Targeting G Protein-Coupled Receptors by Capture Compound Mass Spectrometry: A Case Study with Sertindole.

Unbiased chemoproteomic profiling of small-molecule interactions with endogenous proteins is important for drug discovery. For meaningful results, all protein classes have to be tractable, including G protein-coupled receptors (GPCRs). These receptors are hardly tractable by affinity pulldown from lysates.

A novel approach to detect resistance mechanisms reveals FGR as a factor mediating HDAC inhibitor SAHA resistance in B-cell lymphoma.

Histone deacetylase (HDAC) inhibitors such as suberoylanilide hydroxamic acid (SAHA) are not commonly used in clinical practice for treatment of B-cell lymphomas, although a subset of patients with refractory or relapsed B-cell lymphoma achieved partial or complete remissions. Therefore, the purpose of this study was to identify molecular features that predict the response of B-cell lymphomas to SAHA treatment. We designed an integrative approach combining drug efficacy testing with exome and captured target analysis (DETECT).

Identification of Potential Off-target Toxicity Liabilities of Catechol-O-methyltransferase Inhibitors by Differential Competition Capture Compound Mass Spectrometry.

Structurally related inhibitors of a shared therapeutic target may differ regarding potential toxicity issues that are caused by different off-target bindings. We devised a differential competition capture compound mass spectrometry (dCCMS) strategy to effectively differentiate off-target profiles. Tolcapone and entacapone are potent inhibitors of catechol-O-methyl transferase (COMT) for the treatment of Parkinson's disease.

Dabigatran and dabigatran ethyl ester: potent inhibitors of ribosyldihydronicotinamide dehydrogenase (NQO2).

Recent studies have revealed that compounds believed to be highly selective frequently address multiple target proteins. We investigated the protein interaction profile of the widely prescribed thrombin inhibitor dabigatran (1), resulting in the identification and subsequent characterization of an additional target enzyme. Our findings are based on an unbiased functional proteomics approach called capture compound mass spectrometry (CCMS) and were confirmed by independent biological assays.

Light-triggered conversion of non-ionic into ionic surfactants: towards chameleon detergents for 2-D gel electrophoresis.

Caged non-ionic detergents, comprised of polar oligo(ethylene glycol) and non-polar alkyl chains joined by a photocleavable ortho-nitrobenzyl sulfonate linker have been synthesized and characterized. The light-triggered transformation of such chameleon surfactant from a charge-neutral into a charged form offers great potential to improve 2-D gel electrophoretic separation of complex protein mixtures.

Profiling of methyltransferases and other S-Adenosyl-L-homocysteine-binding proteins by Capture Compound mass spectrometry.

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions. We describe a generic approach based on trifunctional Capture Compounds, in which the initial equilibrium-driven interaction between a small molecule probe and target proteins is irreversibly fixed upon photo-crosslinking between an independent photo-activable reactivity function of the Capture Compound and the surface of the target protein(s). Subsequently, Capture Compound - protein conjugates are isolated from complex biological mixtures via the sorting function of the Capture Compound.

Proteome-wide identification of staurosporine-binding kinases using capture compound mass spectrometry.

The enormous diversity of kinases and their pivotal role in cell signaling have set kinases in the focus of biomedical research. Profiling the kinome of tissues of different origin is essential for biomarker discovery. In drug research, it is necessary to comprehend the specificity profile of a given kinase inhibitor.

Improvement of capture compound mass spectrometry technology (CCMS) for the profiling of human kinases by combination with 2D LC-MS/MS.

An increasingly popular and promising field in functional proteomics is the isolation of proteome subsets based on small molecule-protein interactions. One platform approach in this field are Capture Compounds that contain a small molecule of interest to bind target proteins, a photo-activatable reactivity function to covalently trap bound proteins, and a sorting function to isolate captured protein conjugates from complex biological samples for direct protein identification by liquid chromatography/mass spectrometry (nLC-MS/MS). In this study we used staurosporine as a selectivity group for analysis in HepG2 cells derived from human liver.

