Differentially embedded glioblastoma tumor protein quantitation
Glioblastoma multiforme (GBM) is the most aggressive malignant primary brain tumor with a dismal mean survival even with the current standard of care. Although in vitro cell systems can provide mechanistic insight into the regulatory networks governing GBM cell proliferation and migration, clinical samples provide a more physiologically relevant view of oncogenic signaling networks. However, clinical samples are not widely available and may be embedded for histopathologic analysis. With the goal of accurately identifying activated signaling networks in GBM tumor samples, we investigated the impact of embedding in optimal cutting temperature (OCT) compound followed by flash freezing in LN2 vs. immediate flash freezing (iFF) in LN2 on protein expression and phosphorylationmediated signaling networks. Quantitative proteomic and phosphoproteomic analysis of 8 pairs of tumor specimens revealed minimal impact of the different sample processing strategies and highlighted the large inter-patient heterogeneity present in these tumors. Correlation analyses of the differentially processed tumor sections identified activated signaling networks present in selected tumors and revealed the differential expression of transcription, translation, and degradation associated proteins. This study demonstrates the capability of quantitative mass spectrometry for identification of in vivo oncogenic signaling networks from human tumor specimens that were either OCT-embedded or immediately flash-frozen.
Sample Processing Protocol
Four independent iTRAQ 8plex analyses of differentially embedded GBM tumor samples from 8 patients. iTRAQ8plex samples were fractionated using isoelectric focussing into 5 fractions before LC-MS/MS analysis.
Data Processing Protocol
Data was analysed using an ABI 5600 instrument. Data fies were searched using protein pilot
Johnson H, White FM. Quantitative analysis of signaling networks across differentially embedded tumors highlights interpatient heterogeneity in human glioblastoma. J Proteome Res. 2014 Nov 7;13(11):4581-93 PubMed: 24927040