Netherlands Proteomics Center

Utrecht, Netherlands

Netherlands Proteomics Center

Utrecht, Netherlands
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Birkner J.P.,Groningen Biomolecular science and Biotechnology Institute | Poolman B.,Groningen Biomolecular science and Biotechnology Institute | Poolman B.,Netherlands Proteomics Center | Poolman B.,Zernike Institute for Advanced Materials | Kocer A.,Groningen Biomolecular science and Biotechnology Institute
Proceedings of the National Academy of Sciences of the United States of America | Year: 2012

Mechanosensitive (MS) ion channels are membrane proteins that detect and respond to membrane tension in all branches of life. In bacteria, MS channels prevent cells from lysing upon sudden hypoosmotic shock by opening and releasing solutes and water. Despite the importance of MS channels and ongoing efforts to explain their functioning, the molecular mechanism of MS channel gating remains elusive and controversial. Here we report a method that allows single-subunit resolution for manipulating and monitoring "mechanosensitive channel of large conductance" from Escherichia coli. We gradually changed the hydrophobicity of the pore constriction in this homopentameric protein by modifying a critical pore residue one subunit at a time. Our experimental results suggest that both channel opening and closing are initiated by the transmembrane 1 helix of a single subunit and that the participation of each of the five identical subunits in the structural transitions between the closed and open states is asymmetrical. Such a minimal change in the pore environment seems ideal for a fast and energyefficient response to changes in the membrane tension.


Koo B.-K.,University Utrecht | Spit M.,University Utrecht | Jordens I.,University Utrecht | Low T.Y.,University Utrecht | And 10 more authors.
Nature | Year: 2012

LGR5 + stem cells reside at crypt bottoms, intermingled with Paneth cells that provide Wnt, Notch and epidermal growth factor signals. Here we find that the related RNF43 and ZNRF3 transmembrane E3 ubiquitin ligases are uniquely expressed in LGR5 + stem cells. Simultaneous deletion of the two genes encoding these proteins in the intestinal epithelium of mice induces rapidly growing adenomas containing high numbers of Paneth and LGR5 + stem cells. In vitro, growth of organoids derived from these adenomas is arrested when Wnt secretion is inhibited, indicating a dependence of the adenoma stem cells on Wnt produced by adenoma Paneth cells. In the HEK293T human cancer cell line, expression of RNF43 blocks Wnt responses and targets surface-expressed frizzled receptors to lysosomes. In the RNF43-mutant colorectal cancer cell line HCT116, reconstitution of RNF43 expression removes its response to exogenous Wnt. We conclude that RNF43 and ZNRF3 reduce Wnt signals by selectively ubiquitinating frizzled receptors, thereby targeting these Wnt receptors for degradation. © 2012 Macmillan Publishers Limited. All rights reserved.


Kakar K.,University Utrecht | Zhang H.,University Utrecht | Zhang H.,Netherlands Proteomics Center | Scheres B.,University Utrecht | And 4 more authors.
Nature | Year: 2013

Recent evidence indicates a correlation between orientation of the plant cortical microtubule cytoskeleton and localization of polar cargoes. However, the molecules and mechanisms that create this correlation have remained unknown. Here we show that, in Arabidopsis thaliana, the microtubule orientation regulators CLASP and MAP65 (refs 3, 4) control the abundance of polarity regulator PINOID kinase at the plasma membrane. By localized upregulation of clathrin-dependent endocytosis at cortical microtubule-and clathrin-rich domains orthogonal to the axis of polarity, PINOID accelerates the removal of auxin transporter PIN proteins from those sites. This mechanism links directional microtubule organization to the polar localization of auxin transporter PIN proteins, and clarifies how microtubule-enriched cell sides are kept distinct from polar delivery domains. Our results identify the molecular machinery that connects microtubule organization to the regulation of the axis of PIN polarization. © 2013 Macmillan Publishers Limited. All rights reserved.


Liebensteiner M.G.,Wageningen University | Pinkse M.W.H.,Technical University of Delft | Pinkse M.W.H.,Netherlands Proteomics Center | Schaap P.J.,Wageningen University | And 3 more authors.
Science | Year: 2013

Perchlorate and chlorate anions [(per)chlorate] exist in the environment from natural and anthropogenic sources, where they can serve as electron acceptors for bacteria. We performed growth experiments combined with genomic and proteomic analyses of the hyperthermophile Archaeoglobus fulgidus that show (per)chlorate reduction also extends into the archaeal domain of life. The (per)chlorate reduction pathway in A. fulgidus relies on molybdo-enzymes that have similarity with bacterial enzymes; however, chlorite is not enzymatically split into chloride and oxygen. Evidence suggests that it is eliminated by an interplay of abiotic and biotic redox reactions involving sulfur compounds. Biological (per)chlorate reduction by ancient archaea at high temperature may have prevented accumulation of perchlorate in early terrestrial environments and consequently given rise to oxidizing conditions on Earth before the rise of oxygenic photosynthesis.