Probing small molecule-protein interactions: A new perspective for functional proteomics.

The isolation of proteome subsets on the basis of the interactions of small molecules with proteins is an emerging paradigm in proteomics. Depending on the nature of the small molecule used as a bait, entire protein families can be monitored in biological samples, or new functions can be attributed to previously uncharacterized proteins. With pharmaceutical compounds as baits, drug targets and toxicity-relevant off-targets can be discovered in an unbiased proteomic screen.

Dasatinib, imatinib and staurosporine capture compounds - Complementary tools for the profiling of kinases by Capture Compound Mass Spectrometry (CCMS).

Capture Compound Mass Spectrometry (CCMS) is a platform technology for the functional isolation of subproteomes. Here we report the synthesis of two new kinase Capture Compounds (CCs) based on the tyrosine-kinase specific inhibitors dasatinib and imatinib and compare their interaction profiles to that of our previously reported staurosporine-CCs. CCs are tri-functional molecules: they comprise a sorting function (e.

Profiling of methyltransferases and other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (CCMS).

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity chromatography (1) or Activity Based Protein Profiling (2). Trifunctional Capture Compounds (CCs, Figure 1A) (3) are the basis for a generic approach, in which the initial equilibrium-driven interaction between a small molecule probe (the selectivity function, here S-adenosyl-(L)-homocysteine, SAH, Figure 1A) and target proteins is irreversibly fixed upon photo-crosslinking between an independent photo-activable reactivity function (here a phenylazide) of the CC and the surface of the target proteins. The sorting function (here biotin) serves to isolate the CC - protein conjugates from complex biological mixtures with the help of a solid phase (here streptavidin magnetic beads).

Differential expression level of cytokeratin 8 in cells of the bovine nucleus pulposus complicates the search for specific intervertebral disc cell markers.

Introduction: Development of cell therapies for repairing the intervertebral disc is limited by the lack of a source of healthy human disc cells. Stem cells, particularly mesenchymal stem cells, are seen as a potential source but differentiation strategies are limited by the lack of specific markers that can distinguish disc cells from articular chondrocytes.

Methods: We searched for markers using the differential in-gel electrophoresis proteomic technology to compare proteins of bovine nucleus pulposus cells, phenotypically similar to mature human nucleus cells, with those of bovine articular chondrocytes.

Comprehensive identification of staurosporine-binding kinases in the hepatocyte cell line HepG2 using Capture Compound Mass Spectrometry (CCMS).

The central role of kinases in cell signaling has set them in the focus of biomedical research. In functional proteomics analyses, large- scale profiling of kinases has become feasible through the use of affinity pulldown beads that carry immobilized kinase inhibitors. As an alternative approach to solid phase beads, Capture Compound Mass Spectrometry (CCMS) enables the functional isolation of protein-classes on the basis of small molecule-protein interactions in solution.

GDP-capture compound--a novel tool for the profiling of GTPases in pro- and eukaryotes by capture compound mass spectrometry (CCMS).

The functional isolation of proteome subsets based on small molecule-protein interactions is an increasingly popular and promising field in functional proteomics. Entire protein families may be profiled on the basis of their common interaction with a metabolite or small molecule inhibitor. This is enabled by novel multifunctional small molecule probes.

Capture compound mass spectrometry sheds light on the molecular mechanisms of liver toxicity of two Parkinson drugs.

Capture compound mass spectrometry (CCMS) is a novel technology that helps understand the molecular mechanism of the mode of action of small molecules. The Capture Compounds are trifunctional probes: A selectivity function (the drug) interacts with the proteins in a biological sample, a reactivity function (phenylazide) irreversibly forms a covalent bond, and a sorting function (biotin) allows the captured protein(s) to be isolated for mass spectrometric analysis. Tolcapone and entacapone are potent inhibitors of catechol-O-methyltransferase (COMT) for the treatment of Parkinson's disease.

The cAMP capture compound mass spectrometry as a novel tool for targeting cAMP-binding proteins: from protein kinase A to potassium/sodium hyperpolarization-activated cyclic nucleotide-gated channels.

