1,721,004 research outputs found
A Simple Cross-Linking/Mass Spectrometry Workflow for Studying System-wide Protein Interactions
No full textWe present a cross-linking/mass spectrometry workflow for performing proteome-wide cross-linking analyses within 1 week. The workflow is based on the commercially available mass spectrometry-cleavable cross-linker disuccinimidyl dibutyric urea and can be employed by every lab having access to a mass spectrometer with tandem mass spectrometry capabilities. We provide an updated version 2.0 of the freeware software tool MeroX, available at www.StavroX.com, that allows us to conduct fully automated and reliable studies delivering insights into protein-protein interaction networks and protein conformations at the proteome level. We exemplify our optimized workflow for mapping protein-protein interaction networks in Drosophila melanogaster embryos on a system-wide level. From cross-linked Drosophila embryo extracts, we detected 29931 cross-link spectrum matches corresponding to 7436 unique cross-linked residues in biological triplicate experiments at a 1% false discovery rate. Among these, 1611 interprotein cross-linking sites were identified and yielded valuable information about protein-protein interactions. The 5825 remaining intraprotein cross-links yield information about the conformational landscape of proteins in their cellular environment
Evaluating Imide-Based Mass Spectrometry-Cleavable Cross-Linkers for Structural Proteomics Studies
Full text linkDisuccinimidyl dibutyric urea (DSBU) is a mass spectrometry (MS)-cleavable cross-linker that has multiple applications in structural biology, ranging from isolated protein complexes to comprehensive system-wide interactomics. DSBU facilitates a rapid and reliable identification of cross-links through the dissociation of its urea group in the gas phase. In this study, we further advance the structural capabilities of DSBU by remodeling the urea group into an imide, thus introducing a novel class of cross-linkers. This modification preserves the MS cleavability of the amide bond, granted by the two acyl groups of the imide function. The central nitrogen atom enables the introduction of affinity purification tags. Here, we introduce disuccinimidyl disuccinic imide (DSSI) as a prototype of this class of cross-linkers. It features a phosphonate handle for immobilized metal ion affinity chromatography enrichment. We detail DSSI synthesis and describe its behavior in solution and in the gas phase while cross-linking isolated proteins and human cell lysates. DSSI and DSBU cross-links are compared at the same enrichment depth to bridge these two cross-linker classes. We validate DSSI cross-links by mapping them in high-resolution structures of large protein assemblies. The cross-links observed yield insights into the morphology of intrinsically disordered proteins and their complexes. The DSSI linker might spearhead a novel class of MS-cleavable and enrichable cross-linkers
An immunohistochemical study of endothelin-1 converting enzyme in the human eye
No full textPurpose. The important role of ET-1 in vasoconstriction has been shown for the vasoregulation in the retina, choroid and optic nerve. ET-1 induced vaso-constriction, however, can be treated effectively at the level of endothelin-1 converting enzyme (ECE-1) by ECE-1 inhibitors because ECE-1 converts biologically almost inactive big endothelin to endothelin-1 (ET-1), the most potent vasoconstrictor known. The purpose of this study was to clarify the anatomical distribution of ECE-1 in the human eye. Methods. 11 post-mortem eyes were fixed in 4% formalin and embedded in paraffin. 4 m m thin sections were analyzed immuno-histochemically using a self-produced monoclonal primary antibody against human ECE-1 and a polyclonal a actin antibody for comparison. Results. ECE-1 -IR was demonstrated in the corneal epithelium, vascular smooth muscle and endothelial cells, and in the non-vascular smooth muscle cells of the ciliary body, the dilator and sphincter muscle of the iris. Conclusions. A strong immunoreactivity for ECE-1 can be found in the blood vessels of the retina, optic nerve and choroid. Therefore, it should be possible to treat ET-1-induced vaso-constriction in the eye using ECE-1 inhibitors, especially in diseases like hypertensive vasculopathy, vasospasm, vaso-occlusions or low tension glaucoma
Carboxyl-Photo-Reactive MS-Cleavable Cross-Linkers: Unveiling a Hidden Aspect of Diazirine-Based Reagents
No full textA major challenge in cross-linking/mass spectrometry (MS) is targeting carboxyl functions in proteins under physiological conditions that do not disturb the protein's conformation. Cross-linking of glutamic acid and aspartic acid residues in proteins will greatly expand the scope of structural mass spectrometry. We discovered that carboxyl-reactive cross-linkers have already been employed for many years in cross-linking/MS studies, yet in a completely different context. Diazirine-based cross-linkers, such as photomethionine and succinimidyldiazirine cross-linkers, are currently considered to react nonspecifically upon UV-A photoactivation with all 20 proteinogenic amino acids through a reactive carbene that inserts mainly into C-H bonds. We discovered that the cross-linking capability of diazirines based on X-H (X = C, N, O) insertion is in fact only the tip of the iceberg. Diazirines isomerize to linear diazo compounds that can react with carboxylic acids to yield esters. On top of that, the resulting cross-linked products are MS-cleavable allowing an automated analysis of cross-links via customized software tools. Therefore, diazirines open an entirely new route for photo-cross-linking of carboxylic acids. Previous cross-linking studies using diazirines have to be revisited in the light of these findings
The isotope-labeled, MS-cleavable cross-linker disuccinimidyl dibutyric urea for improved cross-linking/mass spectrometry studies
