internal ion trap space. The trapped ions may be released from the ion trap as they are, or may be released after operations such as selection and cleavage. The ions emitted from the ion trap are subjected to mass analysis by a subsequent mass spectrometer such as TOFMS (Time Of Flight Mass Spectrometer).

  Mass spectrometers using ion traps generally have a narrow mass range that can be measured at once. This is because most of the ions generated by MALDI are monovalent, and the ion trap has a low mass cutoff. That is, when the maximum value of the mass-to-charge ratio (m / z) of ions to be measured is set to a certain value, ions having a mass-to-charge ratio of about 1/2 to 1/3 or less are formed in the ion trap. This is because they cannot be trapped by the quadrupole electric field and pass through the ion trap (Patent Document 1). The lower limit of the mass-to-charge ratio is determined mainly by the value (amplitude) of the radio frequency (RF) voltage applied to the ring electrode of the ion trap immediately after introducing ions into the ion trap. Fine adjustment can be performed by changing the time difference between the laser beam irradiation and the laser irradiation.

  In mass spectrometry using a conventional MALDI ion trap mass spectrometer, any mass range can be dedicated from multiple mass ranges prepared in advance (e.g., ultra-low mass, low mass, medium mass, high mass, ultra-high mass, etc.) The selection is made on the program, and in some cases, the setting parameters, particularly the laser irradiation time, are finely adjusted to optimize the estimated mass of the sample to be measured for measurement.

JP 2004-303719 A ([0015])

  In many cases, the content of the sample is unknown, and an optimal mass range cannot be set in advance. For example, even in a sample to be measured in the medium mass range as a standard, the compound may actually be slightly biased toward the low mass side or conversely toward the high mass side. Therefore, in order to truly optimize the analysis conditions, it is necessary to first confirm the ions to be detected, finely adjust the timing of laser irradiation, and perform measurement again.

  For example, in the case of a mixture sample in which a compound is widely distributed in the medium mass range, the entire sample cannot be measured with high sensitivity no matter what setting is made. For this reason, sequence coverage may be lowered when highly reliable identification cannot be performed by database search using MS data such as peptide mass fingerprinting (PMF).

  The problem to be solved by the present invention is to provide a method and apparatus configuration capable of covering a wider mass range in one measurement in a MALDI ion trap mass spectrometer.

The present invention made to solve the above problems
A MALDI unit that irradiates the sample with a pulsed laser according to the first operating parameter;
An ion trap unit that traps ions generated in the MALDI unit and releases them to the mass spectrometer unit according to a second operating parameter;
In a MALDI ion trap mass spectrometer equipped with
The pulse laser irradiation time is set as the first operating parameter, and the time when the high frequency voltage is applied to the ring electrode of the ion trap portion is set as the second operating parameter, and the pulse laser irradiation time and the high frequency voltage applied to the ring electrode are set. The MALDI unit and the ion trap unit are controlled so that multiple pulse laser irradiations are performed on one sample while changing the time interval from the application time, and the mass spectrum obtained for each pulse laser irradiation is added. Alternatively, a control unit that generates a mass spectrum for the sample by averaging is provided.

One general, the more change the time for applying a voltage to the electrodes of the ion trap, it is easier to change the irradiation time of the pulsed laser.

  Conventionally, when a single sample is analyzed with a MALDI ion trap mass spectrometer, the MALDI unit irradiates the sample with a pulsed laser several times, and a profile that constitutes a mass spectrum that is the result of mass analysis for each irradiation Data was added or averaged. Here, while one sample was analyzed, the parameters defining the operation in the MALDI unit and the parameters defining the operation in the ion trap unit did not change. Therefore, the results measured under the same conditions were added or averaged.

On the other hand, in the MALDI ion trap mass spectrometer according to the present invention, in each of mass spectrometry performed on the same sample a plurality of times, a first operation parameter that is one parameter that defines the operation in the MALDI unit, and the ion trap unit The relationship between the second operation parameter, which is one parameter that defines the operation in the above , that is, the time interval between the pulse laser irradiation time and the high frequency voltage application time to the ring electrode is changed .

A control unit is by performing a plurality of times of pulse laser irradiation while changing the time interval of two times, it is possible to widen the range of mass-to-charge ratios are trapped in the ion trap. That is, a wider mass range can be covered by one mass analysis.

  In this case, the voltage applied to the ring electrode of the ion trap is a high voltage, and it is relatively difficult to change the application time, but it is relatively easy to change the irradiation time of the pulse laser. It is. Therefore, when this method is adopted, it is desirable to adopt a method in which the time for applying the high frequency high voltage to the ring electrode is fixed and the irradiation time of the pulse laser is sequentially changed.

  A MALDI ion trap mass spectrometer according to an embodiment of the present invention will be described with reference to FIG. In the present mass spectrometer 10, a MALDI unit 11 that is an ion source, an ion trap unit 12 that temporarily holds ions generated in the MALDI unit, and ions that are accelerated and released from the ion trap unit fly and are detected. The time-of-flight (TOF) type mass analysis unit 13 and the control unit 14 for controlling these units are included. In addition, the control unit 14 has a function of generating a mass spectrum based on detection data output from the detector 131 of the mass analysis unit 13 and performing subsequent data analysis as necessary.

