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(Toronto, ON—June 29, 2011) In the first study of its kind to date, an Ontario group of scientists including Drs. Nicolas Bisson and Tony Pawson at Mount Sinai Hospital have highlighted the combined power of affinity purification with selected reaction monitoring (AP-SRM) as a new mass spectrometry-based strategy to assess the dynamic nature of protein interactions associated with specific cellular responses—a methodology that will help accelerate scientists’ understanding of signaling pathways implicated in cancer and other complex illnesses.
The study was published online on June 26 in the leading biomedical journal Nature Biotechnology.
To date, scientists have had little quantitative information regarding the kinetics with which signaling networks assemble and dissolve, and had thought protein complexes to be fairly static. It has been especially difficult to assess the role and effects of adaptor proteins that link activated receptors to their cytoplasmic effectors.
“To address this problem, we designed and employed a large-scale mass spectrometry assay coupled with affinity purification of protein complexes, which was used to investigate quantitative changes in proteins interacting with the adaptor protein GRB2, an important hub protein in growth factor signaling,” said Dr. Nicolas Bisson, first author of the study and a post-doctoral fellow at the Samuel Lunenfeld Research Institute of Mount Sinai Hospital. Dr. Bisson noted that the AP-SRM method can measure and quantify large numbers of proteins as well as the dynamics by which they interact.
After determining the network of proteins that interact with GRB2, the researchers used the AB SCIEX QTRAP® 5500 mass spectrometer coupled to a Eksigent nanoLC Ultra + cHiPLC Nanoflex liquid chromatography system to quantify all of the proteins comprising the extended GRB2 interaction network in a single assay. The assay was employed to address the dynamics with which 90 of the GRB2-associated proteins assemble in response to growth factors that signal through tyrosine kinase receptors.
“AB SCIEX has had a long-standing collaboration with the Samuel Lunenfeld Research Institute, which has some of the top biomedical researchers in the world,” said Dave Hicks, Vice President and General Manager of the Pharma and Omics Business at AB SCIEX. “Their trust in using our QTRAP 5500 System and Eksigent technology to develop and conduct an innovative multiple reaction monitoring-based technique for high specificity helped uncover previously unseen kinetic insight into protein interactions, which could lead to pushing the limits in disease research.” 
Traditional mass spectrometry studies, focused solely on identifying proteins at a given point in time, do not provide scientists dynamic and contextual data associated with protein assemblies. This new quantitative analysis allowed the study group to, for the first time, identify the distinct GRB2-containing complexes that form upon stimulation by different growth factors, and how these complexes change over time. 
“These results underscore the precision that can be achieved with SRM measurements in deciphering the dynamic temporal behaviour of protein interaction networks,” said Lorne Taylor, study co-author from the Ontario Proteomics Methods Centre at the Lunenfeld. “With this method, we also revealed that different GRB2-binding partners have distinct kinetics of complex formation. GRB2 shows remarkable versatility in growth factor signaling and is involved in the formation of a myriad of highly specific protein complexes.”
“By focusing on key hub proteins we have identified, this AP-SRM method can be easily extended to obtain a truly quantitative view of the dynamics of signaling networks in cells stimulated by various classes of extracellular signals, said Dr. Bisson. “As well, we can compare these changes, for example, between normal and cancer cells.”
The study was funded by the Ontario Ministry of Research and Innovation, Genome Canada through the Ontario Genomics Institute, Canadian Institutes of Health Research, Terry Fox Research Institute/Ontario Institute for Cancer Research and the Canadian Cancer Society Research Institute.   

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