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Please Note:The technology listed below is not available to the public at this time. This technology requires further development before it is ready for the marketplace. T he VA is currently in the process of identifying potential companies who may be interested to commercially develop the technology. The VA inventors are available to collaborate with interested companies through a Cooperative Research and Development Agreement (CRADA). Through cooperative research initiatives such as these, it is our hope and goal that commercial products will be fully developed and made available to benefit veterans and others.
VA TECHNOLOGY OPPORTUNITY BRIEF
Pressure Assisted Molecular Recovery (PAMR) and Pressure Assisted Antigen
Retrieval (PAAR)
(#08-003)
OPPORTUNITY
The present invention is the use of high pressure to induce the hydration of cross-linked molecular aggregates that result from tissue fixation with formaldehyde. This technology facilitates sample recovery from archived tissue samples for proteomic analysis. It also has potential to significantly reduce the processing time tissue biopsies in the pathology lab.
TECHNOLOGY BACKGROUND AND DESCRIPTION
Analysis of formalin-fixed paraffin-embedded (FFPE) tissues from biopsy libraries has been a challenge for proteomics biomarker studies. This is because formaldehyde cross-links basic amino acid side chains, often destroying tertiary structures essential to antibody staining. The chemical cross-links formed by the process also alters the chemical connectivity of proteins, making analysis of them by mass spectrometry difficult, if not impossible.
It has previously been shown that treatment of FFPE tissue samples with high temperature and altered pH increases the accessibility (or "recovery") of proteins. It is hypothesized that under the correct conditions, hydrolysis of the methylene cross-links between amino acid side chains occurs, and the proteins return to a near-original state. The high temperatures often denature the proteins, though, sometimes making them unable to be recognized by antibodies.
The inventors have demonstrated that by using lower temperatures, but higher pressures, the methylene bonds can be hydrolyzed without thermally-induced denaturation. They have assembled an apparatus using a high-pressure generator, a specimen holder, and a temperature modulator that can perform the hydrolysis on a test sample. They have performed a proof-of-principle experiment showing that FFPE treated lysozyme can be recovered with their technique.
COMPETITIVE ADVANTAGE
Analysis of FFPE tissues from biopsies has been a challenge for proteomics biomarker studies. To date, FFPE tissues are limited candidates for MALDI mass spectrometry tissue profiling because these treatments hamper efficient ionization of the proteins and peptides. There are recent methodological reports demonstrating the principal feasibility of MALDI mass spectrometry on FFPE tissues.
Lemaire et al. (J. Proteome Research, 2007 6:1295-305) present two methods for the direct analysis of FFPE tissues by MALDI-MS. The first is based on the use of a reactive matrix, 2,4-dinitrophenylhydrazine, useful for FFPE tissues stored less than 1 year. The second approach is applicable for all FFPE tissues regardless of conservation time. The strategy is based on in situ enzymatic digestion of the tissue section after paraffin removal. In situ digestion can be performed on a specific area of the tissue as well as on a very small area (microdigestion). Combining automated microdigestion of a predefined tissue array with either in situ extraction prior to classical MS analysis or automated microspotting of MALDI matrix allows the identification of both proteins by nanoLC-nanoESI and MALDI imaging. When adjacent tissue sections are used, it is possible to correlate protein identification and molecular imaging. These combined approaches, along with FFPE tissue analysis provide access to massive amounts of archived samples in the clinical pathology setting.
IP STATUS
Patent pending.
FOR MORE INFORMATION CONTACT
Saleem Sheredos
Program Manager
Technology Transfer Program
Department of Veterans Affairs
Office of Research & Development (12TT)
810 Vermont Avenue, NW
Washington, DC 20420
Phone: 202-380-5080
Fax: 202- 254-0473
E-mail: saleem.sheredos@va.gov
Last Updated - January 23, 2009