Radiant energy – Ionic separation or analysis – With sample supply means
Reexamination Certificate
2003-07-09
2004-11-30
Wells, Nikita (Department: 2881)
Radiant energy
Ionic separation or analysis
With sample supply means
C250S281000, C250S282000, C250S289000, C250S442110, C250S440110, C250S441110
Reexamination Certificate
active
06825466
ABSTRACT:
FIELD OF THE INVENTION
The present invention relates to the field of chemistry and biochemistry and, in particular, to the analytical methods and apparatuses for loading, unloading and analyzing samples by atmospheric pressure matrix assisted laser desorption ionization (AP-MALDI) technique.
PRIOR ART AND DISADVANTAGE OF THE PRIOR ART
Mass spectrometers have become one of the essential tools of the biochemistry lab. Biochemists take advantage of the capabilities of mass spectrometers to determine molecular weights of biomolecules, monitor bioreactions, detect post-translational modifications, perform protein, and oligonucleotide sequencing, and many more applications. During the past decade, one of the methods, which became most successful for the mass spectrometric analysis and investigation of large molecules is a method known as MALDI (Matrix-Assisted Laser Desorption Ionization). This method, which in application to time-of-flight (TOF) mass spectrometry (MS) is known as MALDI-TOF MS, is a relatively novel technique in which a co-precipitate of an UV-light absorbing matrix and a biomolecule is irradiated by a nanosecond laser pulse. The sample (analyte) is suspended or dissolved in a matrix (e.g., in 1000× molar excess).
Most of the laser energy is absorbed by the matrix, which prevents unwanted fragmentation of the biomolecule. Matrices are small organic compounds that are co-crystallized with the analyte. It seems that the presence of the matrix, spares the analyte from degradation, resulting in the detection of intact molecules as large as 1 million Da. The ionized biomolecules are accelerated in an electric field and enter the flight tube. During the flight in this tube, different molecules are separated according to their mass to charge ratio and reach the detector at different times. In this way each molecule yields a distinct signal. The method is used for detection and characterization of biomolecules, such as proteins, peptides, oligosaccharides and oligonucleotides, with molecular masses between 400 and 350,000 Da. It is a very sensitive method, which allows the detection of low (10
−15
to 10
−18
mole) quantities of sample with a mass accuracy of 0.1-0.01% or higher.
Another advantage of MALDI is that this method allows for vaporization and ionization of non-volatile biological samples from a solid-state phase directly into the gas phase.
The most important step in MALDI, is sample preparation. During this step, the matrix and analyte are mixed and the mixture is dried on a probe or as it is more common now, on a sample plate. Upon preparation, the sample plate with samples is loaded into the mass spectrometer.
A laser beam, serves as the desorption and ionization source in MALDI. The matrix plays a key role in this technique by absorbing the laser light energy and causing part of the illuminated substrate to vaporize. A rapidly expanding matrix plume carries some of the analyte into the vacuum with it and aids the sample ionization process. The matrix molecules absorb most of the incident laser energy minimizing sample damage and ion fragmentation (i.e., soft ionization). Nitrogen lasers operating at 337 nm (a wavelength that is well absorbed by most UV matrices) are the most common illumination sources because they are inexpensive and offer the ideal combination of power/wavelength/pulsewidth. However, other UV and even IR pulsed lasers have been used with properly selected matrices.
Once the sample molecules are vaporized and ionized, they are transferred, e.g., into a time-of-flight mass spectrometer (TOF-MS) where they are separated from the matrix ions, and individually detected, based on their mass-to-charge (m/z) ratios and analyzed. High transmission and sensitivity, along with theoretically unlimited mass range are among the inherent advantages of TOF instruments. Detection of the ions at the end of the tube is based on their flight time, which is proportional to the square root of their m/z.
Roughly, the MALDI system can be divided into two groups: VP-MALDI for matrix-assisted laser desorption at vacuum pressure and AP-MALDI for matrix-assisted laser desorption at atmospheric pressure. A characteristic feature of vacuum-pressure ionization sources is that sample ionization occurs inside a mass spectrometer housing under vacuum conditions. In contrast to vacuum ionization, any atmospheric pressure ionization takes place outside a mass spectrometer instrument. It should be noted that different instrument types are used in both cases. However, for sampling atmospheric pressure ions any mass spectrometer must be equipped with Atmospheric Pressure Interface (API) to transfer ions from an external region of atmospheric pressure to a mass analyzer under high vacuum.
Examples of both these systems are given below for more detailed familiarization with the MALDI technique.
U.S. Pat. No. 5,288,644 issued in Feb. 22, 1994 to Ronald C. Beavis, et al. discloses an apparatus and method for the sequencing of genome. The apparatus comprises an automated DNA sampler, which adds a matrix solution from a container to separated DNA samples. A large number of DNA fragment samples, for example 120 samples, may be loaded into a sample tray. The matrix solution may be added automatically to each sample using procedures available on the aforementioned autosampler, and the samples may then be spotted sequentially as sample spots on an appropriate surface, such as the planar surface of the disk rotated by a stepper motor. Sample spot identification is entered into the data storage and computing system, which controls both the autosampler and the mass spectrometer. The location of each spot relative to a reference mark is recorded in the computer. Sample preparation and loading onto the solid surface is done off-line from the mass spectrometer, and multiple stations may be employed for each mass spectrometer, if the time required for sample preparation is longer than the measurement time.
Once the samples in suitable matrix are deposited on the disk, the disk may be inserted into the ion source of a mass spectrometer through the vacuum lock. Any gas introduced in this procedure must be removed prior to measuring the mass spectrum. Loading and pump down of the spectrometer typically requires two to three minutes, and the total time for measurement of each sample to obtain a spectrum is typically one minute or less.
Thus, the above-described VP-MALDI technique requires that the sample plate carrier be loaded into the mass spectrometer through a vacuum lock and even though manipulations with the sample support are carried out automatically and coordinated by the computer, all sampler operations for loading the samples into the mass spectrometer are performed in vacuum. Such a system requires the use of complicated vacuum seals and special drive, transportation, and actuation mechanisms, which have to be vacuum-proof. Furthermore, the sample loading system of U.S. Pat. No. 5,288,644 has a relatively low throughput rate. As the authors of the above invention states, the system is limited to about 50 complete DNA spectra per hour. Furthermore, the system is expensive, as it requires the use of vacuum-proof sample plate carrier handling mechanisms.
U.S. Reissued Patent RE 37,485 filed by Marvin L. Vestal and published on Dec. 25, 2001 describes another mass spectrometer system and method for vacuum-pressure matrix-assisted laser desorption measurements. The system is equipped with a sample plate transport mechanism for automatically inputting and outputting each of the sample plates into and from the sample-receiving chamber of the mass spectrometer.
A sample support or plate used in the aforementioned system comprises a thin, substantially square plate of stainless steel or other suitable electrically conducting material approximately 1.5 mm thick and 50 mm wide. The plate may contain precisely located holes to allow the position and orientation of the plate to be accurately determined relative to a moveable stage, which is required both in the sample loading step
Automated Biotechnology, Inc.
Wells Nikita
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