Proteomics continues to have an increasing role in drug discovery and diagnostics development and also provides a better understanding of disease. Advances in technology are providing innovative methods to enhance protein analysis with an increased focus on sample preparation. Cambridge Healthtech’s recent “Proteomic Sample Prep” conference shed some light on what researchers are focusing on to help bring protein analysis to the next level.
One of the challenges in protein sample prep is avoiding degradation. The most common method currently used for inactivation of denaturation is chemical inhibitors. These only provide a reduction in degradation, however. “The diversity of proteins is large, and it’s not fully known which chemical inhibitors work well for some subgroups, proteases, or other fully active groups,” explained Mats Boren, Ph.D., head of research, Denator Biotechnology (www.denator.com).
“Degradation potentially occurs during the entire work flow unless you stabilize the sample.”
Denator’s Stabilizor T1™ was designed to provide rapid, controlled processing of tissue samples. The beta-stage product was developed by company founders via a research project at Uppsala University and the Karolinska Institute. According to Dr. Boren, it works quickly to stop all biological activity and maintain the primary structure of the protein. It can be used for solid tissue (fresh or frozen) samples less than 5 mm thick.
“The Stabilizor enables efficient detection of endogenous peptides, analyzes phosphorylations in a state as in vivo as possible, lowers intragroup variability, and provides fast detection of novel peptide targets or biomarkers,” said Dr. Boren. The current application is for basic research, but it has the potential for use in diagnostics with nonsolid tissues (e.g., blood and urine). The company anticipates launching the Stabilizor T1 in late August.
Researchers at Millipore (www.millipore.com) have developed a new strategy for Amicon® Ultra Centrifugal Filter Units to deplete some of the high-abundant proteins and enrich for low-abundant proteins, potential targets for biomarkers.
Janice Simler, Ph.D., research scientist, presented information at the meeting on using these filter units with serum. “Separating out the low-abundant proteins, especially in matrices like serum, is one of the challenges for biomarker research. We’re trying to empower people who are doing this type of research with a different method that is something they could easily adapt with the equipment they already have in the lab—a centrifuge,” noted Dr. Simler.
Her group presented a model system of human serum to demonstrate the efficiency of Amicon Ultra Centrifugal Filter Units in separation of high molecular weight proteins from low molecular weight proteins. The units are scalable, allowing researchers to start with 50 or 100 microliters and scale up as needed (4 mL and 15 mL versions are available). “These devices save time because you can concentrate and separate in one step, and do a buffer-exchange within the devices, so you don’t have to do an acetone-precipitation, you can just buffer exchange into the buffer you would need for your 2-D analysis.”
The filter units can be used in several steps of protein research. “We feel you can combine this approach with others in order to get optimal depletion, because right now it doesn’t seem like there’s one unique solution,” added Dr. Simler.
In addition, Amicon Ultra-4 filters allow the researcher access to different protein fractions, covering the whole proteome. “I think it’s key to have the whole proteome available. You may initially think your target is in a certain MW range, and then you realize you have to look at higher MW proteins.”
This method is adaptable to any lab and does not require a new skill set, according to Dr. Simler. “Anyone can easily use this method and optimize it to answer the questions they are looking at—either low-abundance proteins or molecular proteins in the high molecular weight range.”