Clinical OMICs - Issue 3 - (Page 26)

Clinical OMICs CASE STUDY A Global Phosphorylation Assay Ian Pike and Emma Lahert T he use of targeted agents against key signaling kinases is transforming cancer treatment. Drugs such as Herceptin and Zelboraf have increased progression-free survival in breast cancer and melanoma respectively and are far from the only examples. However, the complexity of signaling pathway networks allows tumor cells to adapt under monotherapy using alternative pathways to maintain a proliferative phenotype. Such resistance mechanisms may be inherently present at the onset of treatment (de novo resistance) or may develop in response to the treatment (acquired resistance). This has led researchers to call for the use of more comprehensive analysis of signaling pathways to identify resistance pathways, and to then use this knowledge to select precise combinations of drugs matched to the individual tumor profile1,2. Regulation of pathway activity is achieved through precisely timed IAN PIKE is chief operating officer (ian.pike@proteomics.com) and EMMA LAHERT is product manager at Proteome Sciences. (emma.lahert@proteomics.com) Website: www.proteomics.com. 26 Clinical OMICs May 15, 2014 gene expression overlain with the more subtle regulation of posttranslational modifications such as phosphorylation, glycosylation, and ubiquitination. While genomic tools such as RNAseq and next-generation sequencing may demonstrate gross changes in signaling networks, only a comprehensive analysis of protein post-translational modifications, particularly phosphorylation, across thousands of pathway proteins, can provide the required biological information to enable selection of the most appropriate drug combination and switching of therapies. Current technologies for measuring protein phosphorylation are based on site-specific antibodies and rely on Western blotting, which is labor intensive, or reverse phosphoproteomic arrays, which have issues of cross-talk. These techniques are particularly limited in differentiating between close protein homologs (e.g., Erk1 and Erk 2) or occupancy at adjacent or closely situated phosphorylation sites. Furthermore, only those proteins/sites for which high-quality antibodies are available can be interrogated. To overcome these limitations, Proteome Sciences has combined state of the art developments in mass spec- trometry, chromatography, and isobaric labeling to develop SysQuant, a global phosphoproteomic workflow that monitors thousands of molecular switches regulating hundreds of signaling pathways in up to ten disease tissue biopsies in a single experiment. The SysQuant Workflow SysQuant (Figure 1) requires around 100 mg of fresh or frozen tissue or cultured cells, equivalent to a core needle biopsy or 2 x 15 cm2 dishes, from which the total proteome is extracted. Following tryptic digestion and isobaric TMT labeling, up to 10 samples can be combined into a single analytical mixture for subsequent fractionation and quantification by mass spectrometry. The analytical mixture is first fractionated by strong cation exchange (SCX) followed by selective enrichment of phosphopeptides (IMAC and TiO2) prior to uHPLC-MS/ MS analysis (Easy-nLC 1000 coupled to Orbitrap Fusion Tribrid-both Thermo Scientific). Figure 1 One set of SCX fractions is analyzed directly to monitor total protein levels based on nonmodified peptides. www.clinicalomics.com http://www.proteomics.com/images/Fig_1-SQ-Workflow.png http://www.proteomics.com http://www.clinicalomics.com

Table of Contents for the Digital Edition of Clinical OMICs - Issue 3

Contents

Clinical OMICs - Issue 3

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