Over the course of his distinguished career, Nobel Prize winner Percy Bridgman, Ph.D., investigated the behavior of materials under elevated hydrostatic pressure. These studies, for which he designed his own high-pressure equipment, included the thermodynamic effect of pressure on proteins and other biological substances.
Researchers recently met to discuss the use of ultrahigh pressure in biotech-related studies at a symposium held at Harvard Medical School and co-hosted by the Harvard Catalyst—Laboratory for Innovative Translational Technologies, Harvard Catalyst Central Laboratory, and by the Proteomics Resource of the Harvard School of Public Health. The hosting of this event was apropos as Dr. Bridgman was a professor at Harvard from 1908 to 1954.
Scientists no longer need to design their own instruments, as counter-top high-pressure instruments are now commercially available. “Commercialization of the Barocycler® platform brings pressure-control instrumentation to an average laboratory, opening opportunities for understanding of high-pressure thermodynamics in synthetic chemistry, catalysis, structural biology, biomarker discovery, and drug development,” explained Alexander Lazarev, Ph.D., vp of R&D at Pressure BioSciences.
With pressure-cycling technology (PCT) becoming a common tool for scientific investigations, many of the presentations focused on the physical effects of compression on macromolecules and its applications in proteomics, mass spectrometry, protein extraction, and tissue investigations.
FFPE Tissues
“High-throughput genomic and proteomic methods hold great promise for developing a fundamental knowledge of the molecular characteristics of cancer,” noted Carol Fowler, Ph.D., senior research associate in the department of biophysics at the Armed Forces Institute of Pathology.
Dr. Fowler and her colleagues have used high hydrostatic pressure to expedite the removal of proteins from archival FFPE tissues. Ordinarily, such samples cannot be used to follow the clinical course of tumor development and evolution since the fixation process makes extraction of marker proteins extremely difficult. The alternative of using fresh tissue is not a satisfactory option for logistic reasons.
The Fowler team conducted high-pressure experimental treatments of tissue surrogates at 65–100ºC under 45,000 psi. Complete reversal of formaldehyde-induced protein adducts was observed, and protein recovery was fourfold greater than that obtained from samples processed by conventional procedures, she said. These samples were obtained in quantities sufficient for high-throughput proteomic analysis including 2-D gel electrophoresis and mass spectrometry.
“We noted that when FFPE mouse liver was extracted at elevated pressures at pH 4, 7, or 9, there was an almost twofold increase in the number of unique protein identifications by mass spectrometry, compared to tissue extracted at the same temperature and length of time but without pressure.”
According to Dr. Fowler, preliminary results show that the application of pressure increases the rate of formaldehyde penetration into tissue by more than sevenfold while preserving tissue morphology. These procedures could be used in the future to improve the uniformity of tissue fixation.