330 results on '"Bernhard Kuster"'
Search Results
302. Identification of oligosaccharides by matrix-assisted laser desorption ionization and electrospray MS
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Thomas J. P. Naven, David Harvey, and Bernhard Kuster
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Matrix-assisted laser desorption electrospray ionization ,Chromatography ,Glycoside Hydrolases ,Molecular Structure ,Chemistry ,Electrospray ionization ,Molecular Sequence Data ,Extractive electrospray ionization ,Oligosaccharides ,Mass spectrometry ,Biochemistry ,Capillary electrophoresis–mass spectrometry ,Gas Chromatography-Mass Spectrometry ,Surface-enhanced laser desorption/ionization ,Matrix-assisted laser desorption/ionization ,Carbohydrate Sequence ,Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ,Electrospray ms ,Glycoproteins - Published
- 1996
303. Comparison of fragmentation modes for the structural determination of complex oligosaccharides ionized by matrix-assisted laser desorption/ionization mass spectrometry
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Martin R. Green, Bernhard Kuster, Glen Critchley, Robert Harold Bateman, David Harvey, and Thomas J. P. Naven
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Collision-induced dissociation ,Chemistry ,Organic Chemistry ,Molecular Sequence Data ,Analytical chemistry ,Oligosaccharides ,Galactosides ,Thermal ionization mass spectrometry ,Mass spectrometry ,Tandem mass spectrometry ,Soft laser desorption ,Gas Chromatography-Mass Spectrometry ,Analytical Chemistry ,law.invention ,Matrix-assisted laser desorption/ionization ,Fragmentation (mass spectrometry) ,Carbohydrate Sequence ,Reflectron ,law ,Physics::Atomic and Molecular Clusters ,Nuclear Experiment ,Spectroscopy - Abstract
Fragment ions from underivatized N-linked oligosaccharides ionized by matrix-assisted laser desorption/ionization mass spectrometry were obtained by spontaneous fragmentation on a magnetic sector mass spectrometer, by post-source decay (PSD) on a reflectron time-of-flight (TOF) instrument and by collision-induced dissociation on a magnetic sector instrument fitted with an orthogonal-TOF analyser. Spontaneous fragmentation on the magnetic sector instrument produced ions mainly by glycosidic cleavage together with two abundant ions formed by cross-ring cleavage of the reducing-terminal residue. The PSD spectra were similar, the majority of ions being formed by glycosidic cleavage. Internal fragment ions were abundant. High-energy collision-induced dissociation spectra, recorded with the orthogonal-TOF analyser, differed considerably from the other types of spectra, particularly in the appearance of major fragment ions produced by cross-ring cleavages of most of the constituent monosaccharide residues. These ions allowed much sequence and branching information to be obtained from the oligosaccharide.
- Published
- 1995
304. MALDI Imaging Mass Spectrometry for In Situ Proteomic Analysis of Preneoplastic Lesions in Pancreatic Cancer
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Jörg Kleeff, Jens T. Siveke, Axel Walch, Irene Esposito, Roland M. Schmid, Hannes Hahne, Stefan Maier, Bernhard Kuster, Sandra Rauser, Pawel K. Mazur, E Kalideris, Marija Trajkovic-Arsic, Barbara M. Grüner, and Christoph W. Michalski
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Male ,Proteomics ,MALDI imaging ,Pathology ,Mouse ,endocrine system diseases ,Medizin ,Mice ,Gastrointestinal Cancers ,Basic Cancer Research ,Multidisciplinary ,medicine.diagnostic_test ,Chemistry ,Animal Models ,Middle Aged ,medicine.anatomical_structure ,Oncology ,Medicine ,Female ,Pancreas ,Carcinoma, Pancreatic Ductal ,Research Article ,Lung-cancer ,Cells ,Tumors ,medicine.medical_specialty ,Science ,Mice, Transgenic ,Gastroenterology and Hepatology ,Mass spectrometry imaging ,Pancreatic Cancer ,Model Organisms ,Western blot ,Pancreatic cancer ,Gastrointestinal Tumors ,Biomarkers, Tumor ,Cancer Detection and Diagnosis ,medicine ,Animals ,Humans ,Biology ,Aged ,Intraductal papillary mucinous neoplasm ,Cancers and Neoplasms ,medicine.disease ,Pancreatic Neoplasms ,Matrix-assisted laser desorption/ionization ,Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization - Abstract