In such a MALDI ion trap mass spectrometer, mass analysis of one sample is performed according to the procedure shown in FIG.
In the MALDI unit 11, the sample 111 mixed in the matrix is placed on a sample plate, and the sample 111 is ionized and emitted by irradiating a pulsed laser beam from the N2 laser light source 112 there. The released ions are focused on the opening of the inlet end cap electrode 121 of the ion trap portion 12 by the ion lens 113 and introduced into the ion trap space 122. As described later, a quadrupole electric field is formed in the ion trap space 122, and thus sequentially input ions are held in the ion trap space 122. The held ions are subjected to processing such as cooling, selection, and cleavage as necessary.

  Ions held in the ion trap space 122 are accelerated by applying a voltage to a predetermined electrode, which will be described later, and are released toward the TOF mass spectrometer 13 from the opening of the outlet end cap electrode 123 at once. During this acceleration, ions are given a speed corresponding to their mass-to-charge ratio, and ions with a lower mass-to-charge ratio are accelerated faster. Thereby, the ions are separated by the mass-to-charge ratio while flying in the flight space where the electromagnetic field of the TOF type mass analyzer 13 does not exist, and separated and detected by the detector 131.

  When mass spectrometry is performed once in this way, one mass spectrum is generated with the mass / charge ratio on the horizontal axis and the ion intensity on the vertical axis. In the MALDI unit 11, the sample 111 set on the sample plate is irradiated with laser pulses many times, and a mass spectrum is generated by the process as described above for each irradiation. The control unit 14 adds the mass spectra generated in this way or adds and averages them to generate a mass spectrum related to the sample.

  In the series of procedures as described above, each operation is performed at a timing as shown in FIG. That is, first, the sample 111 is irradiated with a pulse laser at time t0. At this time, no voltage is applied to the inlet end cap (EC) electrode 121 of the ion trap section 12 (grounded), and a positive DC voltage (in the case of positive ions) is applied to the outlet end cap (EC) electrode 123. Apply it. Ions generated by pulsed laser irradiation enter the ion trap space 122 in order from the smallest mass-to-charge ratio (m / z), but from the opening by the voltage (Retard voltage) applied to the exit-side end cap electrode 123. It stays in the ion trap space 122 without exiting to the mass analyzer 13.

  By continuing this state for a predetermined time, all ions are introduced into the ion trap space 122. At the time t1, the outlet end cap electrode 123 is grounded, and a high frequency (RF) voltage is applied to the ring electrode 124 of the ion trap section 12. A pseudo potential is formed in the ion trap space 122 by this RF voltage, and ions are trapped. The trapped ions collide with a gas (a rare gas such as He) introduced in advance into the ion trap space 122, energy converges, and collects at the center of the ion trap space 122 (cooling).

  In the MS mode, application of the RF voltage to the ring electrode 124 is stopped at the time t2 when the cooling process is completed, and a reverse polarity voltage is applied to the inlet side end cap electrode 121 and the outlet side end cap electrode 123 ( In the case of positive ions, a positive voltage is applied to the inlet-side end cap electrode 121 and a negative voltage is applied to the outlet-side end cap electrode 123) to form a strong voltage gradient in the ion trap space 122, and ions are supplied to the mass analyzer 13. Accelerate towards. As a result, the ions obtain an initial velocity corresponding to the mass-to-charge ratio, jump out of the openings of the outlet end cap electrode 123 all at once, and fly in the flight space of the mass analyzer 13 at a constant velocity. In the case of the apparatus of FIG. 1, the ions are turned back by the reflecting portion (reflectron) at the end of the mass analyzing unit 13 and flew for a longer distance, and the detector 131 has a small mass-to-charge ratio (from a large initial velocity). Detected sequentially. The control unit 14 generates a mass spectrum from the input detection signal intensity of ions and the time of flight.

  After the mass analysis by one pulse laser irradiation is thus completed, the voltage at the inlet end cap electrode 121 and the outlet end cap electrode 123 is returned to the standby state value (the value at time t0) at time t3, and the next pulse Laser irradiation is performed. Such a pulse laser irradiation is repeated many times (usually several tens to several hundreds), and each mass spectrum obtained thereby is added or averaged by the control unit 14, whereby mass analysis data of the sample 111 is obtained. Get.

In the MS / MS mode and the MS ⁿ mode, the precursor ions are selected and the collision-induced dissociation process using gas is performed before ions are released from the ion trap unit 12 at time t2 (and before cooling).

  In the MALDI ion trap mass spectrometer 10 according to the present invention, when this pulse laser irradiation is repeated, an RF voltage is applied to the ring electrode 124 and the voltage of the outlet end cap electrode 123 is grounded as shown in FIG. The pulse laser irradiation times t01, t02, t03,...

  The mass-to-charge ratio m / z of ions trapped in the ion trap space 122 is theoretically determined by the magnitude and frequency of the RF voltage, but ionization in the MALDI unit 11, that is, laser irradiation time t0, and RF voltage application (RF The time between the start-up time t1 is also significantly affected, and as the time from the laser irradiation time t0 to the RF start-up time t1 increases, ions having a larger mass-to-charge ratio are trapped in the ion trap space 122. Become so.

