Radiant energy – Ionic separation or analysis – Ion beam pulsing means with detector synchronizing means
Reexamination Certificate
1999-12-01
2001-10-09
Berman, Jack (Department: 2881)
Radiant energy
Ionic separation or analysis
Ion beam pulsing means with detector synchronizing means
Reexamination Certificate
active
06300627
ABSTRACT:
The invention relates to time-of-flight mass spectrometers for the measurement of daughter ion spectra (also called fragment ion spectra or MS/MS spectra) and corresponding measurement methods.
According to the invention, the ions of an ion source are initially accelerated only to an intermediate level of energy, allowing them to decompose at that energy level by metastable decomposition or by collisionally induced fragmentation (CID). The ions are then accelerated in a second step to a high energy level. Light fragment ions gain a higher velocity than heavier fragment ions or non-decomposed parent ions. The spectrum of fragment ions can be detected separated by mass in either linear or reflector mode. An ion selector at the low energy level selects a single type of parent ion in order to avoid superpositions with fragment ions of other parent ions. A particularly preferred embodiment raises the potential of ions, for there second acceleration, during their flight through a small electrically isolated flight path chamber.
PRIOR ART
The conventional method of time-of-flight mass spectrometry generates the ions in pulses, e.g. by shots of laser light, within the ion source at a constant high voltage of 6 to 30 kilovolts. The ions being expelled from the ion source are accelerated in the acceleration region between the ion source and the base electrode, then pass through an aperture in the base electrode into a field-free flight region, and finally hit an time-resolving ion detector where they are measured. The measured arrival time of the ions at the detector can be used to determine their mass m (or rather their mass-to-charge ratio m/e) from their identical kinetic energy. For the purpose of simplification, reference is here always made to the mass m, even though mass spectrometry is only involved in measuring the mass-to-charge ration m/e, whereby z is the number of elementary charges of the ion. Since many types of ionization, for example MALDI, mainly provide ions with a single charge only (z=1), there is literally no difference.
As the ions originating from the ion source frequently possess an initial energy which is not the same for all the ions, higher acceleration methods of 20 to 30 kilovolts have become common, because then the spread of the initial energy of the ions has a less detrimental effect on mass resolution. For even better levels of mass resolution the velocity-focusing method with a two-stage Mamyrin ion reflector has proven successful whereby the ions are reflected into a second linear, field-free flight region. In the first stage of the reflector, the ions are considerably decelerated, while in the second stage they are only decelerated slightly. Faster ions penetrate farther into the weak deceleration field of the second stage than slower ions so they cover a longer distance, which, if the two deceleration fields are set correctly, can accurately compensate for the faster velocity of flight and therefore increase the mass resolving power.
One of the most frequently used ion sources in time-of-flight mass spectrometry utilizes matrix-assisted laser desorption for ionization (MALDI). The samples are located in a matrix substance on a sample support plate. The ions generated by a laser light pulse lasting 1 to 20 nanoseconds leave the surface with a higher spread of velocities.
Since this rather wide spread of velocities can no longer be properly focused by a reflector, another method for improving the mass resolution, a delayed acceleration of the ions with respect to the laser pulse, has proven successful for MALDI. The basic principle for this increase in mass resolution under conditions of initial energy spread of the ions has already been known for over 40 years now. The method was published in the paper by W. C. Wiley and I. H. McLaren, “Time-of-Flight Mass Spectrometer with Improved Resolution”, Rev. Scient. Instr. 26, 1150, 1955. The method was termed “time lag focusing” by the authors. Most recently it has been investigated under various names (“space-velocity correlation focusing” or “delayed extraction” for instance) in scientific papers with regard to MALDI ionization; it is also available in commercial time-of-flight mass spectrometers.
The reflector of a time-of-flight mass spectrometer can, however, also be used to investigate fragment ions which are generated in the field-free ion path from selected ions. The selected type of ions is frequently called “parent ions” or “precursor ions”. The decomposition may be caused by internal energy of the ions gained in the ionization process itself or by collisions in a gas filled collision cell.
If parent ions decompose into fragment ions in the field-free region after acceleration, all the fragment ions continue to fly at the same velocity v as their parent ions but they carry considerably less kinetic energy E
k
=mv
2
/2 due to their smaller mass. They penetrate to a much lesser extent into the second deceleration field of the reflector, return much earlier, and are measured mass-separated at the end of the second field-free flight region.
In the MALDI process of ionization, the ions in the vapor cloud generated by the laser pulse are subjected to very many collisions, which increase the inner energy of the ions by multiple but mild excitation of intra-molecular oscillations. Consequently a number of these ions become “metastable”, which means these ions decompose with a half life in the order of several microseconds so a detection of decomposition ions in the mass spectrometer becomes possible. Detection of fragment ions which occur in the first field-free flight region of the mass spectrometer by the reflector of a time-of-flight spectrometer has become known as the PSD method (PSD =post source decay). On the other hand, the parent ions in flight can also pass through a collision-gas filled cell in the drift region and thus form collision-induced fragment ions which can be detected in the same manner (CID=collisionally induced decomposition).
The method of measuring PSD or CID fragment ions by means of the reflector has serious disadvantages. Detection of ions is restricted to a relatively small energy range, about 25% 30% in usual versions of commercially available equipment. Ions always have to pass through the strong deceleration field of the first reflector stage to be reflected with velocity focusing. However, this first deceleration field already consumes a good ⅔ of the original acceleration energy, thus light ions do not pass this region. The full mass spectrum has to be measured step-wise. From parent ions with a mass of 3,200 atomic mass units, only fragment ions of about 2,400 to 3,200 atomic mass units can be scanned in a first step of spectrum acquisition, fragment ions between 1,800 and 2,400 mass units can be scanned in a second spectrum acquisition, fragment ions between 1,350 and 1,800 mass units can be scanned in a third spectrum acquisition, and so forth. For a medium-sized peptide about 10-15 scans are necessary if the entire spectrum of fragment ions is to be measured. All these spectra must be adjusted to one another by a complex mass calibration method. Only then can these partial sections of the spectrum be collated in the data system to make up an artificially generated composite spectrum.
The number of individual spectra can in principle be reduced if the reflector is lengthened considerably. Then the first deceleration field can be reduced. However, then the ion spends the largest part of its life between generation in the ion source and its measurement in the ion detector in precisely this reflector. This causes most of the decompositions to take place not in the first field-free flight region but in the reflector. These ions are then distributed as background ions over the entire spectrum and thus cause substantial background noise which leads to a bad signal-to-noise ratio and impairs detection of the decomposed ions.
A better method was proposed in U.S. Pat. No. 5 464 985 (T. J. Cornish and R. J. Cotter). Here the reflector did not
Franzen Jochen
Holle Armin
Koster Claus
Berman Jack
Bruker Daltonik GmbH
Smith II Johnnie L
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