August 1, 2010 (Vol. 30, No. 14)

Decades-Old Technology Being Rediscovered to Bolster Proteomics Research

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.

Commercialization of Pressure BioSciences’ Barocycler platform makes pressure-control instrumentation available to the average laboratory for investigations in the fields of synthetic chemistry, catalysis, structural biology, biomarker discovery, and drug development.

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.

N-Linked Glycan Preparation

Zoltan Szabo, Ph.D., senior research scientist at the Barnett Institute at Northeastern University, uses PCT for glycan analysis. Glycans are ordinarily N-linked and are evaluated using capillary electrophoresis and liquid chromatography. While assessment of glycosylation in proteins is critical to the development of biologic drugs, it is invariably slow and difficult due to long deglycosylation times, as long as overnight, and the additional cleanup step required after labeling.

Dr. Szabo ran through a laundry list of alternatives for accelerating glycan analysis, including microwave-assisted deglycosylation of N-linked glycans, immobilized enzyme reactors in capillary columns, integrated microfluidic chips, and his approach of choice—PCT.

Dr. Szabo and his colleagues used PCT to accelerate N-linked glycan release by peptides using the enzyme N-Glycosidase F, also known as PNGase F. This amidase cleaves between the innermost N-acetylglucosamine and asparagine residues of complex oligosaccharides to separate them from N-linked glycoproteins. The high pressure facilitates conformation changes of the target glycoprotein, increasing the accessibility of the endoglycosidase to the cleavage sites. The investigators determined that pressure cycling did not lead to loss of sialic acid residues.

“In model proteins, our strategy of pressure cycling from atmospheric levels up to pressures as high as 30 kPsi leads to rapid and complete release of N-linked glycans.” 

Attacking Membrane Proteins

Membrane proteins represent one of the most appealing targets for cancer detection and therapy. Ovarian cancer is especially pernicious, given the low five-year survival rate of 46%. However, if the cancer is detected before it has spread outside the ovary, the five-year survival rates jumps to 93%, according to the American Cancer Society. But these tantalizing possibilities are tempered by the reality that the extraction of proteins from membranes is complex and challenging, given the inculcation of the proteins throughout a tenacious lipid bilayer.

Although a range of procedures has  been introduced over the years, traditional membrane extraction protocols are cumbersome and slow, according to Luke Schneider, Ph.D., CSO at Target Discovery. For this reason, he and his co-workers have applied PCT to the extraction of membrane proteins from ovarian cancer tissues.

The ProteoSolve™-TD system is configured to lyse cells under 35,000 psi, releasing proteins that are difficult to purify under standard extraction procedures. The process is essentially a pressure-driven lipid crystallization as a result of the effects of pressure on the temperature of the membrane phase change. ProteoSolve-TD buffers support the solubility of the exuded proteins. The rapidity of the phase change and the use of multiple cycles improves recovery and prohibits re-equilibration.

Dr. Schneider has investigated the compatibility of the procedures with standard proteomic technologies including immunoaffinity capture procedures, SDS-PAGE, and 2-D gel separation. Also with mass spectrometry becoming a simple and economic analytical option for proteomics programs, there is an increasing demand for coupling it to the Proteosolve technology.

Whereas much work will be required to explore the limits of this approach to cancer antigen detection, the efforts of Dr. Schneider and his colleagues suggest that this may represent a new and fruitful approach to developing novel assays for diagnosis and therapy of malignancies.

Luke Schneider, Ph.D., CSO of Target Discovery, cryogenically grinds an ovarian metastatic tumor sample in preparation for protein extraction.

Trypsin Digestion

“Why pressure?” queried Paul Pevsner, M.D., associate professor of pathology at the University of Missouri, in his discussion of the application of PCT to a variety of direct tissue-extraction protocols. Dr. Pevsner said that denaturation occurs when protein-protein and protein-solvent interactions are disrupted. High pressure exposes a protein’s hydrophobic groups to the penetration of water molecules, with a resulting destabilization of the hydrogen bonds that hold the secondary and tertiary structure of the protein together. The end result of this process is an unfolding and destabilization of the protein molecule. 

The approach reduces trypsin-digestion time from hours to minutes, yielding results that are identical with those obtained through standard digestion for four hours at 55ºC. The samples were prepared for liquid chromatography purification followed by identification through mass spectrometry.

“Pressures above 35,000 will denature trypsin, combined with temperatures up to 60ºC, resulting in increased susceptibility to proteolytic digestions,” Dr. Pevsner explained. “This allows us to escape the long digestion intervals required by conventional proteolysis treatments.”

Care must be taken to avoid artifacts introduced by trypsin autodigestions. For this reason the Pevsner research team kept the trypsin-to-substrate ratio low and maintained temperatures below 55ºC. This approach minimizes the appearance of trypsin peptides, which introduces artifacts into the analysis. In a series of mass spectrometry proof-of-principle experiments employing MALDI/TOF/ TOF, the group was able to identify the proteins cytochrome C and bovine serum albumin in processed samples.

Melkamu Getie-Kebtie, Ph.D., a post-doc in the division of cell and gene therapy at the Center for Biologics Evaluation and Research of the FDA, discussed his lab’s evaluation of ultrahigh pressure technology to achieve identification and quantification of the three major viral proteins on the surface of the influenza A virus.

Using an in-gel trypsin digestion procedure, Dr. Getie-Kebtie found it was possible to release and identify the proteins without loss of downstream identification and quantification information.

“This method is based on the discovery of an unexpected relationship between mass spectrometry signal response and protein concentration—that is, the average response for the three most intense tryptic peptides per mole of protein is constant within a coefficient of variation of less than 10 percent.”

By employing label-free mass spec with added internal standards, the group was able to an obtain adequate level of precision. “The mass spectrometry-based quantification with PCT, can be a viable option when specific antibodies are unavailable.”

Thermodynamic Principles

“We reduce sample time from hours to minutes,” said Daniel López-Ferrer, Ph.D., director of the biological separations and mass spectrometry group at the Pacific Northwest National Laboratory. “Sample preparation has become a significant bottleneck in proteomics analysis, and pressure cycling offers a way to unplug the pipeline.”

Dr. López-Ferrer described the standard protocol for preparation of protein samples for mass spec analysis. A solution of enzymes and a protein sample are added to a reaction tube, and the mixture is incubated on a heating block for 24 hours to digest the proteins down to peptides. In contrast, using the Barocycler, the same mixture is subjected to pressure cycling and the peptides are available for analysis in one hour.

As an example of the application of this approach, he detailed a study of tissues infected with Yersinia pestis, the bacterium responsible for bubonic plague. Lung and spleen samples from infected and uninfected tissues were digested and broken down using ultrahigh pressure. Because of the quality of the extraction procedure, the samples are highly reproducible, showing distinct differences between the experimental and control samples.

Proteomics has been bedeviled by irreproducible and inconsistent profiles since its inception, making the isolation of true biomarkers and disease-related proteins virtually impossible. The development of a fast, online pressure digestion system offers the opportunity for a new level of investigation of proteome markers in the search for new therapeutic agents. According to Dr. López-Ferrer, “we can couple a fast on-line digestion system with liquid chromatography in an automated system. This allows us to automate proteomic workflows, as well as proteomic analysis of small samples.”

K. John Morrow Jr., Ph.D. ([email protected]), is president of Newport Biotech and a contributing editor for GEN. Web:

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