To address the challenges of conventional proteomic approaches, such as limited sample size, poor assay reproducibility and unreliable data quantification, two analysis platforms have been established in our core: 1) Capillary nano-immunoassay (CNIA) system - the Simple Western (ProteinSimple, CA). It is an automated capillary immunoassay system, which employs high-resolution MW (size-based) or isoelectric-focusing (IEF, charge-based) separation, followed by target-specific immunoprobing to profile proteins and respective post-translational modification isoforms. The capillary platform allows sample analysis with nanogram level of proteins, which makes it attractive for extremely small and precious samples, such as stem cells, primary cells, micro-dissected samples, fine needle aspirates, and other patient specimens etc. 2) Luminex multiplexing immunoassays - a multiplex in-solution ELISA analysis system, based on the Luminex xMAP beads technology, which combines advanced fluidics, optics, and digital signal processing with proprietary microsphere technology to deliver multiplexed assay capabilities with small sample consumption. These technologies have been successfully applied in both discovery research and clinical practice for precise and accurate measurement of signaling molecules and their activations, as well as mid-throughput quantitative proteomic analysis. Highly quantitative data, good assay sensitivity and reproducibility have been observed in both analysis platforms. Through collaborations with investigators from different branches/laboratories of CCR/NCI/NIH, we have demonstrated that the system is appealing for clinical applications filling gaps in protein analysis, such as a) the low successful rate of assay transfer from discovery research to clinical practice and b) the lack of efficient and high-performance methods for real-time disease management. In addition, because of very small sample requirements, the systems enable the measurement of protein level responses to drug treatment in specimens collected in a non- or minimally- invasive manner. We have successfully applied the CNIA assays in a variety of projects including cell signaling profiling, biomarker development, targeted therapy evaluations and investigation on drug resistance mechanism etc. A panel of about two hundred capillary-immunoassays, covering key signaling 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 novel platform at CCR for comprehensive and quantitative proteomic analysis with minute sample consumption. The IEF analysis platform of the CNIA assays offers the capability to separate, detect and quantify different post-translationally modified states of a protein without using modification-specific antibodies. Assays and protocols have also been developed and validated for patient specimen analysis and applied in several clinical trial projects. The effort greatly facilitated the clinical investigations on monitoring drug response at the protein level, and helped the investigators to further modify treatment strategies. Our efforts have demonstrated that the CNIA system provides quantitative and precise measurement of signaling molecules and their activation status, and have led to multiple publications and presented at national and international meetings, as well as established our core as a leader in the field of CNIA technology. Recent implementation of the Luminex multiplexing immunoassay system allows our core to accommodate the demand for affordable, high performance, good-throughput multiplex in-solution ELISA assays. The technology has been selected to be deployed for biobank profiling research in the Human Protein Atlas (HPA) project. While most of our CNIA assays are for cell lysate analysis, the Luminex technology provides complementary assays on cytokine, metabolite, immune response, serum/plasma biomarker analysis etc. in cell supernatant and plasma/serum samples. Working with CCR Office of Science and Technology Resource (OSTR), we have explored/evaluated potential applications of other innovative proteomic analysis technologies as well, such as Nanostring in proteomic analysis, Singulex immunoassay for low abundance biomarker detection, SOMAscan technology to study global protein expression, and single cell western analysis etc. Using a new generation of protein-capture SOMAmer (Slow Off-rate Modified Aptamer) reagents, the SOMAscan proteomic assay simultaneously measures over one thousand analytes within a small amount of protein sample. The technology is emerging as a highly sensitive, multiplexed and quantitative proteomic tool for biomarker discovery and validation. A joint collaborative project was established with the technology vendor SomaLogic (Boulder, CO) to study differential protein response to ligand stimulations and revealed the importance of monitoring signaling response at both the transcriptomic and proteomic levels. We are also in the process of acquiring a single-cell western system which performs Western analysis on thousands of single cells in parallel, offering a tool for single cell signaling study and population heterogeneity dissection. A collaborative relationship is maintained with the investigators. We are actively involved and offer our expertise on different project advancement stages, such as project feasibility discussion, experiment design, method development, data evaluation and summary etc. We also incorporated high standard Good Laboratory Practice (GLP) concepts into the core's operation for better data quality and reliability, as well as assay readiness for bench to bed-side transfer. Attention is devoted to operational efficiency to ensure that the CCR resources invested are efficiently utilized and accessible to all PIs, with accountability and tracking of scientific impact for the CCR. A web-based interface (https://cptr.cancer.gov/) has been established that allows a) better accessibility for the investigators to our services; and b) more efficient project management and advancement. The following services have been established and offered at our core: 1) Protein characterization and post-translational modification analysis; 2) Cell signaling profiling and mid-throughput quantitative proteomics; 3) Biomarker development and analysis; 4) Pharmcodynamic and molecular diagnostic analysis, target therapy evaluation etc;
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