Radiant energy – Ionic separation or analysis – With sample supply means
Reexamination Certificate
2000-03-15
2003-02-04
Berman, Jack (Department: 2881)
Radiant energy
Ionic separation or analysis
With sample supply means
Reexamination Certificate
active
06515280
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to a method and a device for matrix-assisted laser desorption ionization.
PRIOR ART
Conventional methods for the ionization of substances for analysis by mass spectrometry, where a solid substance is heated, for example, transferred to the gaseous phase and ionized there by electron collision, cannot be applied to the large organic and biological molecules. Electron collision with energetic electrons (typically 70 eV) leads to a substantial fragmentation of this species, whereby only small fragments can be observed. On the other hand, even if energy is only supplied at a slow rate, as is always the case when heating a solid sample, large organic molecules decompose before they can vaporize. Only if energy supply takes place at an extremely fast rate, as is the case with a laser beam, for example, the usually slower decomposition process of the molecules does not occur at all.
Laser desorption ionization was already used in the last decade to transfer large organic molecules to the gaseous phase and to ionize them. A special type of laser desorption ionization (LDI) is matrix-assisted laser desorption ionization (MALDI). The detailed review article by F. Hillenkamp, M. Karas, R. Beavis, and B. Chait in “Analytical Chemistry”, Volume 63, year 1991, on pages 1193A-1203A, reports about this technology. In MALDI the analyte molecules are mixed with a so-called matrix. The analyte/matrix molar ratio is 1:10
2
to 1:10
4
. The laser energy is absorbed by matrix molecules and passed on to analyte molecules. The latter thus receive the necessary energy to enter the gaseous phase and are thereby partially ionized. The ionization usually takes place by a protonation. The substances which are mostly used as a matrix are proton donors. In special cases, alkali-metal salts or silver salts are also added to achieve alkali-metal or silver attachment. With some samples both protonated analyte molecules and small quantities of sodium adducts are also observed. The latter often form due to the presence of sodium chloride residues in biological samples.
Experience shows that the ions formed by the MALDI process have kinetic energy which is not negligible and which can be up to or over 10 eV. Since in the classical time-of-flight mass spectrometry the MALDI-generated ions are normally extracted and accelerated at voltages of between 15 and 30 kV, an energy spread of about 10 eV is relatively unimportant here. However, in ion trap mass spectrometers like the Fourier transform ion cyclotron resonance mass spectrometers (FTICRs) the ions produced in an external ion source must be transferred to the trap and captured there. Therefore, the extraction of the MALDI-generated ions no longer takes place here at a potential difference of several kilovolts. However, in the range of low ion extraction potential, which does not exceed 10-20 V, a fluctuation of excess energy in the region of 10 eV is too high and therefore causes enormous difficulties. It leads to a considerable intensity variation of the obtained mass signals and therefore to irreproducible analytical results.
In Fourier transform ion cyclotron resonance mass spectrometry (FTICR-MS) one attempts by various methods to capture the ions in the trap with as few losses as possible and first of all effectively reduce their energy for the ICR measurements. This happens, for instance, by dynamic trapping, where an inert gas is also pulsed into the ion cyclotron resonance trap (ICR trap) in order to absorb the kinetic energy of the ions by collisions with gas molecules or atoms.
One can also attempt to cool the ions in the ion source. This method uses an increased static pressure in the source so that the ions generated by MALDI lose their energy immediately by collisions. According to a different method the MALDI process can even be performed at atmospheric pressure. The U.S. Pat. No. 5,663,561 describes such a device for atmospheric pressure laser desorption ionization. In this case matrix substances are used which undergo photolytic of thermolytic decomposition. However, as opposed to MALDI, the gases formed during this atmospheric pressure desorption are not intended to ionize the large analyte molecules. The selection of matrix molecules therefore only depends on their capability to liberate the large molecules by desorption. Analyte molecules catapulted into the gaseous phase are then ionized for example by a corona discharge. The corona discharge primarily forms nitrogen ions which, in turn, ionize water molecules in moist air, which then perform the ionization of the analyte molecules.
In FTICR mass spectrometry with an external MALDI ion source the dynamic trapping of the ions that are formed in a low voltage MALDI source requires an electrical “opening and closing” of the ICR trapping plate facing the ion source. This is usually combined with an increase of the trapping potential of the rear trapping plate. Capturing higher-energy ions in an ion trap is always problematic. A loss-free capture is especially difficult if a swarm of ions with a broad energy spread arrives from such a MALDI source. In FTICR mass spectrometry one also uses frequently a pulsed inert gas in the ICR trap. Collisions with these gas molecules remove the excess energy of the MALDI-generated ions. Thus, reduced-energy ions are obtained which can be resonantly excited and detected in the ICR trap. However, FTICR mass spectrometry requires a very good vacuum of around ≦10
−9
mbar in the analyzer range, particularly in order to achieve the high resolution. In FTICR one avoids operating at pressures above 10
−8
mbar since the broadening of the ion cyclotron resonance signals disturbs the measurements. If the capture of the ions is associated with a gas pulse, one has to wait for a time period after each gas pulse-assisted ion trapping until the pulsed gas is pumped out. This time period can be 5-10 seconds or even longer, thus, a much longer time is required to add up a multitude of spectra.
These problems appear if the ions in MALDI source are transferred to the ICR trap by a low voltage extraction and acceleration. Therefore, it seems to be simpler to absorb the excess kinetic energy of the MALDI-generated ions already in the source by collisions with gas molecules.
The alternative to increasing the pressure in the ICR trap is to statically increase the pressure in the ion source of an FTICR mass spectrometer. As described above, the collisions with gas molecules would already remove the excess energy from MALDI-generated ions at the location of their formation. A MALDI ion source with statically increased pressure, in connection with time-of-flight mass spectrometry though, was described in the publication by A. N. Krutchinsky, A. V. Loboda, V. L. Spicer, R. Dworschak, W. Ens, and K. G. Standing in “Rapid Communications in Mass Spectrometry”, Volume 12, year 1998, on pages 508 to 518. However, when a statically increased source pressure is applied to FTICR mass spectrometry in order to achieve a higher analyte yield for MALDI, it leads also to a higher static pressure in the ICR trap. A certain increase in trap pressure occurs despite a differentially pumped system if the source pressure rises to levels such as 0.01 or 0.1 mbar, which in turn can considerably affect the performance of the FTICR system (broader peak, reduced resolution).
Ultimately, the methods proposed so far using a high static pressure in the laser desorption ion source, e.g. atmospheric pressure (4 or 5 orders of magnitude higher than a source pressure increased statically to 0.1 or 0.01 mbar) are not intended for classic MALDI processes. They are new techniques associated with atmospheric pressure ionization with the aid of an additional reactant gas, whereby the classic matrix substances are not normally used.
OBJECTIVE OF THE INVENTION
It is the objective of the invention to find a device and a method for absorbing the excess kinetic energy of the ions formed in a MALDI ion source, immediately after their formation and locally in the ion
Berman Jack
Bruker Daltonik GmbH
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