The identification of new biomarkers for preneoplastic pancreatic lesions (PanINs, IPMNs) and early pancreatic ductal adenocarcinoma (PDAC) is crucial due to the diseasés high mortality rate upon late detection. To address this task we used the novel technique of matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) on genetically engineered mouse models (GEM) of pancreatic cancer. Various GEM were analyzed with MALDI IMS to investigate the peptide/protein-expression pattern of precursor lesions in comparison to normal pancreas and PDAC with cellular resolution. Statistical analysis revealed several discriminative m/z-species between normal and diseased tissue. Intraepithelial neoplasia (PanIN) and intraductal papillary mucinous neoplasm (IPMN) could be distinguished from normal pancreatic tissue and PDAC by 26 significant m/z-species. Among these m/z-species, we identified Albumin and Thymosin-beta 4 by liquid chromatography and tandem mass spectrometry (LC-MS/MS), which were further validated by immunohistochemistry, western blot, quantitative RT-PCR and ELISA in both murine and human tissue. Thymosin-beta 4 was found significantly increased in sera of mice with PanIN lesions. Upregulated PanIN expression of Albumin was accompanied by increased expression of liver-restricted genes suggesting a hepatic transdifferentiation program of preneoplastic cells. In conclusion we show that GEM of endogenous PDAC are a suitable model system for MALDI-IMS and subsequent LC-MS/MS analysis, allowing in situ analysis of small precursor lesions and identification of differentially expressed peptides and proteins. © 2012 Grüner et al.
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- 2012
305. Systematic Identification of the HSP90 Regulated Proteome
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Amin Moghaddas Gholami, Bernhard Kuster, and Zhixiang Wu
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Kinase ,Biology ,Geldanamycin ,Biochemistry ,ISG15 ,Hsp90 ,Analytical Chemistry ,chemistry.chemical_compound ,chemistry ,Unfolded protein binding ,Proteome ,polycyclic compounds ,biology.protein ,Signal transduction ,Protein kinase A ,Molecular Biology - Abstract
HSP90 is a central player in the folding and maturation of many proteins. More than two hundred HSP90 clients have been identified by classical biochemical techniques including important signaling proteins with high relevance to human cancer pathways. HSP90 inhibition has thus become an attractive therapeutic concept and multiple molecules are currently in clinical trials. It is therefore of fundamental biological and medical importance to identify, ideally, all HSP90 clients and HSP90 regulated proteins. To this end, we have taken a global and a chemical proteomic approach in geldanamycin treated cancer cell lines using stable isotope labeling with amino acids in cell culture and quantitative mass spectrometry. We identified >6200 proteins in four different human cell lines and ∼1600 proteins showed significant regulation upon drug treatment. Gene ontology and pathway/network analysis revealed common and cell-type specific regulatory effects with strong connections to unfolded protein binding and protein kinase activity. Of the 288 identified protein kinases, 98 were downregulated upon geldanamycin treatment including >50 kinases not formerly known to be regulated by HSP90. Protein turn-over measurements using pulsed stable isotope labeling with amino acids in cell culture showed that protein down-regulation by HSP90 inhibition correlates with protein half-life in many cases. Protein kinases show significantly shorter half lives than other proteins highlighting both challenges and opportunities for HSP90 inhibition in cancer therapy. The proteomic responses of the HSP90 drugs geldanamycin and PU-H71 were highly similar suggesting that both drugs work by similar molecular mechanisms. Using HSP90 immunoprecipitation, we validated several kinases (AXL, DDR1, TRIO) and other signaling proteins (BIRC6, ISG15, FLII), as novel clients of HSP90. Taken together, our study broadly defines the cellular proteome response to HSP90 inhibition and provides a rich resource for further investigation relevant for the treatment of cancer.
