JP2007115556A - Mass spectrometry - Google Patents

Abstract

<P>PROBLEM TO BE SOLVED: To provide a mass spectromety that identifies the kinds of particles by measuring the masses of the particles, for not only measuring the masses of the ions from time-of-flight of ions or neutral particles (hereinafter, to be referred to as "particles"), but also analyzing the masses of fragment particles generated by the dissociation of precursor ions or particles. <P>SOLUTION: A detector measures both the flight time and the kinetic energy of particles. The precursor ions are dissociated by the flight time, and the fragment particles are dissociated by the kinetic energy. <P>COPYRIGHT: (C)2007,JPO&INPIT

Description

  The present invention relates to a mass spectrometer that identifies the type of particle by measuring the mass of the particle. In particular, in time-of-flight mass spectrometry, not only the mass of ions is measured from the time of flight of ions or neutral particles (hereinafter abbreviated as “particles”), but also fragments generated by dissociation of precursor ions or particles. Used to perform mass analysis of particles. It is effective for detecting disease markers and the like.

  The mass spectrometer includes an ionization unit, an acceleration unit, a mass separation unit, and a particle detection unit. A device that uses the time-of-flight method for the mass separation unit is called a time-of-flight mass spectrometer, and has the feature that there is no limit on the mass range that can be analyzed simultaneously, in principle, for particles having different masses. In the ionization unit, the particles to be measured are taken out in a vacuum and ionized, and then kinetic energy is given at a constant acceleration voltage in the acceleration unit. After acceleration, it moves at a constant speed. The time of flight of the ion moving at a constant velocity is proportional to the square root of the value (m / z) obtained by dividing the mass of the ion by the valence of the ion. Therefore, if the valence is known, the mass can be known from the flight time (linear mode).

When excessive energy is accumulated in ions during extraction in a vacuum, acceleration, or constant velocity motion, ions may break during flight, resulting in a plurality of fragment particles. In addition, fragmented particles may occur at the moment when excessive energy is applied. The mass pattern of the fragmented particles has the structural information of the precursor particles, and can be used, for example, for identification of peptides and proteins and determination of amino acid sequences. In the case of a peptide, the sequence can be analyzed from the fragmentation pattern of the N-terminal ion and C-terminal ion, and the amino acid sequence can be known. The mass measurement of ions contained in the fragment particles is performed on the time-of-flight axis in the same manner as the precursor ions using a reflectron that reflects the ions (reflector mode, see “Non-Patent Document 1” below).
B. Spengler, Journal of Mass Spectrometry, vol. 32, 1019 (1997).

  When the fragment ions are measured in the reflector mode, they appear as time-of-flight peaks different from the precursor ions on the mass spectrum having the horizontal axis as the time of flight. For this reason, for example, in a sample in which a plurality of proteins are mixed, a plurality of precursor ion peaks, precursor ion peaks with different valences, and fragment ion peaks generated from the precursor ions are superimposed, making it difficult to assign peaks. There are many cases. In addition, particles that can be analyzed after fragmentation occurs are only ions contained in the fragment particles, and neutral particles cannot be analyzed. Neutral particles can be detected in the linear mode. However, since particles fragmented during constant velocity motion have the same flight time as the precursor ions, the precursor ions cannot be distinguished from the fragment particles in the flight time.

  In the present invention, the problem is solved by using a particle detector capable of measuring the time and kinetic energy at which particles reach the detector in the linear mode. The mass of the precursor ion is separated on the time axis, and the mass of the fragment particles is separated on the energy axis.

  According to the present invention, precursor ions can be analyzed based on the time of flight, and analysis of all particles of ions and neutral particles fragmented by kinetic energy becomes possible. Since the measurement is performed in the linear mode, a plurality of proteins can be analyzed simultaneously even in a sample in which a plurality of proteins having different masses are mixed. In addition, since all the ions and neutral particles of the fragment particles can be analyzed, the detection sensitivity of fragment particles is improved compared to the conventional method, which can analyze only fragment ions that remain charged after fragmentation. Can be made.

  In the present invention, various fragmentation methods can be used. In post-source decomposition (PSD), the ion source becomes unstable due to the excess energy imparted during acceleration, and the precursor ions fragment during free flight after acceleration. In collision-induced dissociation (CID), accelerated precursor ions collide with atoms such as argon to cause fragmentation. In laser dissociation (LD) and infrared multiphoton absorption dissociation (IRMPD), light energy is absorbed by precursor ions and fragmentation occurs.

  As a method for measuring the energy of these fragment particles, there is a method using a magnetic field or an electric field for ions. As described below, the present invention measures not only ions but also the energy of neutral particles by a solid state detector without using a magnetic field or an electric field. The best mode of the present invention will be described below.

