FZD4 Marks Lateral Plate Mesoderm and Signals with NORRIN to Increase Cardiomyocyte Induction from Pluripotent Stem Cell-Derived Cardiac Progenitors.

The identification of cell surface proteins on stem cells or stem cell derivatives is a key strategy for the functional characterization, isolation, and understanding of stem cell population dynamics. Here, using an integrated mass spectrometry- and microarray-based approach, we analyzed the surface proteome and transcriptome of cardiac progenitor cells (CPCs) generated from the stage-specific differentiation of mouse and human pluripotent stem cells. Through bioinformatics analysis, we have identified and characterized FZD4 as a marker for lateral plate mesoderm.

Targeted protein identification, quantification and reporting for high-resolution nanoflow targeted peptide monitoring.

Mass spectrometry-based targeted proteomic assays are experiencing a surge in awareness due to the diverse possibilities arising from the re-application of traditional LC-SRM technology. The FDA-approved quantitative LC-SRM-pipeline in drug discovery motivates the use to quantitatively validate putative proteomic biomarkers. However, complexity of biological specimens bears a huge challenge to identify, in parallel, specific peptides and proteins of interest from large biomarker candidate lists.

Target identification by chromatographic co-elution: monitoring of drug-protein interactions without immobilization or chemical derivatization.

Bioactive molecules typically mediate their biological effects through direct physical association with one or more cellular proteins. The detection of drug-target interactions is therefore essential for the characterization of compound mechanism of action and off-target effects, but generic label-free approaches for detecting binding events in biological mixtures have remained elusive. Here, we report a method termed target identification by chromatographic co-elution (TICC) for routinely monitoring the interaction of drugs with cellular proteins under nearly physiological conditions in vitro based on simple liquid chromatographic separations of cell-free lysates.

Identification of enzyme-converted peptide products from single cells using capillary electrophoresis and liquid chromatography-mass spectrometry.

Single-cell analysis using chemical methods, otherwise known as chemical cytometry, promises to provide significant leaps in understanding signaling processes which result in cellular behavior. Sensitive methods for chemical cytometry such as capillary electrophoresis can detect and quantify multiple targets; however, conclusive identification of detected analytes is required for useful data to be obtained. Here, we demonstrate a method for determining the identity of enzyme-converted peptide products from single cells using a combination of capillary electrophoresis and liquid chromatography-mass spectrometry (LC-MS).

Synthetic peptide arrays for pathway-level protein monitoring by liquid chromatography-tandem mass spectrometry.

Effective methods to detect and quantify functionally linked regulatory proteins in complex biological samples are essential for investigating mammalian signaling pathways. Traditional immunoassays depend on proprietary reagents that are difficult to generate and multiplex, whereas global proteomic profiling can be tedious and can miss low abundance proteins. Here, we report a target-driven liquid chromatography-tandem mass spectrometry (LC-MS/MS) strategy for selectively examining the levels of multiple low abundance components of signaling pathways which are refractory to standard shotgun screening procedures and hence appear limited in current MS/MS repositories.

A lentiviral functional proteomics approach identifies chromatin remodeling complexes important for the induction of pluripotency.

Protein complexes and protein-protein interactions are essential for almost all cellular processes. Here, we establish a mammalian affinity purification and lentiviral expression (MAPLE) system for characterizing the subunit compositions of protein complexes. The system is flexible (i.

Single amino acid resolution of proteolytic fragments generated in individual cells.

The complex nature of enzyme regulation mandates that enzyme activity profiles be measured in the context of the intact cell. Single-cell capillary electrophoresis (CE) coupled with laser-induced fluorescence is a powerful approach for quantitation and separation of analytes present in small samples and single live cells; however, it does not allow for the definitive identification of the reaction products. On the other hand, mass spectrometry (MS) is able to identify analytes but still lacks the requisite sensitivity for most single-cell analysis applications.

Molecular mechanisms of chronic kidney transplant rejection via large-scale proteogenomic analysis of tissue biopsies.

The most common cause of kidney transplant failure is the poorly characterized histopathologic entity interstitial fibrosis and tubular atrophy (IFTA). There are no known unifying mechanisms, no effective therapy, and no proven preventive strategies. Possible mechanisms include chronic immune rejection, inflammation, drug toxicity, and chronic kidney injury from secondary factors.

Large-scale characterization and analysis of the murine cardiac proteome.

Recent advances in mass spectrometry and bioinformatics have provided the means to characterize complex protein landscapes from a wide variety of organisms and cell types. Development of standard proteomes exhibiting all of the proteins involved in normal physiology will facilitate the delineation of disease mechanisms. Here, we examine the wild-type cardiac proteome using data obtained from a subcellular fractionation protocol in combination with a multidimensional protein identification proteomics approach.

High-resolution biomarker discovery: Moving from large-scale proteome profiling to quantitative validation of lead candidates.

Diverse proteomic techniques based on protein MS have been introduced to systematically characterize protein perturbations associated with disease. Progress in clinical proteomics is essential for personalized medicine, wherein treatments will be tailored to individual needs based on patient stratification using noninvasive disease monitoring procedures to reveal the most appropriate therapeutic targets. However, breakthroughs await the successful development and application of a robust proteomic pipeline capable of identifying and rigorously assessing the relevance of multiple candidate proteins as informative diagnostic and prognostic indicators or suitable drug targets involved in a pathological process.

Evaluation of data-dependent versus targeted shotgun proteomic approaches for monitoring transcription factor expression in breast cancer.

In breast cancer, there is a significant degree of molecular diversity among tumors. Multiple perturbations in signal transduction pathways impinge on transcriptional networks that in turn dictate malignant transformation and metastatic progression. Detailed knowledge of the sequence-specific transcription factors that become activated or repressed within a tumor and comparison of their relative levels of expression in cancer versus normal tissue should therefore provide insight into disease mechanisms, improving patient stratification and facilitating personalized treatment.

Isolation and angiogenesis by endothelial progenitors in the fetal liver.

Endothelial progenitor cells (EPCs) have significant therapeutic potential. However, the low quantity of such cells available from bone marrow and their limited capacity to proliferate in culture make their use difficult. Here, we present the first definitive demonstration of the presence of true EPCs in murine fetal liver capable of forming blood vessels in vivo connected to the host's vasculature after transplantation.