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- 2012
306. 1094 Probiotic Structure Function Analysis Reveals Prtp-Encoded Lactocepin to Mediate Anti-Inflammatory Effects via Selective Degradation of PRO-Inflammatory Chemokines
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Gaspar Pérez Martínez, Tomas Hrncir, Hannes Hahne, Lothar Steidler, Christine Bäuerl, Gabriele Hörmannsperger, Bernhard Kuster, Monika Weiher, Dirk Haller, Carl-Alfred Alpert, Marie-Anne von Schillde, and Karolien van Huynegem
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Chemokine ,Hepatology ,biology ,medicine.drug_class ,Chemistry ,Structure function ,Gastroenterology ,Anti-inflammatory ,law.invention ,Microbiology ,Probiotic ,Selective degradation ,law ,medicine ,biology.protein ,Lactocepin - Published
- 2012
307. Quantitative Chemical Proteomics Reveals New Potential Drug Targets in Head and Neck Cancer
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Reidar Grénman, Stephan M. Feller, Simone Lemeer, Axel Walch, Bernhard Kuster, Jessica Doondeea, Amin Moghaddas Gholami, Susanne M. Gollin, Karl Kramer, Zhixiang Wu, M. Janning, and Suzanne A. Eccles
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Proteomics ,SQUAMOUS-CELL CARCINOMA ,GROWTH-FACTOR RECEPTOR ,MASS-SPECTROMETRY ,TYROSINE KINASE ,RADIATION SENSITIVITY ,THERAPEUTIC TARGET ,ANTITUMOR-ACTIVITY ,EPHA2 RECEPTOR ,LUNG-CANCER ,IN-VITRO ,Cell Survival ,Cell Cycle Proteins ,Protein Serine-Threonine Kinases ,Lapatinib ,Biochemistry ,Analytical Chemistry ,Gefitinib ,Receptor-Interacting Protein Serine-Threonine Kinase 2 ,Cell Line, Tumor ,medicine ,Humans ,NIMA-Related Kinases ,Molecular Targeted Therapy ,Protein Kinase Inhibitors ,Molecular Biology ,biology ,Kinase ,Receptor, EphA2 ,Research ,Carcinoma ,Nuclear Proteins ,Cancer ,Janus Kinase 1 ,Protein-Tyrosine Kinases ,Proto-Oncogene Proteins c-met ,medicine.disease ,Head and neck squamous-cell carcinoma ,Tongue Neoplasms ,ErbB Receptors ,Dasatinib ,Wee1 ,src-Family Kinases ,Tissue Array Analysis ,Gene Knockdown Techniques ,Cancer research ,biology.protein ,RNA Interference ,Erlotinib ,Protein Kinases ,medicine.drug - Abstract
Tumors of the head and neck represent a molecularly diverse set of human cancers, but relatively few proteins have actually been shown to drive the disease at the molecular level. In order to identify new targets for individualized diagnosis or therapeutic intervention, we performed a kinase centric chemical proteomics screen and quantified 146 kinases across 34 head and neck squamous cell carcinoma (HNSCC) cell lines using intensity-based label-free mass spectrometry. Statistical analysis of the profiles revealed significant inter-cell line differences for 42 kinases (p
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- 2011
308. O2-03-05 Proteomic analysis of amyloid precursor protein processing complexes in human neuroblastoma cells by tandem affinity purification
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Adele Rowley, Pierre-Olivier Angrand, Martin Stein, Eithne O'Sullivan, Jyoti S. Choudhary, Susie Thomas, Pamela Völkel, Brian Heffernan, Bernhard Kuster, Julien Gagneur, Gerard Drewes, Heinz Ruffner, Carsten Hopf, Lisa Pickard, and Raffaella Mangano
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Neuroblastoma cell ,Tandem affinity purification ,Aging ,biology ,Chemistry ,General Neuroscience ,Amyloid precursor protein ,biology.protein ,Neurology (clinical) ,Geriatrics and Gerontology ,Molecular biology ,Developmental Biology - Published
- 2004
309. [Untitled]
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Toby J. Gibson, Rune Linding, Scott Cameron, Bernhard Kuster, Nikolaj Blom, Thomas Sicheritz-Pontén, Francesca Diella, Christine Gemünd, and Allegra Via
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inorganic chemicals ,Database ,Kinase ,Applied Mathematics ,Biology ,computer.software_genre ,environment and public health ,Biochemistry ,Computer Science Applications ,Post-transcriptional modification ,Eukaryotic Linear Motif resource ,enzymes and coenzymes (carbohydrates) ,chemistry.chemical_compound ,chemistry ,Structural Biology ,Phosphoserine ,bacteria ,Phosphorylation ,Protein phosphorylation ,Signal transduction ,Protein kinase A ,Molecular Biology ,computer - Abstract
Post-translational phosphorylation is one of the most common protein modifications. Phosphoserine, threonine and tyrosine residues play critical roles in the regulation of many cellular processes. The fast growing number of research reports on protein phosphorylation points to a general need for an accurate database dedicated to phosphorylation to provide easily retrievable information on phosphoproteins. Phospho.ELM http://phospho.elm.eu.org is a new resource containing experimentally verified phosphorylation sites manually curated from the literature and is developed as part of the ELM (Eukaryotic Linear Motif) resource. Phospho.ELM constitutes the largest searchable collection of phosphorylation sites available to the research community. The Phospho.ELM entries store information about substrate proteins with the exact positions of residues known to be phosphorylated by cellular kinases. Additional annotation includes literature references, subcellular compartment, tissue distribution, and information about the signaling pathways involved as well as links to the molecular interaction database MINT. Phospho.ELM version 2.0 contains 1703 phosphorylation site instances for 556 phosphorylated proteins. Phospho.ELM will be a valuable tool both for molecular biologists working on protein phosphorylation sites and for bioinformaticians developing computational predictions on the specificity of phosphorylation reactions.
