Radiant energy – Ionic separation or analysis – With sample supply means
Reexamination Certificate
2002-05-24
2004-03-16
Lee, John R. (Department: 2881)
Radiant energy
Ionic separation or analysis
With sample supply means
C250S287000
Reexamination Certificate
active
06707037
ABSTRACT:
BACKGROUND OF THE INVENTION
Matrix Assisted Laser Desorption Ionization (MALDI) has become an important ionization technique for use in mass spectrometry. MALDI ion sources are typically configured to produce ions in vacuum pressure that is lower than 10
−4
torr. Ions are produced in MALDI ionization by impinging a pulse of laser light onto a target on which a sample solution has been deposited with an appropriate matrix. The resulting ions produced from a MALDI laser pulse are directed into a mass spectrometer where they are mass to charge analyzed. Time-Of-Flight (TOF) mass analyzers are particularly well suited to mass to charge analyze MALDI generated ions. Ions produced from a MALDI pulse in the TOF vacuum region are accelerated into the TOF flight tube and mass analyzed. Techniques such as delayed extraction or reverse acceleration have been employed to improve the resolution when acquiring low vacuum pressure MALDI TOF mass spectra. TOF mass analyzers are capable of separating and detecting ions over a wide mass to charge range, which is essential when analyzing higher molecular weight compounds. MALDI ion sources have also been interfaced to other mass spectrometer types including Fourier Transform Mass Spectrometers (FTMS) and three dimensional quadrupole ion traps (Ion Traps).
Several recipes are available for optimizing a sample and MALDI matrix combination for a given laser wavelength. Typically a nitrogen laser may be used with a DHB matrix. The matrix is chosen to absorb the laser wavelength and transfer the laser power to the matrix to achieve rapid heating of the sample. The rapid heating desorbs and ionizes the sample that was initially dissolved and dried in the matrix solution and a portion of the sample molecules are ionized in the desorption process. To prepare a sample for MALDI ionization, sample solution and matrix solution are combined, deposited on a MALDI probe and dried prior to insertion of the probe into the MALDI ion source. Various conductive and dielectric materials such as glass, metal, silicon and plastics have been configured for use as the MALDI probe substrate. Hydrophobic substrate materials have been used to avoid spreading and thinning of the sample and matrix solution when it is deposited on the probe. It is desirable to concentrate the sample in as small a volume as possible on the MALDI probe to increase the sample ion yield per laser pulse. The MALDI probe substrate should not react with the sample, contribute minimum background peaks in the mass spectrum and allow sufficient binding of sample and matrix to prevent sample loss during MALDI probe handling. When conditioned silicon surfaces are used as MALDI targets, the use of a matrix solution can be eliminated. In some of the embodiments of the invention described below, the additional constraint of using a dielectric MALDI probe material allows the configuration of MALDI probe targets positioned within multipole ion guides or ion funnels causing minimum distortion of Electric fields.
Ions produced from MALDI ion sources configured in the low vacuum pressure region of TOF mass analyzers can be pulsed directly into the TOF MS flight tube for mass analysis. This configuration minimizes any constraint on the mass to charge range that can be analyzed but may limit the resolving power and mass measurement accuracy that can be achieved. Ions that are produced from a MALDI matrix have an uncorrelated energy and spatial spread in the pulsing region of a TOF mass analyzer, resulting in reduced resolving power and mass measurement accuracy in TOF ion mass to charge analysis. Although delayed extraction or reverse field extraction of MALDI produced ions has reduced the effects of ion energy and spatial spread, the techniques have a limit as to how much improvement can be achieved. Also delayed extraction must be carefully tuned to minimize distortion of ion signal intensities in the mass to charge range of interest. The kinetic energy spread of MALDI produced ions also reduces the ion transport and capture efficiency in FTMS and ion trap mass analyzers resulting in decreased sensitivity. Mass to charge selection and fragmentation experiments known as MS/MS experiments may be achieved by using MALDI post source decay or by the configuration of gas collision cells in TOF mass analyzer flight tubes. Ion fragmentation and MS/MS TOF experiments have been achieved using these TOF techniques at some sacrifice to resolving power, mass measurement accuracy and, in some configurations, sensitivity. In an effort to improve mass to charge measurement, resolving power, mass to charge selection precision and efficiency and fragmentation efficiency in MS/MS analysis of MALDI produced samples, MALDI ion sources have been configured in atmospheric pressure and in intermediate vacuum pressure regions of mass analyzers.
Introducing MALDI samples into an atmospheric (AP) or intermediate vacuum pressure (IP) MALDI ion source facilitates sample handling by eliminating the need to load MALDI samples into low vacuum pressure. Laiko et al. in U.S. Pat. No. 5,965,884 and in Anal. Chem. 2000, 72, 652-657 describe the configuration of an atmospheric pressure MALDI Ion source interfaced to an orthogonal pulsing TOF mass analyzer. Krutchincsky et al. J. Am. Soc. Mass Spectrom 2000, 11, 493-504, describe the configuration of MALDI ion source in the second vacuum pumping stage of a hybrid quadrupole/quadrupole/orthogonal pulsing TOF (QTOF) mass analyzer that includes an atmospheric pressure Electrospray ion source. In the atmospheric and vacuum pressure MALDI mass spectrometers described, the ions traverse at least one multipole ion guide prior to being pulsed into the TOF mass analyzer. The mass to charge range of ions that can be analyzed is limited to the range of mass to charge values that can be transmitted with stable ion trajectories through the downstream ion guides. Ion guides positioned in the first or second vacuum pumping stages have pressures maintained sufficiently high to cause multiple ion to neutral background collisions. Elevated background pressures in multipole ion guides cause damping of ion kinetic energies as the ions traverse an ion guide length. The energy damping creates a primary ion beam with a narrow energy spread and a controlled average kinetic energy. Ion mass to charge selection and collisional induced dissociation fragmentation can be achieved in single or multiple ion guide assemblies prior to TOF mass to charge analysis. The upstream ion kinetic energy damping processes result in improved TOF resolving power and ion mass to charge measurement accuracy in orthogonal pulsing TOF. MALDI ionization at atmospheric and intermediate vacuum pressure may yield differences in ion populations when compared with low vacuum pressure MALDI ionization. Neutral to ion collisions occurring in atmospheric pressure and intermediate vacuum pressure MALDI ion source regions reduce the internal energy of the newly formed ion, minimizing post source decay. Subsequent MS/MS functions can be conducted in downstream multipole ion guides, ion traps, FTMS censor TOF-TOF mass analyzers is user controlled through selected experimental methods. The decoupling of the MALDI ionization, ion mass to charge selection, ion fragmentation and subsequent ion mass to charge analysis steps allows independent optimization of each analytical step.
Laiko et al. describe the configuration of a sample MALDI probe positioned near the orifice into vacuum of an API TOF MS instrument so that a portion of the ions produced can be transported into vacuum. A DC field is applied between the MALDI sample target and the orifice into vacuum to direct ions toward the orifice. A gas flow directed over the probe surface was added to push ions produced near the probe surface toward the orifice into vacuum. Laiko reports that substantial sensitivity losses occurred when using the atmospheric pressure MALDI ion source compared with a MALDI ion source configured in the pulsing region of a TOF mass analyzer. Most of the loss of signal was attrib
Analytica of Branford, Inc.
Hughes James P.
Lee John R.
Levisohn, Berger & Langsam LLP
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