Chemistry: analytical and immunological testing – Including sample preparation
Reexamination Certificate
2001-08-16
2004-11-30
Soderquist, Arlen (Department: 1743)
Chemistry: analytical and immunological testing
Including sample preparation
C250S281000, C250S282000, C250S288000, C436S086000, C436S087000, C436S088000, C436S089000, C436S090000, C436S091000, C436S092000, C436S093000, C436S094000, C436S173000, C436S175000, C436S176000, C436S181000
Reexamination Certificate
active
06825045
ABSTRACT:
BACKGROUND OF THE INVENTION
1. Field of the Invention
The present invention generally relates to a system and method for desorption and ionization of analytes in a medium. More particularly, the present invention relates to a system and method for infrared matrix-assisted laser desorption and ionization mass spectrometry of analytes in a medium such as polyacrylamide gels.
2. Description of the Related Art
The recent sequencing of the human genome is accelerating the need to understand the variety, characteristics, and functions of the numerous proteins expressed by the genomes of humans and other organisms in response to internal and external stimuli. The characterization of proteins, such as their structure and function, is commonly referred to as proteomics. Mass spectrometry (“MS”) has proven to be an invaluable tool for proteomics, making possible the accurate profiling of proteins, polypeptides, peptides and other factors by precision measurements of molecular ion masses. Polyacrylamide gel electrophoresis (“PAGE”) and related electrophoretic techniques are invaluable tools for the analysis of proteins, nucleic acids and other factors. Using PAGE techniques coupled with experimental manipulation and/or labeling techniques, it is possible to characterize structurally and functionally a variety of cellular determinants.
The ability to combine the preliminary separation provided by PAGE and resolution of MS into a coupled PAGE-MS system is highly desirable. Specifically, matrix-assisted laser desorption and ionization (MALDI) mass spectrometry is generally acknowledged to be an integral part of any integrated strategy for proteomics, as discussed in the article “Proteomics: quantitative and physical mapping of cellular proteins,” Blackstock et al., Trends in Biotech 17: 121-127 (1999). However, previous attempts to examine proteins by PAGE-MS or MALDI-MS required one or more of following limitations: (1) extraction of the protein from the polyacrylamide gel followed by routine sample preparation for MS; (2) transfer of the protein from the gel onto a membrane and permeation of the sample and membrane with a matrix for MALDI-MS; and/or (3) dehydration of the polyacrylamide gel and permeation or coating of the gel with a MALDI matrix.
The article “Mass spectrometry of whole proteins eluted from sodium dodecyl sulfate-polyacrylamide electrophoresis gels,” Cohen et al., Anal. Biochem. 247(2): 257-267 (1997), describes the use of mass spectrometry in the analysis of proteins eluted from SDS-PAGE gels using a 4-HCCA matrix and a fixed-wavelength laser operating in the ultraviolet range.
Researchers have described a method for identifying proteins from two-dimensional gels by electroblotting the proteins from the gels onto a membrane in the article “Identifying proteins from two-dimensional gels by molecular mass searching of peptide fragments in protein sequence databases,” Henzel et al., Proc. Natl. Acad. Sci. USA 90: 5011-5015 (1993). The proteins were located by staining with Coomassie brilliant blue. Protein spots of interest were eluted from the membrane, trypsin digested, and analyzed by capillary high-performance liquid chromatography (HPLC), peptide mapping, and automated protein sequencing using the Edmond method. Mass spectrometry was performed by reconstituting an aliquot of the tryptic digest with a MALDI matrix and applying the sample to a mass spectrometer probe tip. The laser for the mass spectrometer was operated in the ultraviolet range at 337 nanometers (nm). The proteins and peptide fragments were identified by searching for the mass spectra of the ionization products in peptide fragment and protein sequence databases.