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- 2004
310. The gut microbiota promotes hepatic fatty acid desaturation and elongation in mice
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Alida Kindt, Gerhard Liebisch, Thomas Clavel, Dirk Haller, Gabriele Hörmannsperger, Hongsup Yoon, Daniela Kolmeder, Alexander Sigruener, Sabrina Krautbauer, Claudine Seeliger, Alexandra Ganzha, Sabine Schweizer, Rosalie Morisset, Till Strowig, Hannelore Daniel, Dominic Helm, Bernhard Küster, Jan Krumsiek, and Josef Ecker
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Science - Abstract
The role of the gut microbiota in hepatic lipid metabolism is controversial and incompletely understood. Here the authors perform multi-omics analyses of altered lipid metabolic processes in germ-free and specific pathogen-free mice, revealing how the gut microbiota affects hepatic fatty acid desaturation and elongation.
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- 2018
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311. Sucrose-Induced Proteomic Response and Carbohydrate Utilization of Lactobacillus sakei TMW 1.411 During Dextran Formation
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Roman M. Prechtl, Dorothee Janßen, Jürgen Behr, Christina Ludwig, Bernhard Küster, Rudi F. Vogel, and Frank Jakob
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Lactobacillus sakei ,proteomics ,genomics ,sucrose ,dextran ,metabolism ,Microbiology ,QR1-502 - Abstract
Lactobacillus (L.) sakei belongs to the dominating lactic acid bacteria in indigenous meat fermentations, while diverse strains of this species have also been isolated from plant fermentations. We could recently show, that L. sakei TMW 1.411 produces a high molecular weight dextran from sucrose, indicating its potential use as a dextran forming starter culture. However, the general physiological response of L. sakei to sucrose as carbohydrate source has not been investigated yet, especially upon simultaneous dextran formation. To address this lack of knowledge, we sequenced the genome of L. sakei TMW 1.411 and performed a label-free, quantitative proteomics approach to investigate the sucrose-induced changes in the proteomic profile of this strain in comparison to its proteomic response to glucose. In total, 21 proteins were found to be differentially expressed at the applied significance criteria (FDR ≤ 0.01). Among these, 14 were associated with the carbohydrate metabolism including several enzymes, which enable sucrose and fructose uptake, as well as, their subsequent intracellular metabolization, respectively. The plasmid-encoded, extracellular dextransucrase of L. sakei TMW 1.411 was expressed at high levels irrespective of the present carbohydrate and was predominantly responsible for sucrose consumption in growth experiments using sucrose as sole carbohydrate source, while the released fructose from the dextransucrase reaction was more preferably taken up and intracellularly metabolized than sucrose. Genomic comparisons revealed, that operons coding for uptake and intracellular metabolism of sucrose and fructose are chromosomally conserved among L. sakei, while plasmid-located dextransucrase genes are present only in few strains. In accordance with these findings, all 59 different L. sakei strains of our strain collection were able to grow on sucrose as sole carbohydrate source, while eight of them exhibited a mucous phenotype on agar plates indicating dextran formation from sucrose. Our study therefore highlights the intrinsic adaption of L. sakei to plant environments, where sucrose is abundant, and provides fundamental knowledge regarding the use of L. sakei as starter culture for sucrose-based food fermentation processes with in-situ dextran formation.