  FIG. 1 shows an embodiment of a quantum particle detector using a superconducting tunnel junction. The ion source is matrix-assisted laser desorption / ionization (MALDI) using an ultraviolet laser, and the precursor ions acquire excess energy immediately after ionization or ionization, and fragmentation occurs after acceleration by an electric field. Precursor ions and fragment particles that have not been fragmented move at the same speed and simultaneously enter the detector. The energy of ions or neutral particles is measured by the detector. Measure in linear mode. Measurement examples of bovine serum albumin (abbreviated as BSA) having a mass of 66.4 kDa are shown in FIGS. 2 to 4 show the experimental results under conditions of about 8 μJ per laser pulse, and FIG. 5 shows about 12 μJ. The voltage for accelerating the ions is 17.5 kV.

Figure 2 plots points for each collision event on the m / z value calculated from the time the ion accelerated at a constant voltage flies from the ion source to the detector and the kinetic energy coordinates measured by the detector. It is a scatter diagram. The horizontal axis represents the value of m when z = 1 is assumed for convenience. A BSA ⁺ event group appears at the 66.4 kDa position, and a BSA ⁺⁺ event group appears at the 33.2 kDa position. The kinetic energy of BSA ⁺⁺ accelerated at a constant voltage is observed to be larger than that of BSA ⁺ .

FIG. 3 is a histogram of m / z values calculated from the time required for ions accelerated at a constant voltage to fly from the ion source to the detector, and is a so-called mass spectrum. The horizontal axis represents the value of m when z = 1 is assumed for convenience. A BSA ⁺ peak appears at 66.4 kDa, and a BSA ⁺⁺ peak appears at 33.2 kDa.

  FIG. 4 is a histogram for the kinetic energy of FIG. 1 consisting only of collision events (events between 60 kDa and 80 kDa) during the time when the monovalent ions of BSA reach the detector in FIG. In addition to the peak of the precursor ion, an event with a small kinetic energy appears in the range indicated as PSD particles. This is an event of particles fragmented after ions pass through the acceleration. This fragmentation is called post-source decomposition (PSD) and is believed to collide with particles during acceleration and gain excess energy. The flying speed of the particles that have undergone post-source decomposition is almost the same as that of the precursor ions, but since the mass is smaller than that of the precursor ions, the kinetic energy is reduced and appears at a position of kinetic energy lower than the peak of the precursor ions.

  FIG. 5 shows the case where the laser power is increased (about 12 μJ), and the precursor ion hardly forms a peak, and the event of fragmented PSD particles accounts for the majority. The mass of these PSD particles can be determined from the detected kinetic energy. The PSD particle mass pattern has the precursor ion structure information. It can be used for identification of precursor ions and determination of amino acid sequences.

FIG. 6 shows an embodiment in which precursor ions are fragmented by collision-induced dissociation (CID) after the acceleration unit. The ion source is MALDI using an ultraviolet laser, and precursor ions accelerated by an electric field collide with atoms in a CID cell into which a gas is introduced. This collision causes fragmentation. Precursor ions and fragment particles that have not been fragmented move at the same speed and simultaneously enter the detector. The energy of ions or neutral particles is measured by the detector.

FIG. 2 is a layout of an experimental device according to the first embodiment of the present invention. Data 1 of Example 1 according to the present invention. Data 2 of Example 1 according to the present invention. Data 3 of Example 1 according to the present invention. Data 4 of Example 1 which concerns on this invention. The experimental apparatus layout of Example 2 which concerns on this invention.

Claims (5)

The mass of the molecule to be measured is specified by the time-of-flight method, and the fragment particles derived from the specified molecule to be measured are extracted from the fragment particles generated by the fragmentation during the flight. A mass spectrometric method comprising identifying a molecule to be measured by separating a mass based on kinetic energy. 2. The mass spectrometric method according to claim 1, wherein when the fragment particles are detected, the energy of both ions and neutral particles is measured to enable mass analysis of the fragment particles. . 2. The mass spectrometric method according to claim 1, wherein the fragmentation is caused by post-source decomposition (PSD), collision-induced dissociation (CID), electron capture dissociation (ECD), laser dissociation (LD) or infrared multiphoton. A mass spectrometric method characterized by absorption dissociation (IRMPD). 2. The mass spectrometric method according to claim 1, wherein the particle detector that measures time of flight and kinetic energy is a quantum particle detector that extracts an electric signal by electronic excitation. 2. The mass spectrometric method according to claim 1, wherein the particle detector for measuring the time of flight and the kinetic energy is a thermal particle detector for measuring a temperature rise of an absorber with which particles collide. Method.

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