“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.”
“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.”