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- 2018
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312. Preparation of 5-Hydroxymethylfurfural Part I. Dehydration of Fructose in a Continuous Stirred Tank Reactor
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H. J. C. Der Van Steen and Bernhard Kuster
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Chromatography ,Organic Chemistry ,Extraction (chemistry) ,Continuous stirred-tank reactor ,Fructose ,Residence time (fluid dynamics) ,medicine.disease ,chemistry.chemical_compound ,chemistry ,5-hydroxymethylfurfural ,Scientific method ,medicine ,Dehydration ,Food Science - Abstract
A continuous process for the manufacture of 5-hydroxy methylfurfural (HMF), using a stirred tank reactor with simultaneous reaction and extraction was studied. Methylisobutylketone (MIBK) was used as the extraction agent. The experiments were carried out at elevated temperatures and moderate acidity. Other process parameters were the initial concentration of D-fructose, the residence time, and the ratio of MIBK and H2O. Though the effect of extraction was limited due to the fact that the formation of HMF was faster than its breakdown, the use of MIBK as a cosolvent appeared to have a beneficial influence on the course of the reaction. The productivity at short residence times is lowered due to the stability of intermediate products.
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- 1977
313. PAS-cal: a Generic Recombinant Peptide Calibration Standard for Mass Spectrometry
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Joscha Breibeck, Andreas J. Reichert, Stefan Maier, Adam Serafin, Arne Skerra, and Bernhard Kuster
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Spectrometry, Mass, Electrospray Ionization ,Protein mass spectrometry ,Protein Conformation ,Recombinant Fusion Proteins ,Electrospray ionization ,Amino Acid Motifs ,Peptide ,Recombinant polypeptide ,Protein Engineering ,Mass spectrometry ,Protease cleavage ,Protein structure ,Structural Biology ,Endopeptidases ,medicine ,Trypsin ,Spectroscopy ,chemistry.chemical_classification ,Chromatography ,Peptide sequence tag ,Reference Standards ,Peptide Fragments ,Recombinant Proteins ,Molecular Weight ,Matrix-assisted laser desorption/ionization ,PASylation ,chemistry ,Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ,Calibration ,Proteolysis ,Peptides ,Hydrophobic and Hydrophilic Interactions ,Research Article ,medicine.drug - Abstract
We describe the design, preparation, and mass-spectrometric characterization of a new recombinant peptide calibration standard with uniform biophysical and ionization characteristics for mass spectrometry. “PAS-cal” is an artificial polypeptide concatamer of peptide cassettes with varying lengths, each composed of the three small, chemically stable amino acids Pro, Ala, and Ser, which are interspersed by Arg residues to allow site-specific cleavage with trypsin. PAS-cal is expressed at high yields in Escherichia coli as a Small Ubiquitin-like MOdifier (SUMO) fusion protein, which is easily purified and allows isolation of the PAS-cal moiety after SUMO protease cleavage. Upon subsequent in situ treatment with trypsin, the PAS-cal polypeptide yields a set of four defined homogeneous peptides in the range from 2 to 8 kDa with equal mass spacing. ESI-MS analysis revealed a conveniently interpretable raw spectrum, which after deconvolution resulted in a very simple pattern of four peaks with similar ionization signals. MALDI-MS analysis of a PAS-cal peptide mixture comprising both the intact polypeptide and its tryptic fragments revealed not only the four standard peptides but also the singly and doubly charged states of the intact concatamer as well as di- and trimeric adduct ion species between the peptides, thus augmenting the observable m/z range. The advantageous properties of PAS-cal are most likely a result of the strongly hydrophilic and conformationally disordered PEG-like properties of the PAS sequences. Therefore, PAS-cal offers an inexpensive and versatile recombinant peptide calibration standard for mass spectrometry in protein/peptide bioanalytics and proteomics research, the composition of which may be further adapted to fit individual needs. Figure ᅟ
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314. Universal Spectrum Explorer: A Standalone (Web-)Application for Cross-Resource Spectrum Comparison
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Mathias Wilhelm, Eric W. Deutsch, Tobias Kockmann, Yasset Perez-Riverol, Patroklos Samaras, Christian Panse, Tobias Schmidt, Leon Bichmann, Bernhard Kuster, Viktoria Dorfer, Bart Van Puyvelde, University of Zurich, and Wilhelm, Mathias
