Student Speaker: Leroi V. DeSouza Potential Glioblastoma Multiforme markers: From discovery, to screening, to functional characterization We have successfully used the iTRAQ technique to discover markers for various forms of cancer including endometrial, head and neck, renal and glioblastoma multiforme (GBM). An analysis on resected tissue from GBM patients yielded a number of candidates, some of which were verified by independent techniques such as Western and RT-PCR analyses. Using targeted analysis by multiple reaction monitoring (MRM) we subsequently demonstrated that a number of these proteins were also detectable in plasma. This in turn suggests the feasibility of screening of plasma to monitor the expression levels of markers. Similarly an iTRAQ analysis on GBM explants expressing EGFR wt and those that express the EGFRvIII mutant also revealed a number of differentially expressed proteins. An in-depth investigation performed on one such protein, collapsin response mediator protein 1 (CRMP1), verified the differential expression through RT-PCR, Western, and immunohistochemical analyses. Further, a functional analysis involving quantitative RT-PCR analysis, followed by transwell migration assays on U87 and U373 cells after knock down of CRMP1 suggests a role as a negative regulator of invasion. Additionally, rescue of CRMP1 expression in U87vIII and U373vIII cells, both of which exhibit a lower expression of CRMP1 than the parental cells, showed an approximately 35% decrease in invasion compared with cells transfected with control vectors. The above is an ideal demonstration of the utility of iTRAQ analyses enabling not just the discovery of potential biomarkers, but also providing a basis for the generation of hypotheses regarding functional roles of select proteins.