APPLICATIONS OF TECHNOLOGY:
- Biomedical research, drug design
- More efficient than target affinity purification
- May be scaled up for high throughput
- Identifies multiple protein complexes simultaneously
- Low false positive and false negative rates
Scientists at Berkeley Laboratory and the University of California, San Francisco have invented a high-throughput method for purifying and identifying protein complexes in wild-type cells without the use of affinity tags and genetic manipulation. This “tagless” strategy detects polypeptides in endogenous protein complexes and then identifies the polypeptides by mass spectrometry.
The invention overcomes the limitations of target affinity purification—the most commonly used method for identifying protein complexes. Target affinity purification can only be applied to biological systems that are amenable to genetic manipulations; may destabilize or alter the protein-protein interactions being studied, causing false positive and false negative results; and requires that a separate genetic strain be constructed, cultured, and analyzed for each polypeptide. Because the tagless Berkeley Lab/UCSF technology does not require genetic modification, it can be applied to a wide variety of cells and organisms. It is also less likely to alter endogenous protein associations. This invention is more efficient because it uses a single, large culture for the simultaneous analysis of several polypeptides and complexes.
The process begins by growing large batches of cells in culture, lysing them, and separating the crude cell extracts into thousands of fractions via multidimensional chromatographic steps run in parallel. Polypeptides that are associated in endogenous protein complexes migrate together (co-elute) through a multi-step separation scheme. The polypeptides in the fractions are identified, and their relative abundances are calculated by mass spectrometry. The elution profiles of the polypeptides are then analyzed to identify those that cluster at an experimentally established confidence level. Researchers tested the technology and found that it reliably assigned 37 polypeptide components to 13 known E. coli protein complexes; 95% of the components were grouped by coelution and 57% by a prototype cluster analysis.
DEVELOPMENT STAGE: Proven principle.
STATUS: Patent pending. Available for licensing or collaborative research.
FOR MORE INFORMATION:
Dong M., L.L. Yang, K. Williams, S.J. Fisher, S.C. Hall, M.D. Biggin, J. Jin, and H.E. Witkowska. “A ‘tagless’ strategy for identification of stable protein complexes genome-wide by multidimensional orthogonal chromatographic separation and iTRAQ reagent tracking,” Journal of Proteome Research 7:1836-1849 (2008).
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REFERENCE NUMBER: JIB-2538