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0301 basic medicine ,1303 Biochemistry ,Computer science ,Interface (computing) ,610 Medicine & health ,10071 Functional Genomics Center Zurich ,1600 General Chemistry ,Peptide ,Biochemistry ,01 natural sciences ,Tandem mass spectrum ,Vector graphics ,03 medical and health sciences ,Software ,Tandem Mass Spectrometry ,Web application ,030304 developmental biology ,chemistry.chemical_classification ,0303 health sciences ,Internet ,Information retrieval ,030102 biochemistry & molecular biology ,business.industry ,010401 analytical chemistry ,General Chemistry ,Visualization ,0104 chemical sciences ,Identifier ,030104 developmental biology ,chemistry ,Mass spectrum ,570 Life sciences ,biology ,Table (database) ,The Internet ,business ,Peptides - Abstract
Here we present the Universal Spectrum Explorer (USE), a web-based tool based on IPSA for cross-resource (peptide) spectrum visualization and comparison (https://www.proteomicsdb.org/use/). Mass spectra under investigation can either be provided manually by the user (table format), or automatically retrieved from online repositories supporting access to spectral data via the universal spectrum identifier (USI), or requested from other resources and services implementing a newly designed REST interface. As a proof of principle, we implemented such an interface in ProteomicsDB thereby allowing the retrieval of spectra acquired within the ProteomeTools project or real-time prediction of tandem mass spectra from the deep learning framework Prosit. Annotated mirror spectrum plots can be exported from the USE as editable scalable high quality vector graphics. The USE was designed and implemented with minimal external dependencies allowing local usage and integration into other websites (https://github.com/kusterlab/universal_spectrum_explorer).
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315. Mass Spectrometry-Based Proteomics in Preclinical Drug Discovery
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Bernhard Kuster, Markus Schirle, and Marcus Bantscheff
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Drug ,Proteomics ,Drug-Related Side Effects and Adverse Reactions ,media_common.quotation_subject ,Clinical Biochemistry ,Drug Evaluation, Preclinical ,Computational biology ,Biology ,Bioinformatics ,Biochemistry ,Mass Spectrometry ,Protein Interaction Mapping ,Drug Discovery ,Animals ,Molecular Biology ,media_common ,Pharmacology ,Mass spectrometry based proteomics ,Drug discovery ,Proteins ,General Medicine ,Pharmaceutical Preparations ,Proteins metabolism ,Molecular Medicine ,Identification (biology) ,Biomarkers ,Protein Binding - Abstract
Preclinical stages in the drug discovery process require a multitude of biochemical and genetic assays in order to characterize the effects of drug candidates on cellular systems and model organisms. Early attempts to apply unbiased proteomic techniques to the identification of protein targets and off-targets as well as to elucidate the mode of action of candidate drug molecules suffered from a striking discrepancy between scientific expectations and what the technology was able to deliver at the time. Dramatic technological improvements in mass spectrometry-based proteomic and chemoproteomic strategies have radically changed this situation. This review, therefore, highlights proteomic approaches suitable for preclinical drug discovery illustrated by recent success stories.
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316. Defining the carrier proteome limit for single-cell proteomics
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Chien-Yun Lee, Florian P. Bayer, Christopher M. Rose, Bernhard Kuster, Tommy K. Cheung, and Atticus McCoy
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0303 health sciences ,Computer science ,Software tool ,Cell Biology ,Computational biology ,Mass spectrometry ,Proteomics ,Biochemistry ,Multiplexing ,03 medical and health sciences ,Isobaric labeling ,Data quality ,Proteome ,Limit (mathematics) ,Molecular Biology ,030304 developmental biology ,Biotechnology - Abstract
Single-cell proteomics by mass spectrometry (SCoPE-MS) is a recently introduced method to quantify multiplexed single-cell proteomes. While this technique has generated great excitement, the underlying technologies (isobaric labeling and mass spectrometry (MS)) have technical limitations with the potential to affect data quality and biological interpretation. These limitations are particularly relevant when a carrier proteome, a sample added at 25-500× the amount of a single-cell proteome, is used to enable peptide identifications. Here we perform controlled experiments with increasing carrier proteome amounts and evaluate quantitative accuracy, as it relates to mass analyzer dynamic range, multiplexing level and number of ions sampled. We demonstrate that an increase in carrier proteome level requires a concomitant increase in the number of ions sampled to maintain quantitative accuracy. Lastly, we introduce Single-Cell Proteomics Companion (SCPCompanion), a software tool that enables rapid evaluation of single-cell proteomic data and recommends instrument and data analysis parameters for improved data quality.
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317. Two serines in the distal C-terminus of the human ß1-adrenoceptor determine ß-arrestin2 recruitment.