The profiling of subproteomes from complex mixtures on the basis of small molecule interactions shared by members of protein families or small molecule interaction domains present in a subset of proteins is an increasingly important approach in functional proteomics. Capture Compound Mass Spectrometry (CCMS) is a novel technology to address this issue. CCs are trifunctional molecules that accomplish the reversible binding of target protein families to a selectivity group (small molecule), covalent capturing of the bound proteins by photoactivated cross-linking through a reactivity group, and pullout of the small molecule-protein complexes through a sorting function, e.

Jmjd6 catalyses lysyl-hydroxylation of U2AF65, a protein associated with RNA splicing.

The finding that the metazoan hypoxic response is regulated by oxygen-dependent posttranslational hydroxylations, which regulate the activity and lifetime of hypoxia-inducible factor (HIF), has raised the question of whether other hydroxylases are involved in the regulation of gene expression. We reveal that the splicing factor U2 small nuclear ribonucleoprotein auxiliary factor 65-kilodalton subunit (U2AF65) undergoes posttranslational lysyl-5-hydroxylation catalyzed by the Fe(II) and 2-oxoglutarate-dependent dioxygenase Jumonji domain-6 protein (Jmjd6). Jmjd6 is a nuclear protein that has an important role in vertebrate development and is a human homolog of the HIF asparaginyl-hydroxylase.

A quantitative strategy to detect changes in accessibility of protein regions to chemical modification on heterodimerization.

We describe a method for studying quantitative changes in accessibility of surface lysine residues of the PB1 subunit of the influenza RNA polymerase as a result of association with the PA subunit to form a PB1-PA heterodimer. Our method combines two established methods: (i) the chemical modification of surface lysine residues of native proteins by N-hydroxysuccinimidobiotin (NHS-biotin) and (ii) the stable isotope labeling of amino acids in cell culture (SILAC) followed by tryptic digestion and mass spectrometry. By linking the chemical modification with the SILAC methodology for the first time, we obtain quantitative data on chemical modification allowing subtle changes in accessibility to be described.

Immunocapture and identification of cell membrane protein antigenic targets of serum autoantibodies.

There is increasing interest in the role of antibodies targeting specific membrane proteins in neurological and other diseases. The target(s) of these pathogenic antibodies is known in a few diseases, usually when candidate cell surface proteins have been tested. Approaches for identifying new antigens have mainly resulted in the identification of antibodies to intracellular proteins, which are often very useful as diagnostic markers for disease but unlikely to be directly involved in disease pathogenesis because they are not accessible to circulating antibodies.

Studies of the ATPase activity of the ABC protein SUR1.

The ATP-sensitive potassium (K(ATP)) channel couples glucose metabolism to insulin secretion in pancreatic beta-cells. It comprises regulatory sulfonylurea receptor 1 and pore-forming Kir6.2 subunits.

Analysis of the dorsal spinal cord synaptic architecture by combined proteome analysis and in situ hybridization.

The proteomic analysis of tissue samples is an analytical challenge, because identified gene products not only have to be assigned to subcellular structures, but also to cell subpopulations. We here report a strategy of combined subcellular proteomic profiling and in situ hybridization to assign proteins to subcellular sites in subsets of cells within the dorsal region of rat spinal cord. With a focus on synaptic membranes, which represent a complex membrane protein structure composed of multiple integral membrane proteins and networks of accessory structural proteins, we also compared different two-dimensional gel electrophoresis systems for the separation of the proteins.

Identification of versican as an isolectin B4-binding glycoprotein from mammalian spinal cord tissue.

Nociceptors are specialized nerve fibers that transmit noxious pain stimuli to the dorsal horn of the spinal cord. A subset of nociceptors, the nonpeptidergic C-fibers, is characterized by its reactivity for the plant isolectin B4 (IB4) from Griffonia simplicifolia. The molecular nature of the IB4-reactive glycoconjugate, although used as a neuroanatomical marker for more than a decade, has remained unknown.