A technique described in “Matrix-assisted laser desorption ionization mass-spectrometry of proteins electroblotted after polyacrylamide-gel electrophoresis,” Strupat et al., Anal. Chem. 66: 464-470 (1994), was used to obtain MS signals of proteins physically transferred from a polyacrylamide gel to a membrane, wherein the samples were then treated with succinic acid, using a 100-nanosecond (ns) pulse from an Er:YAG laser operated at a fixed wavelength of 2.94 micrometer (&mgr;m). These researchers attempted to do MALDI-MS, using an Er: YAG laser, directly from the polyacrylamide gel, both with and without the addition of exogenous matrix, but were unsuccessful.
Additional publications describe minor variations of these same techniques. The article “Characterization of SDS-PAGE-separated proteins by matrix-assisted laser desorption/ionization mass spectrometry,” Liang et al., Anal. Chem. 68: 1012-1018 (1996), describes MALDI-MS performed on proteins on nitrocellulose membranes using a frequency-tripled Nd:YAG laser operating at a fixed wavelength of 355 nm.
The paper “Mass spectrometry of proteins directly from polyacrylamide gels,” Ogorzalek Loo et al., Anal. Chem. 68:1910-1917 (1996), described preparing a protein sample on a polyacrylamide gel, identifying bands by staining or comparison to an identical stained gel, dehydrating the gel, adding a MALDI matrix (sinapinic acid) to gel positions of interest, irradiating the MALDI matrix with 337 nm laser light, and analyzing the products in a time-of-flight (TOF) mass spectrometer. The same group of researchers later described a variant of MALDI-MS performed on slices of polyacrylamide gel soaked in sinapinic acid in the article “High sensitivity mass spectrometric methods for obtaining intact molecular weights from gel-separated proteins,” Loo et al., Electrophoresis 20: 743-748 (1999).
Among other disadvantages, all of these techniques at least require considerable sample handling procedures that are impractical for adaptation to future designs for high-throughput sample analysis procedures. Therefore, there exists a need for an improved system and method for desorption and ionization of analytes in a medium. In particular, there is a need for an improved system and method that can allow direct high-speed sampling and analysis of proteins from polyacrylamide and other electrophoretic gels.
SUMMARY OF THE INVENTION
The present invention is related to a system and method for desorption and ionization of analytes in an ablation medium. In one aspect of the present invention, there is provided a method for desorption and ionization of analytes including the steps of preparing a sample comprising analytes in a medium having at least one component, selecting a resonant vibrational mode of at least one component of the medium, selecting a laser tuned to emit light substantially at the wavelength of the selected vibrational mode, and irradiating the sample with the laser light to cause medium ablation and desorption and ionization of the analytes. The method further includes the steps of passing the ionized analytes through a mass spectrometer, and obtaining a mass spectrum of the ionized analytes. The medium can be an electrophoresis medium that is in the form of an electrophoresis gel. The medium may also be chosen from, but is not limited to, materials such as cellulose acetate, paper, agarose an the like.
In another aspect, the present invention relates to a method for desorption and ionization of analytes including the steps of preparing a sample having analytes and a polyacrylamide medium having at least one component, selecting a resonant vibrational mode of at least one component of the medium, selecting a laser tuned to emit light substantially at the wavelength of the selected vibrational mode, and irradiating the sample with laser light to cause medium ablation and desorption and ionization of the analytes. In one embodiment, the sample is irradiated by laser light delivered in pulses, each pulse having duration of less than 5.0 picoseconds (ps), where the pulses are separated in time by more than 100 ps. The method further includes the steps of passing the ionized analytes through a mass spectrometer, and obtaining a mass spectrum of the ionized analytes.
In a further aspect, the present invention relates to a method for desorption and ionization of analytes including the steps of preparing
Baltz-Knorr Michelle Lee
Ermer David R.
Haglund, Jr. Richard F.
Morris Manning & Martin
Soderquist Arlen
Tingkang Xia, Esq. Tim
Vanderbilt University
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