1. Field of the Invention
The present invention relates to a mass spectroscopy spectrum analysis system using a mass spectrometer, and to a system for automatically determining an optimum flow of mass spectroscopy within a measurement time in order to identify the chemical structure of biopolymers, such as polypeptides or sugars, with high precision and efficiency.
2. Background Art
In a general mass spectroscopy, a sample as the object of measurement is ionized, and a variety of resultant ions are delivered to a mass spectrometer for measuring the ion intensity for each mass-to-charge ratio m/z, which is the ratio of the mass number m of ion to the valence z. As a result, a mass spectrum is obtained, which consists of a peak of the measured ion intensity (ion peak) for each mass-to-charge ratio m/z value. Such a mass spectroscopic analysis of the ionized sample in a first dissociation step is called MS1. In tandem mass spectrometer, in which multiple-stage isolation is possible, an ion peak having a specific mass-to-charge ratio m/z is selected (the selected ion species is called a parent ion) from the ion peaks detected by MS1, and the thus selected ion is dissociated and broken up by collision with gas molecules or the like. The resultant dissociated ion species is then subjected to mass spectroscopy, thereby obtaining a mass spectrum in a similar manner. The n-stage dissociation of the parent ion and the subjecting of the dissociated ion species to mass spectroscopy are referred to as MSn+1. Thus, in the tandem mass spectrometer, the parent ion is dissociated in multiple stages (1, 2, . . . , n stages), and the mass number of the ion species generated in each stage is analyzed (MS2, MS3, . . . , MSn+1).
(1) Most of the mass spectrometers capable of tandem analysis are equipped with a data-dependent function whereby, when selecting the parent ion for MS2 analysis from the ion peaks in MS1, the ion peaks are selected in decreasing intensities (such as the ion peaks in the top 10 strongest-intensities) as the parent ions, and then they are subjected to dissociation and mass spectroscopy (MS2).
(2) The ion-trapping type mass spectrometer manufactured by Finningan is equipped with a Dynamic Exclusion function whereby, when selecting a parent ion for MS2 analysis from the ion peaks in MS1, the ion species having a mass-to-charge ratio m/z value that is designated by the user in advance is excluded from the selection as a parent ion.
(3) Known examples relating to the determination of correspondence between a measured ion species and an ion species that has been measured include the following:
Patent Document 1: JP Patent Publication (Kokai) No. 2001-249114 A
Patent Document 2: JP Patent Publication (Kokai) No. 10-142196 A 1998
In Patent Document 1, a characteristic peak in the first-stage spectrum data and the spectrum data in the second stage of the corresponding ion species are stored in a database. In the subsequent measurements, spectrum data obtained by mass spectroscopy in the second stage of a sample as the object of measurement is compared with the second-stage spectrum data in the database in order to determine the degree of correspondence. Data components with the highest degree of correspondence is outputted as the comparison result.
In Patent Document 2, a measurement is continuously carried out during a multiple-stage dissociation measurement without conducting a sample injection process during measurement so that an ion intensity fluctuation due to injection between the MSn and MSn+1 data can be prevented. In this way, the need for the addition of a standard sample can be eliminated, thereby enabling an efficient quantitative analysis. The routine returns to MSn+1 or proceeds to the next MS1 measurement, depending on whether or not the data corresponds to the designated ion data that has been already collected in the MSn and MSn+1 data analysis.
Reviews of Modern Physics, Vol. 62 (1990), pp. 531-540, provides a basic description of an ion trap. A cross section of a basic configuration of the ion trap is shown in FIG. 15. The ion trap, which is a quadrupole ion trap, is made up of two end-cap electrodes and a single ring electrode. An RF voltage is applied to these electrodes such that a quadrupole electric field is formed at the center of these electrodes, thus enabling the trapping of gaseous ions three dimensionally. By continuously varying the RF voltage, the mass of the ions that are discharged can be controlled. A quadrupole pole is made up of four parallel poles. By applying a RF voltage to the electrodes, gaseous ions can be two dimensionally trapped at the center of the electrodes. By controlling the RF voltage that is applied, it becomes possible to discharge ions with a specific mass or, conversely, trap only those ions with a specific mass.
A tandem mass spectroscopy (MS/MS) can be conducted using a quadrupole ion trap, as described in the U.S. Pat. No. Re. 34000. In this apparatus, those ions for which no analysis is required are discharged prior to MS/MS. Namely, the removal of the ions for which no analysis is required is not conducted prior to the primary mass spectroscopy. A RF voltage that resonates with the ions is then applied in order to increase the kinetic energy. As a result of these operations, dissociated ions (fragment ions) are created by the collision induced dissociation (CID) with remaining molecules. By subjecting these fragment ions to mass spectroscopy (tandem mass spectroscopy), the mass of the fragment ions can be determined. In this case, it is necessary to initially conduct a mass spectroscopy without involving a CID (primary mass spectroscopy) in order to determine the ions as the object of a tandem mass spectroscopy (MS/MS, or a secondary mass spectroscopy). It is also possible to repeat a similar operation to further conduct a tandem mass spectroscopy (MSn) on a specific dissociated ion.
Recently, mass spectroscopic methods are often employed for an exhaustive analysis of proteins. Analytical Chemistry, Vol. 73 (2001), pp. 5683-5690, describes examples of analysis called a shotgun analysis. In this technique, a peptide mixture prepared by subjection a protein to enzymatic digestion is separated using a liquid chromatograph, and a separated sample is then subjected to a tandem mass spectroscopy using a quadrupole ion-trap mass spectrometer. With reference to the determined mass of the ion and that of the fragment ion, a database of proteins or genes is searched in order to identify a protein. In case the types of the peptide mixture are too numerous, each peptide might not be completely separated in the liquid chromatograph, and a plurality of kinds of peptides might be simultaneously introduced into the mass spectrometer. This gives rise to the need for automatic tandem analysis called data-dependent analysis. Specifically, the band width of a separated sample separated in a liquid chromatograph is in the order of one minute, and the number of kinds of ions that can be subjected to tandem mass spectrometer at one time is limited to five. In many cases, the ions with greater ion intensities are preferentially subjected to tandem mass spectroscopy, although this depends on the setting of the data-dependent analysis.
A technical material for the quadrupole ion-trap mass spectrometer manufactured by ThermoFinnigan (www.thermo.com/eThermo/CMA/PDFs/Articles/articlesFile—10918.pdf) describes a dynamic exclusion function. Prior to the start of analysis, the masses of those ions to be excluded from tandem mass analysis are entered and then a list is prepared. By this operation, it becomes possible to exclude those ions put on the list as the objects of data-dependent analysis (tandem mass spectrometer). When this function is to be employed, a conventional mass spectroscopy is conducted first without involving the CID, and then the mass of the ions to be detected is determined. Next, priorities of the ions as the objects of tandem mass spectroscopy are determined in the detected ions, whereupon those ions put on the list are excluded from the objects of data-dependent analysis (tandem mass spectroscopy).