Snijder J.,University Utrecht | Snijder J.,Netherlands Proteomics Center | Heck A.J.R.,University Utrecht | Heck A.J.R.,Netherlands Proteomics Center
Annual Review of Analytical Chemistry | Year: 2014

Analysis of the size and mass of nanoparticles, whether they are natural biomacromolecular or synthetic supramolecular assemblies, is an important step in the characterization of such molecular species. In recent years, electrospray ionization (ESI) has emerged as a technology through which particles with masses up to 100 MDa can be ionized and transferred into the gas phase, preparing them for accurate mass analysis. Here we review currently used methodologies, with a clear focus on native mass spectrometry (MS). Additional complementary methodologies are also covered, including ion-mobility analysis, nanomechanical mass sensors, and charge-detection MS. The literature discussed clearly demonstrates the great potential of ESI-based methodologies for the size and mass analysis of nanoparticles, including very large naturally occurring protein assemblies. The analytical approaches discussed are powerful tools in not only structural biology, but also nanotechnology. © Copyright ©2014 by Annual Reviews. All rights reserved.


Uetrecht C.,University Utrecht | Uetrecht C.,Netherlands Proteomics Center | Uetrecht C.,Uppsala University | Heck A.J.R.,University Utrecht | Heck A.J.R.,Netherlands Proteomics Center
Angewandte Chemie - International Edition | Year: 2011

Over a century since its development, the analytical technique of mass spectrometry is blooming more than ever, and applied in nearly all aspects of the natural and life sciences. In the last two decades mass spectrometry has also become amenable to the analysis of proteins and even intact protein complexes, and thus begun to make a significant impact in the field of structural biology. In this Review, we describe the emerging role of mass spectrometry, with its different technical facets, in structural biology, focusing especially on structural virology. We describe how mass spectrometry has evolved into a tool that can provide unique structural and functional information about viral-protein and protein-complex structure, conformation, assembly, and topology, extending to the direct analysis of intact virus capsids of several million Dalton in mass. Mass spectrometry is now used to address important questions in virology ranging from how viruses assemble to how they interact with their host. © 2011 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim.


Altelaar A.F.M.,University Utrecht | Altelaar A.F.M.,Netherlands Proteomics Center | Munoz J.,University Utrecht | Munoz J.,Netherlands Proteomics Center | And 3 more authors.
Nature Reviews Genetics | Year: 2013

Next-generation sequencing allows the analysis of genomes, including those representing disease states. However, the causes of most disorders are multifactorial, and systems-level approaches, including the analysis of proteomes, are required for a more comprehensive understanding. The proteome is extremely multifaceted owing to splicing and protein modifications, and this is further amplified by the interconnectivity of proteins into complexes and signalling networks that are highly divergent in time and space. Proteome analysis heavily relies on mass spectrometry (MS). MS-based proteomics is starting to mature and to deliver through a combination of developments in instrumentation, sample preparation and computational analysis. Here we describe this emerging next generation of proteomics and highlight recent applications. © 2013 Macmillan Publishers Limited.


Rosati S.,University Utrecht | Rosati S.,Netherlands Proteomics Center | Yang Y.,University Utrecht | Yang Y.,Netherlands Proteomics Center | And 4 more authors.
Nature Protocols | Year: 2014

The molecular complexity of biopharmaceuticals puts severe demands on the bioanalytical techniques required for their comprehensive structural characterization. Mass spectrometry (MS) has gained importance in the analysis of biopharmaceuticals, taking different complementary approaches ranging from peptide-based sequencing to direct analysis of intact proteins and protein assemblies. In this protocol, we describe procedures optimized to perform the analysis of monoclonal antibodies (mAbs) at the intact protein level under pseudo-native conditions, using native MS. Some of the strengths of native MS in the analysis of biopharmaceuticals are its analysis speed, sensitivity and specificity: for most experiments, the whole protocol requires one working day, whereby tens of samples can be analyzed in a multiplexed manner, making it suitable for high-throughput analysis. This method can be used for different applications such as the analysis of mixtures of mAbs, drug-antibody conjugates and the analysis of mAb post-translational modifications, including the qualitative and quantitative analysis of mAb glycosylation. © 2014 Nature America, Inc. All rights reserved.


Altelaar A.F.M.,University Utrecht | Altelaar A.F.M.,Netherlands Proteomics Center | Heck A.J.R.,University Utrecht | Heck A.J.R.,Netherlands Proteomics Center
Current Opinion in Chemical Biology | Year: 2012

Here we review recent developments and trends in sample preparation, pre-fractionation, chromatography and mass spectrometry contributing towards the ultra-sensitive global analysis of proteins. Highly sensitive MS-based proteomics is not only beneficiary for the proteome analysis of single cells, an aim which is getting into reach, but also clearly relevant for the analysis of (a) subcellular organelles, (b) specific low-abundant cell-types such as adult stem cells, and (c) smaller but more homogeneous cell populations sorted or dissected from (diseased) tissue. © 2011 Elsevier Ltd.


Rose R.J.,University Utrecht | Rose R.J.,Netherlands Proteomics Center | Damoc E.,Thermo Fisher Scientific | Denisov E.,Thermo Fisher Scientific | And 3 more authors.
Nature Methods | Year: 2012

The analysis of intact protein assemblies in native-like states by mass spectrometry offers a wealth of information on their biochemical and biophysical properties. Here we show that the Orbitrap mass analyzer can be used to measure protein assemblies of molecular weights approaching one megadalton with sensitivity down to the detection of single ions. Minor instrumental modifications enabled the measurement of various protein assemblies with outstanding mass-spectral resolution. © 2012 Nature America, Inc. All rights reserved.

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