Radiant energy – Ionic separation or analysis – Ion beam pulsing means with detector synchronizing means
Reexamination Certificate
2002-01-15
2004-10-19
Lee, John R. (Department: 2881)
Radiant energy
Ionic separation or analysis
Ion beam pulsing means with detector synchronizing means
C250S288000
Reexamination Certificate
active
06806465
ABSTRACT:
FIELD OF THE INVENTION
The invention relates to a time-of-flight (TOF) miniature mass spectrometer (MMS), and more particularly to an automated TOF MMS collection, measurement and analysis system for acquisition of mass spectra.
DESCRIPTION OF THE RELATED ART
One of the most powerful laboratory tools for analyzing a broad spectrum of chemical and biological material is the mass spectrometer. Mass spectrometry is a proven technique for analyzing many types of environmental samples. Mass spectrometry is used to determine the masses of molecules formed following their vaporization and ionization. Detailed analysis of the mass distribution of the molecule and its fragments leads to molecular identification. Mass spectrometry is especially suited for aerosol analysis because micrometer-sized heterogeneous particles contain only about 10
−12
moles of material and thus requires a sensitive technique such as mass spectrometry for proper analysis. Liquid samples can be introduced into a mass spectrometer by electrospray ionization (1), a process that creates multiple charged ions. However, multiple ions can result in complex spectra and reduced sensitivity.
A preferred technique, matrix assisted laser desorption time-of-flight mass spectrometry (MALDI-TOF-MS), has become popular in the analysis of biological polymers for its excellent characteristics, such as ease of sample preparation, predominance of singly charged ions in mass spectra, sensitivity and high speed. Time-of-flight MALDI-TOF-MS is established as a method for mass determination of biopolymers and substances such as peptides, proteins, and DNA fragments. The analytical sensitivity of TOF MS is such that under the right conditions only a few microliters of analyte solution at concentrations down to the attomolor (10
−12
moles) range are required to obtain a mass spectrum. The MALDI-MS technique is based on the discovery in the late 1980s that desorption/ionization of large, nonvolatile molecules such as proteins can be effected when a sample of such molecules is irradiated after being co-deposited with a large molar excess of an energy-absorbing “matrix” material, even though the molecule does not strongly absorb at the wavelength of the laser radiation. The abrupt energy absorption initiates a phase change in a microvolume of the absorbing sample from a solid to a gas while also inducing ionization of the sample molecules. Detailed descriptions of the MALDI-TOF-MS technique and its applications may be found in review articles by E. J. Zaluzed et al. (Protein Expression and Purifications, Vol. 6, pp. 109-123 (1995)) and D. J. Harvey (Journal of Chromatography A, Vol. 720, pp. 429-4446 (1996)), each of which is incorporated herein by reference.
In brief the matrix and analyte are mixed to produce a solution with a matrix:analyte molar ratio of approximately 10,000:1. A small volume of this solution, typically 0.5-2. microliters, is applied to a stainless steel probe tip and allowed to dry. During the drying process the matrix codeposits from solution with the analyte. Matrix molecules, which absorb most of the laser energy, transfer that energy to analyte molecules to vaporize and ionize them. Once created, the analyte ions the ions formed at the probe tip are accelerated by the electric field toward a detector through a flight tube, which is a long (on the order of 0.15 to 1 m) electric field-free drift region. Since all ions receive the same amount of energy, the time required for ions to travel the length of the flight tube is dependent on their mass to charge ratio. Thus, low-mass ions have a shorter time of flight (TOF) than heavier ions. All the ions that reach the detector as the result of a single laser pulse produce a transient TOF signal. Typically, ten to several hundred transient TOF mass spectra are averaged to improve ion counting statistics. The mass of an unknown analyte is determined by comparing its experimentally determined TOF to TOF signals obtained with ions of known mass. The MALDI-TOF-MS technique is capable of determining the mass of proteins of between 1 and 40 kDa with a typical accuracy of +−0.1%, and a somewhat lower accuracy for proteins of molecular mass above 40 kDa. The ability to generate UV-MALDI mass spectra is critically dependent upon the co-crystallization or very close special proximity of the analyte and a molar excess of the matrix compound. In routine practice, a small volume of matrix solution that delivers a one thousand-fold molar excess of matrix is manually mixed with a small volume of the analyte solution which then dries on a sample stage. A spatially heterogeneous distribution of analyte and matrix typically develops as the droplet dries to form a sample spot. Under laboratory conditions, the incident laser is rastered across the sample to identify so called “sweet spots” that preferentially yield for an abundance of analyte ions. Although a motorized x-y stage may be incorporated for automated searching for the spot providing the best spectrum, this procedure can be a time consuming step.
MALDI is typically operated as an offline ionization technique, where the sample, mixed with a suitable matrix, is deposited on the MALDI target to form dry mixed crystals and, subsequently, placed in the source chamber of the mass spectrometer. Although solid samples provide excellent results, the sample preparation and introduction into the vacuum chamber requires a significant amount of time. Even simultaneous introduction of several solid samples into a mass spectrometer or off-line coupling of liquid-phase separation techniques with a mass spectrometer do not use TOF mass spectrometer time efficiently.
To improve on these procedures, microfabricated targets have recently been developed for automated high throughput MALDI analysis. In these designs, pL-nL sample volumes can be deposited into a microfabricated well with dimensions similar to the spot size of the desorbing laser beam about 100 micrometers to 1,000 micrometers diameter). Thus, the whole sample spot can be irradiated and the search for the “sweet spot” eliminated. Analysis of short oligonucleotides has been demonstrated with about 3.3 s required to obtain a good signal to noise ratio for each sample spot. Although the total analysis time, including the data storage, takes nearly an hour, theoretically all 96 samples could be recorded in about five minutes.
While the miniaturization of the sample target simplifies the static MALDI analysis, on-line coupling would allow continuous analysis of liquid samples including direct sample infusion and the monitoring of chromatographic and electrophoretic separations. Compared to ESI, MALDI provides less complex spectra and, potentially, higher sensitivity. There have been numerous reports in the literature about the MALDI analysis of flowing liquid samples. In one arrangement, the sample components exiting a CE separation capillary were continuously deposited on a membrane presoaked with the matrix and analyzed after drying. In other cases, the liquid samples were analyzed directly inside the mass spectrometer using a variety of matrices and interfaces. MALDI was then performed directly off rapidly dried droplets. In another design, a continuous probe, similar to a fast atom bombardment (FAB) interface, was used for the analysis of a flowing sample stream with liquid matrix. Glycerol was used to prevent freezing of the sample. Other attempts for liquid sample desorption were also made using fine dispersions of graphite particles and liquid matrices instead of a more conventional matrices. More recently, an outlet of the capillary electrophoresis column was placed directly in the vacuum region of the TOF mass spectrometer. The sample ions, eluting in a solution of CuCl.sub.2, were desorbed by a laser irradiating the capillary end. On line spectra of short peptides separated by CE were recorded. Attempts to use ESI to introduce liquid sample directly to the evacuated source of a mass spectrometer have also been reported.
Standard MALDI sample preparation tec
Anderson Charles W.
Bryden Wayne A.
Chappell Ronald G.
Ecelberger Scott A.
Ko Harvey W.
Cooch Francis A.
Lee John R.
Leybourne James J.
The Johns Hopkins University
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