  As shown in FIG. 3, by gradually shifting the pulse laser irradiation times t01, t02, t03,..., The range of the mass-to-charge ratio of ions trapped in the ion trap section 12 for each irradiation is gradually changed. Can be shifted. Therefore, mass analysis is performed on one sample 111 by shifting the pulse laser irradiation time in this way, and the data obtained thereby are accumulated (addition or addition averaging), so that ions of a wide mass-to-charge ratio range can be obtained. Mass spectrometry can be performed.

  In order to change the time between the pulse laser irradiation time and the RF voltage application time, a method of changing the RF voltage application time by fixing the pulse laser irradiation time is also conceivable, but the RF voltage is a high voltage. In practice, it is easier to change the pulse laser irradiation time than to change the application time.

  The data which measured the bovine serum albumin trypsin digest using the MALDI ion trap type | mold mass spectrometer AXIMA-QIT (AXIMA is a registered trademark) by Shimadzu Corporation which is one Example of this invention are shown. FIG. 4B is a mass spectrum when the time between pulse laser irradiation time t0 and RF voltage application time t1 is a standard value of 23 μsec, and the mass-to-charge ratio m / z is 1200 or less. It can be seen that there are very few ions detected in the part. On the other hand, when the time t0 of the pulse laser irradiation is delayed by 6 μsec and the time t0 to t1 is shortened, the mass spectrum as a whole is shifted to the low mass side as shown in FIG. Many ions are detected even when m / z is 1200 or less.

  For the same sample, irradiation time t0 was shifted little by little, and a plurality of pulse lasers were irradiated. When the obtained profile data were averaged, a mass spectrum over a wide mass range was obtained as shown in FIG.

1 is a schematic configuration diagram of a MALDI ion trap mass spectrometer that is an embodiment of the present invention. FIG. The timing chart of the operation | movement for every pulse laser irradiation performed in the MALDI ion trap type | mold mass spectrometer of the said embodiment. The timing chart of the operation | movement in the case of many times of pulse laser irradiation performed in the MALDI ion trap type | mold mass spectrometer of the said embodiment. It is a mass spectrum of bovine serum albumin trypsin digest collected using a MALDI ion trap mass spectrometer which is an embodiment of the present invention, and (b) is a standard time between pulse laser irradiation and RF voltage application. (A) is when the pulse laser irradiation time is delayed. A mass spectrum of digested bovine serum albumin trypsin collected using a MALDI ion trap mass spectrometer which is an embodiment of the present invention, and a mass spectrum collected many times by gradually changing the irradiation time of the pulse laser. Summed and averaged.

Explanation of symbols

DESCRIPTION OF SYMBOLS 10 ... MALDI ion trap type | mold mass spectrometer 11 ... MALDI part 111 ... Sample 112 ... N2 laser light source 113 ... Ion lens 12 ... Ion trap part 121 ... Inlet side end cap electrode 122 ... Ion trap space 123 ... Outlet side end cap electrode 124 ... Ring electrode 13 ... TOF type mass analysis part 131 ... Detector 14 ... Control part

Claims (3)

A MALDI unit that irradiates the sample with a pulsed laser according to the first operating parameter;
An ion trap unit that traps ions generated in the MALDI unit and releases them to the mass spectrometer unit according to a second operating parameter;
In a MALDI ion trap mass spectrometer equipped with
The pulse laser irradiation time is set as the first operating parameter, and the time when the high frequency voltage is applied to the ring electrode of the ion trap portion is set as the second operating parameter, and the pulse laser irradiation time and the high frequency voltage applied to the ring electrode are set. The MALDI unit and the ion trap unit are controlled so that multiple pulse laser irradiations are performed on one sample while changing the time interval from the application time, and the mass spectrum obtained for each pulse laser irradiation is added. Or a control unit that generates a mass spectrum for the sample by averaging.
A MALDI ion trap mass spectrometer. 2. The MALDI ion trap mass spectrometer according to claim 1, wherein the time interval is changed by fixing a time at which a high frequency voltage is applied to the ring electrode and changing only the pulse laser irradiation time . 3. A MALDI unit that irradiates the sample with a pulsed laser according to the first operating parameter;
An ion trap unit that traps ions generated in the MALDI unit and releases them to the mass spectrometer unit according to a second operating parameter;
A mass spectrometry method using a MALDI ion trap mass spectrometer comprising:
The pulse laser irradiation time is set as the first operating parameter, and the time when the high frequency voltage is applied to the ring electrode of the ion trap portion is set as the second operating parameter, and the pulse laser irradiation time and the high frequency voltage applied to the ring electrode are set. The MALDI unit and the ion trap unit are controlled so that multiple pulse laser irradiations are performed on one sample while changing the time interval from the application time, and the mass spectrum obtained for each pulse laser irradiation is added. Alternatively, a mass spectrometry method using a MALDI ion trap mass spectrometer is characterized in that a mass spectrum of the sample is generated by averaging.

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