Two capillary based immunoassay systems (ProteinSimple,Santa Clara, CA) have been recently established in our core. First, the Nano(fluidic)Pro(teomics) technology (also called, charge-based Simple Western system) is a capillary-based iso-electic-focusing (IEF) immunoassay system, which employs high-resolution IEF separation of proteins by charge followed by target-specific immune-probing 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 and response status of individual phospho-isoforms that are not assessable by traditional Western blotting. Good assay reproducibility has been obtained with the NanoPro system. Second, the size-based Simple Western system is a fully automated, gel free, blot free capillary-based Western system. All steps after 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 Westerns, and offers highly quantitative data and excellent reproducibility. These novel capillary-based immunoassay platforms provide comprehensive and quantitative analysis in the characterization of proteins and their phosphorylation isoforms. They also allow the use of nanogram amounts of material for analysis, thus enabling protein analysis in extremely small samples, such as stem cells, primary cells, fine needle aspirates and other patient specimens, which in many cases do not provide sufficient sample amounts to be analyzed by conventional protein technology. Through collaborations with investigators from different branches/laboratories of CCR/NCI/NIH, we have successfully applied the technology to profile cell signaling events in a variety of projects from basic research to clinical studies. A panel of about two hundred simple Western immunoassays, covering major key signal pathways from receptor activation, down-stream signaling transduction, transcriptional regulation, cell cycle control to apoptosis etc., has been developed/established in our core, providing a noval platform for quantitative proteomic analysis. We have also established clinically feasible assays for pharmacodynamic assessment and targeted therapy evaluation. We have developed / validated assays and protocols for patient specimen analysis in several clinical trial projects, including lung cancer, breast cancer, ovarian cancer, multiple myeloma etc... Our efforts have led to multiple manuscript preparation and publications, as well as presentations in multiple national and international meetings. For example, the study of "Molecular characterization of clinical trial samples from a phase II study of AZD6244, MEK Inhibitor in multiple myeloma biopsies" (collaborated with Dr. Landgren from Metabolism Branch, CCR/NCI) was selected for oral presentation at America Society of Hematology (ASH) annual meeting (San Diego CA, Dec. 2011) and presented at NIH clinical center grand rounds (Bethesda MD, Oct. 2012), and 2013 NCI intramural PI-retreat (Washington DC, Jan. 2013);and the work of "Application of NanoPro to study dynamic onco-protein phosphorylation in non-small cell lung cancer and for evaluating drug response to targeted therapies" (collaborated with Dr. Giaccone) was selected for extended poster discussion in ASCO-EORTC-NCI joint meeting: Markers in Cancer (Hollywood FL, Oct. 2012). 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. The effort has resulted in enhanced assay performance and readiness for clinical applications, and as well as making our core a leader in these novel technologies as we support CCR science.

National Institute of Health (NIH)
National Cancer Institute (NCI)
Scientific Cores Intramural Research (ZIC)
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National Cancer Institute Division of Basic Sciences
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Kedei, Noemi; Chen, Jin-Qiu; Herrmann, Michelle A et al. (2014) Molecular systems pharmacology: isoelectric focusing signature of protein kinase C? provides an integrated measure of its modulation in response to ligands. J Med Chem 57:5356-69
Chen, Jin-Qiu; Heldman, Madeleine R; Herrmann, Michelle A et al. (2013) Absolute quantitation of endogenous proteins with precision and accuracy using a capillary Western system. Anal Biochem 442:97-103
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Bakhsheshian, Joshua; Hall, Matthew D; Robey, Robert W et al. (2013) Overlapping Substrate and Inhibitor Specificity of Human and Murine ABCG2. Drug Metab Dispos 41:1805-12