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.