Two capillary based immunoassay systems have been established in the Collaborative Protein Technology Resource (CPTR) core. First, the Nano(fluidic)Pro(teomics) technology is a capillary-based isoelectric-focusing(IEF) immunoassay system, which employs high-resolution IEF separation of proteins by charge followed by target-specific immunoprobing to detect and quantify multiple protein phosphorylation isoforms. Nanopro assesses protein phosphorylation both qualitatively and quantitatively, and provides more specific and unique information on the activation status of signaling molecules that are not readily assessed by traditional western blotting. Good reproducibility has been obtained with the NanoPro system. Second, the Simple Western system is a fully automated, gel free, blot free western system. All steps following sample preparation are fully automated, including sample loading, size-based protein separation, immunoprobing, washing, detection and data analysis. The system greatly minimizes the variability caused by manual processes in traditional western, and highly quantitative data and excellent reproducibility have been obtained with the system. A panel of capillary immunoassays has been developed and validated in our core, covering major key signal pathways from receptor activation, down-stream signaling transduction, transcriptional regulation, cell cycle control to apoptosis etc.. Through collaborations with investigators from different branches/laboratories of NIH, we have successfully applied the technology to profile cell signaling events in a variety of projects from basic research to clinical studies (see below the list of conference presentations and manuscripts in preparation). The small required sample size and comprehensive signaling molecule characterization, plus highly quantative data and good assay reproducibility of the technology are creating new opportunities for pharmacodynamic biomarker assessment in clinical studies. Assays and protocols have been developed and used to evaluate target therapy responses in peripheral blood mononuclear cells (PMBCs), CD138+ multiple myeloma cells from bone marrow aspirates and solid tumors in multiple clinical trial samples. We have also established a close working relationship with the technology developer, ProteinSimple for continuing improvement of the assay systems to be more robust and reliable, which have resulted in enhanced assay performance and readiness for clinical applications, and as well as making our core a leader in these novel technologies. Conference Presentations in 2011/2012:1.Herrmann MA, Kim YS, Lee MJ, Alarcon SV, Paul K. Goldsmith PK, Giaccone G, Trepel JB, Chen JQ, A novel assay for predictive pharmacodynamic biomarker analysis and evaluating signaling responses to molecular targeted therapies, 2012 Clinical biomarkers &new frontiers in cancer summit, poster 2.Noonan AM, Chen J, Herrmann MA, Annunziata CM, Birinapant, a Novel Smac Mimetic, Activates Apoptosis in NF-kB Dependent Gynecologic Cancer Cell Lines, abstract submitted to 24th EORTC-NCI-AACR Symposium on Molecular Targets and Cancer Therapeutics 3.Chen J, Lee JH, Herrmann MA, Wang Y, Park KS, Heldman M, Goldsmith PM, Giaccone G, A nano-fluidic immunoassay system to develop proteomic responsive biomarkers in non-small cell lung cancer, 2012 ASCO- EORTC-NCI joint meeting: Markers in Cancer, selected for poster discussion 4.Chen J, Heldman M, Herrmann MA, Kedei N, Blumberg PM, Goldsmith PM, A fully automated capillary western system for absolute quantitation of endogenous PKC proteins, an approach to correlate protein quantity with function, 2012 ASCO- EORTC-NCI joint meeting: Markers in Cancer, poster 5.Kang Z, Chen J, Herrmann MA, Jiang Y, Meltzer PS, Cao L. Genome-wide shRNA screening identifies candidate proteins modulating the extrinsic apoptotic pathway, 2012, 103st AACR, poster #6942 6.Noonan AM, Chen J and Annunziata CM. Activity of the SMAC-mimetic Birinapant (TL32711) in Ovarian Cancer Cell Lines, 2012 Keystone Meeting, NF-kappaB Signaling and Biology: From Bench to Bedside, poster 7.Zingone A, Korde N, Chen J, Xi L, Raffeld M, Holkova B, Kmieciak M, Sullivan D, Doyle A, Maric I, Calvo K, Yancey MA, Mulquin M, Annunziata C, Grant S, Landgren O. Molecular characterization and clinical correlations of MEK1/2 inhibition (AZD6244) in relapsed or refractory multiple myeloma: interim analysis from a phase II study, 2011 American Society of Hematology (ASH) annual meeting, selected for oral presentation. Manuscripts: Published: see publication list.Submitted: 1.Xin HW, Ambe CM, Hari DM, Wiegand GW, Miller TC, Chen J, Anderson AJ, Ray S, Mullinax JE, Koizumi T, Langan RC, Burka D, Herrmann MA, Goldsmith PK, Stojadinovic A, Rudloff U, Thorgeirsson SS, Avital I. Label-retaining liver cancer cells demonstrate relative resistance to sorafenib (submitted to Gut)2.Ou L, Huppi K, Gehlhaus K, Dubois W, Patel J, Simmons J, Chen J, Mackiewicz M, Jones T, Pitt JJ, Martin S, Goldsmith PK, Mock B, Caplen NJ. Kinome focused chemosensitizing RNAi screens of rapamycin in breast cancer cells identify rational drug combination strategies (submitted to Molecular Cancer Therapeutics) In Preparation:1.Zingone A, Holkova B, Korde N, Chen J, Hermann MA, Xi L, Raffeld M, Kmieciak M, Sullivan D, Doyle A, Maric I, Calvo K, Yancey MA, Zuchlinski D, Mulquin M, Costello R, Manasanch E, Kwok M, Roschewski M, Kuehl M, Staudt L, Annunziata C, Grant S, Landgren O, MEK1/2 inhibition in relapsed or refractory multiple myeloma: molecular and clinical correlations (to be submitted to JAMA) 2.Kedei N, Lewin NE, Geczy T, Selezneva J, Chen J, Herrmann MA, Heldman MR, Lim L, Mannan P, Garfield SH, Poudel YB, Cummins TJ, Rudra A, Blumberg PM, Keck GE, The less lipophilic and synthetically more accessible bryostatin 7 mechanistically closely resembles bryostatin 1 (to be submitted to ACS Chemical Biology) 3.Chen J, Lee JH, Herrmann MA, Park K-S, Heldman M, Goldsmith PK, Wang Y, Giaccone G. A Nano-fluidic proteomic assay to study dynamic oncoprotein phosphorylation/activation in non-small cell lung cancer in vitro and in vivo 4.Xin HW, Ambe CM, Tyler MC, Chen J, Anderson AJ, Wiegand GW, Godbout J, Ray S, Mullinax JE, Koizumi T, Langan1 RC, Herrmann MA, Goldsmith PK, Thorgeirsson SS, Avital I. Metformin synergizes liver cancer therapy with sorafenib by inhibiting MEK, ERK, JNK and PKC, but doesn?t selectively target stem-like label-retaining cancer cells 5.Chen J, Heldman M, Herrmann MA, Kedei N, Blumberg PM, Goldsmith PM, A fully automated capillary Western system for absolute quantitation of endogenous PKC proteins, an approach to correlate protein quantity with function
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