Assay development
Profiling Cell Signaling Networks at Single-cell ResolutionSignaling network responses can be highly heterogeneous across cells in a tissue because of many sources of genetic and non-genetic variance. The emergence of multiplexed single-cell technologies has made it possible to evaluate this heterogeneity. In this review, we categorize currently established single-cell signaling network profiling approaches by their methodology, coverage, and application, and we discuss the advantages and limitations of these technologies. We describe the computational tools for network characterization using single-cell data and discuss potential confounding factors that may affect single-cell analyses.
Absolute Quantification of Apolipoproteins Following Treatment with Omega-3 Carboxylic Acids and Fenofibrate Using a High Precision Stable Isotope-labeled Recombinant Protein Fragments Based SRM AssayApplications of LC-SRM in clinical research are still limited. SIS PrEST are a novel class of standards added prior to trypsinization. We have developed a semi-automated sample preparation workflow and a SIS PrEST LC-SRM/MS Tier 2 assay for absolute quantification of 13 apolipoproteins in human plasma and applied it on clinical samples from the EFFECT I study. We demonstrate, for the first time, that SIS PrEST can be applied for exploratory biomarker research in clinical settings and capture drug effects.
A Platform for Extracellular Interactome Discovery Identifies Novel Functional Binding Partners for the Immune Receptors B7-H3/CD276 and PVR/CD155The Conditioned Media AlphaScreen represents a new platform for high throughput and sensitivity detection of interactions between plasma membrane proteins. The interleukin receptor IL20RA is identified as a binding partner for the orphan checkpoint inhibitor B7-H3. Further, a new functional interaction between the KIR2DL5 and the immune receptor PVR regulates natural killer cell cytolysis of tumor cells. This technology represents a versatile approach for receptor interactome discovery that provides insights into plasma membrane protein biology.
Multi-omics Biomarker Pipeline Reveals Elevated Levels of Protein-glutamine Gamma-glutamyltransferase 4 in Seminal Plasma of Prostate Cancer PatientsMulti-omics biomarker pipeline generated 19 proteins which were verified by a multiplex quantitative SRM assay in seminal plasma samples of 152 prostate cancer and 67 negative biopsy patients. Verification revealed a prostate-specific, secreted and androgen-regulated protein-glutamine gamma-glutamyltransferase 4 (TGM4), which detected prostate cancer on biopsies with AUC=0.66, as measured by an in-house immunoassay in seminal plasma. Perspectives of seminal plasma proteins as biomarkers of prostate cancer were reviewed.
Intact Transition Epitope Mapping – Targeted High-Energy Rupture of Extracted Epitopes (ITEM-THREE)ITEM-THREE enables rapid epitope mapping. Sample consumption is minimized and in-solution handling reduced to mixing of antibody and antigen peptide solutions. After immune complex formation in solution, epitope mapping is performed in the gas phase using the mass spectrometer for sophisticated ion manipulation and filtering. Because amino acid sequence information is obtained from the epitope peptide, unknown antigens can be identified. Knowing the epitope broadens the application of antibodies to unspecified target proteins from any organism.
High-throughput Identification of FLT3 Wild-type and Mutant Kinase Substrate Preferences and Application to Design of Sensitive In Vitro Kinase Assay SubstratesKinase activity assays are an invaluable tool used to develop new small molecule inhibitors to combat cancer and other human diseases, however, only a small fraction of the human kinome is well characterized. Lack of biological substrate information limits the capability of computational approaches to design sensitive peptide probes for FLT3 and clinically relevant mutant kinases. In this report, we used phosphoproteomics and the KINATEST-ID pipeline (31) to design a panel of FLT3 artificial peptide substrates (FAStides) to design an ELISA-based activity assay.