Chemistry: analytical and immunological testing – Biological cellular material tested
Reexamination Certificate
2000-10-06
2003-03-11
Wallenhorst, Maureen M. (Department: 1743)
Chemistry: analytical and immunological testing
Biological cellular material tested
C436S064000, C436S173000, C435S040500, C435S040510, C435S040520, C435S029000, C250S281000, C250S282000, C250S288000
Reexamination Certificate
active
06531318
ABSTRACT:
TECHNICAL FIELD
The invention relates to a specific and a sensitive technique for exploring cell physiology.
BACKGROUND
Due to recent advances in genomics more attention is being paid to the protein products of genes as the engines of cell physiology and targets for drug development. The study of protein gene products is termed proteomics. Pathologists often require studies of specific protein expression by various cell types by the techniques of immunohistochemistry and flow cytometry. The recent advent of laser capture micro-dissection has enabled pathologists and researchers in genomics and proteomics to separate clusters of cells or even individual cells of interest from a background of millions of other cells. The collected cells could be directly visualized to verify their identity and purity.
Genetics of the separated cells, typically, is studied using PCR, but to explore the physiology of these cells, and in particular global protein expression, more laborious and less sensitive tools such as Western Blots were necessary.
SUMMARY
The invention is based on the discovery that by selecting specific clusters of cells of interest from sections of a tissue sample by embedding them in a polymer film; removing the polymer film from the tissue sample; and characterizing the specific cells within the polymer film by mass spectroscopy, one obtains the beneficial results of a highly specific and sensitive technique for exploring cell physiology.
The highly specific and sensitive technique for exploring cell physiology includes selecting specific cells by laser capture microdissection (LCM) and characterizing the specific cells by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry (MALDI-MS).
In one aspect, the invention features a method of determining a mass spectrum, and thereafter a mass spectrometric biochemical profile, of a tissue sample by covering a tissue sample with a polymer (e.g., attached to a substrate), identifying one or more specific cells of the tissue sample for mass spectrometric analysis, melting a portion of the polymer to cause the portion of the polymer to adhere onto the specified cells of the tissue sample and allowing the polymer to solidify, removing the polymer (or at least a portion), the melted portions of which contain the specified cells, attaching the polymer to a mass spectrometric target, applying a matrix composition to the specified cells, and determining the mass spectrum of the specified cells by matrix assisted laser desorption ionization mass spectrometry. For example, the polymer can be removed (e.g., peeled) from the substrate and then attached (e.g., bonded with glue) to the target.
Mass spectrometric biochemical profiles are mass spectra, or derivatives of the mass spectra, of mixtures of biomolecules (taken from a tissue sample) that indicate the identities of some or all of the molecular constituents of the mixture. In applications to some part of or all of living organisms, the mass spectrometric biomolecular profiles reveal, either directly or indirectly, some aspect of the identity or the physiological state of the organism. For example, as described in further detail below, the mass spectrometric biochemical profiles of laser capture microdissected breast tissues indicate the presence or absence of pathological physiology (cancer) of the tissues. The profiles are created from the mass spectra, e.g., by removing background or irrelevant data, normalizing the spectra, or otherwise processing the spectra to produce useful data that can be used to distinguish diseased from normal tissues, e.g., by comparing the profiles with libraries of profiles of normal and diseased tissues.
In another aspect, the invention features a method of determining a mass spectrum of a tissue sample by selecting specific cells of the tissue sample by laser capture microdissection, and determining the mass spectrum of the selected cells by matrix assisted laser desorption ionization mass spectrometry. Determining the mass spectrum of the selected cells can include applying a matrix composition to the selected cells. Determining the mass spectrum also can include ionizing the selected cells on a polymer, e.g., a thermoplastic polymer. Embodiments of these aspects of the invention can include one or more of the following features. The matrix composition can include a proton donor acid. The proton donor acid can be selected from the group consisting of sinapinic acid, 2,5 dihydroxybenzoic acid, alpha-cyano-4-hydroxy cinnamic acid, nicotinic acid, and ferrulic acid. The matrix composition also can include acetonitrile. The mass spectrum further can be determined by ionizing the selected cells on the polymer, or by separating gas-phase ions by time-of-flight (TOF) measurements, ion-trap measurements, magnetic-sector measurements, quadrupole mass filter measurements, or ion cyclotron resonance measurements.
In another aspect, the invention features methods of characterizing tissue samples by selecting specific cells of the tissue sample by laser capture microdissection, determining the mass spectrum, and then a mass spectrometric biochemical profile, of the selected cells by matrix assisted laser desorption ionization mass spectrometry, and comparing the mass spectrum or mass spectrometric biochemical profile of the selected cells against a standard library of mass spectra or spectrometric biochemical profiles of tissue samples. The invention also includes methods of determining whether a tissue sample includes cancer cells, by characterizing the tissue sample as described herein, wherein the library of mass spectra includes spectra of both normal and cancerous tissues.
The tissue samples can be selected from the group consisting of normal breast stroma, normal breast epithelial, breast carcinoma in situ, invasive breast carcinoma, and metastatic lymph node carcinoma. The tissue sample can be a biopsy, e.g., a frozen biopsy, a resected tissue section, and can be normal or diseased.
Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although methods and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, suitable methods and materials are described below. All publications, patent applications, patents, and other references mentioned herein are incorporated by reference in their entirety. In case of conflict, the present specification, including definitions, will control. In addition, the materials, methods, and examples are illustrative only and not intended to be limiting.
The invention provides one or more of the following advantages. The LCM/MALDI-MS technique is useful to rapidly produce high resolution mass spectra of specific cells from a tissue sample and, in some embodiments, individual cells. Moreover, mass spectra of the tissue samples are recorded by ionizing the sample on a polymer substrate thereby eliminating the need of adhering tissue samples directly to metal target plates.
The technique also allows the study of specific cell types in their native environments, i.e., the specific cells are maintained in their respective cellular location. More importantly, LCM typically separates cells along cell boundaries, and allows visual confirmation that only the desired cells are collected.
Furthermore, the new method allows pooling of material from similar cells to improve capacity of mass spectroscopic detection for low abundance molecular species.
REFERENCES:
patent: 5808300 (1998-09-01), Caprioli
patent: 5843657 (1998-12-01), Liotta et al.
patent: 5985085 (1999-11-01), Baer et al.
patent: 6071610 (2000-06-01), Jarrell et al.
patent: 6184973 (2001-02-01), Baer et al.
patent: 6215550 (2001-04-01), Baer et al.
James, “Breakthroughs and Views of Genomes and Proteomes”,Biochemical and Biophysical Research Communications, 231:1-6, 1997.
Jungblut et al., “Proteomics in human disease: Cancer, heart and infectious diseases”,Electrophoresis, 20:2
Leopold Peter
Palmer-Toy Darryl E.
Sarracino David
Sgroi Dennis
Fish & Richardson PC
The General Hospital Corporation
Wallenhorst Maureen M.
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