No full textPrevious studies have shown the benefits of the aminereactive, CID-MS/MS-cleavable cross-linker disuccinimidyl dibutyric urea (DSBU) for structural proteomics studies via cross-linking/MS (XL-MS). To further facilitate the automation of XL-MS experiments, we synthesized a deuterated (D12) version of the DSBU cross-linker combining the advantages of MS-cleavable linkers and isotope labeling. The rationale of conducting XL-MS with a mixture of unlabeled and stable isotope-labeled DSBU is to obtain characteristic mass differences at the MS level indicating cross-linked species. These cross-linked species can then be selected for fragmentation by collisional activation. At the MS/MS level, the characteristic 26-u doublets arising from cleavage of the central urea group in DSBU confirm the amino acid sequences of cross-linked peptides as well as the exact cross-linking sites. D12-labeled DSBU was tested on three systems with increasing complexity: (i) bovine serum albumin as purified protein, (ii) Escherichia coli ribosome as large, multimeric protein assembly, and (iii) Drosophila embryo extract as complete proteome. We demonstrate the benefits arising from the use of isotope-labeled DSBU for an automated assignment of cross-linked products. Combining isotope labeling and MS cleavability in one cross-linker resulted in higher cross-link identification numbers especially for highly complex protein mixtures
Going Beyond Counting First Authors in Author Co-citation Analysis
Full text linkThe present study examines one of the fundamental aspects of author co-citation analysis (ACA) - the way co-citation
counts are defined. Co-citation counting provides the data on which all subsequent statistical analyses and mappings
are based, and we compare ACA results based on two different types of co-citation counting - the traditional type that
only counts the first one among a cited work's authors on the one hand and a non-traditional type that takes into
account the first 5 authors of a cited work on the other hand. Results indicate that the picture produced through this non-traditional author co-citation counting contains more coherent author groups and is therefore considerably clearer. However, this picture represents fewer specialties in the research field being studied than that produced through the traditional first-author co-citation counting when the same number of top-ranked authors is selected and analyzed. Reasons for these effects are discussed
Structural analysis of 70S ribosomes by cross-linking/mass spectrometry reveals conformational plasticity
Full text linkThe ribosome is not only a highly complex molecular machine that translates the genetic information into proteins, but also an exceptional specimen for testing and optimizing cross-linking/mass spectrometry (XL-MS) workflows. Due to its high abundance, ribosomal proteins are frequently identified in proteome-wide XL-MS studies of cells or cell extracts. Here, we performed in-depth cross-linking of the E. coli ribosome using the amine-reactive cross-linker disuccinimidyl diacetic urea (DSAU). We analyzed 143 E. coli ribosomal structures, mapping a total of 10,771 intramolecular distances for 126 cross-link-pairs and 3,405 intermolecular distances for 97 protein pairs. Remarkably, 44% of intermolecular cross-links covered regions that have not been resolved in any high-resolution E. coli ribosome structure and point to a plasticity of cross-linked regions. We systematically characterized all cross-links and discovered flexible regions, conformational changes, and stoichiometric variations in bound ribosomal proteins, and ultimately remodeled 2,057 residues (15,794 atoms) in total. Our working model explains more than 95% of all cross-links, resulting in an optimized E. coli ribosome structure based on the cross-linking data obtained. Our study might serve as benchmark for conducting biochemical experiments on newly modeled protein regions, guided by XL-MS. Data are available via ProteomeXchange with identifier PXD018935
The First MS-Cleavable, Photo-Thiol-Reactive Cross-Linker for Protein Structural Studies
No full textCleavable cross-linkers are gaining increasing importance for chemical cross-linking/mass spectrometry (MS) as they permit a reliable and automated data analysis in structural studies of proteins and protein assemblies. Here, we introduce 1,3-diallylurea (DAU) as the first CID-MS/MS-cleavable, photo-thiol-reactive cross-linker. DAU is a commercially available, inexpensive reagent that efficiently undergoes an anti-Markovnikov hydrothiolation with cysteine residues in the presence of a radical initiator upon UV-A irradiation. Radical cysteine cross-linking proceeds via an orthogonal “click reaction” and yields stable alkyl sulfide products. DAU reacts at physiological pH and cross-linking reactions with peptides, and proteins can be performed at temperatures as low as 4 °C. The central urea bond is efficiently cleaved upon collisional activation during tandem MS experiments generating characteristic product ions. This improves the reliability of automated cross-link identification. Different radical initiators have been screened for the cross-linking reaction of DAU using the thiol-containing compounds cysteine and glutathione. Our concept has also been exemplified for the biologically relevant proteins bMunc13-2 and retinal guanylyl cyclase-activating protein-2
Molecular Details of Retinal Guanylyl Cyclase 1/GCAP-2 Interaction
Full text linkThe rod outer segment guanylyl cyclase 1 (ROS-GC1) is an essential component of photo-transduction in the retina. In the light-induced signal cascade, membrane-bound ROS-GC1 restores cGMP levels in the dark in a calcium-dependent manner. With decreasing calcium concentration in the intracellular compartment, ROS-GC1 is activated via the intracellular site by guanylyl cyclase-activating proteins (GCAP-1/-2). Presently, the exact activation mechanism is elusive. To obtain structural insights into the ROS-GC1 regulation by GCAP-2, chemical cross-linking/mass spectrometry studies using GCAP-2 and three ROS-GC1 peptides were performed in the presence and absence of calcium. The majority of cross-links were identified with the C-terminal lobe of GCAP-2 and a peptide comprising parts of ROS-GC1's catalytic domain and C-terminal extension. Consistently with the cross-linking results, surface plasmon resonance and fluorescence measurements confirmed specific binding of this ROS-GC peptide to GCAP-2 with a dissociation constant in the low micromolar range. These results imply that a region of the catalytic domain of ROS-GC1 can participate in the interaction with GCAP-2. Additional binding surfaces upstream of the catalytic domain, in particular the juxtamembrane domain, can currently not be excluded
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