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Laura Hinz, Andrea Ahles, Benjamin Ruprecht, Bernhard Küster, and Stefan Engelhardt
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Medicine ,Science - Abstract
G protein-coupled receptors (GPCRs) undergo phosphorylation at several intracellular residues by G protein-coupled receptor kinases. The resulting phosphorylation pattern triggers arrestin recruitment and receptor desensitization. The exact sites of phosphorylation and their function remained largely unknown for the human β1-adrenoceptor (ADRB1), a key GPCR in adrenergic signal transduction and the target of widely used drugs such as β-blockers. The present study aimed to identify the intracellular phosphorylation sites in the ADRB1 and to delineate their function. The human ADRB1 was expressed in HEK293 cells and its phosphorylation pattern was determined by mass spectrometric analysis before and after stimulation with a receptor agonist. We identified a total of eight phosphorylation sites in the receptor's third intracellular loop and C-terminus. Analyzing the functional relevance of individual sites using phosphosite-deficient receptor mutants we found phosphorylation of the ADRB1 at Ser461/Ser462 in the distal part of the C-terminus to determine β-arrestin2 recruitment and receptor internalization. Our data reveal the phosphorylation pattern of the human ADRB1 and the site that mediates recruitment of β-arrestin2.
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- 2017
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318. Design, Synthesis, and Evaluation of WD-Repeat-Containing Protein 5 (WDR5) Degraders
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Andreas Krämer, Elmar Wolf, Frank Löhr, Dalia Barsyte-Lovejoy, Martin Eilers, Mathias Diebold, Magdalena M. Szewczyk, Christoph A. Sotriffer, Cheryl H. Arrowsmith, Lena-Marie Berger, Stephanie Heinzlmeir, Jakob Gebel, Bernhard Kuster, Volker Dötsch, Stefan Knapp, Bikash Adhikari, Anja Dölle, Janik Weckesser, and Nicola Berner
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Male ,Scaffold protein ,Dihydropyridines ,Antineoplastic Agents ,Ligands ,01 natural sciences ,Structure-Activity Relationship ,03 medical and health sciences ,Drug Discovery ,Humans ,WDR5 ,Gene ,Transcription factor ,Cells, Cultured ,030304 developmental biology ,0303 health sciences ,Dose-Response Relationship, Drug ,Molecular Structure ,biology ,Chemistry ,WD Repeat-Containing Protein 5 ,Biphenyl Compounds ,Intracellular Signaling Peptides and Proteins ,0104 chemical sciences ,Cell biology ,010404 medicinal & biomolecular chemistry ,Histone ,Drug Design ,Histone methyltransferase ,biology.protein ,Molecular Medicine ,Female ,Linker - Abstract
Histone H3K4 methylation serves as a post-translational hallmark of actively transcribed genes and is introduced by histone methyltransferase (HMT) and its regulatory scaffolding proteins. One of these is the WD-repeat-containing protein 5 (WDR5) that has also been associated with controlling long noncoding RNAs and transcription factors including MYC. The wide influence of dysfunctional HMT complexes and the typically upregulated MYC levels in diverse tumor types suggested WDR5 as an attractive drug target. Indeed, protein-protein interface inhibitors for two protein interaction interfaces on WDR5 have been developed. While such compounds only inhibit a subset of WDR5 interactions, chemically induced proteasomal degradation of WDR5 might represent an elegant way to target all oncogenic functions. This study presents the design, synthesis, and evaluation of two diverse WDR5 degrader series based on two WIN site binding scaffolds and shows that linker nature and length strongly influence degradation efficacy.
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319. Identification of oligosaccharides by matrix-assisted laser desorption ionization and electrospray MS
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Harvey, D. J., Naven, T. J. P., and Bernhard Kuster
320. Generating high quality libraries for DIA MS with empirically corrected peptide predictions
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Tobias Schmidt, Bernhard Kuster, Siegfried Gessulat, Kristian E. Swearingen, Brian C. Searle, Chris Barnes, and Mathias Wilhelm
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Proteomics ,0301 basic medicine ,Empirical data ,Proteomics methods ,Proteome ,Computer science ,Science ,General Physics and Astronomy ,Computational biology ,Proteome informatics ,Article ,General Biochemistry, Genetics and Molecular Biology ,Workflow ,Market fragmentation ,03 medical and health sciences ,Peptide Library ,Tandem Mass Spectrometry ,parasitic diseases ,Humans ,lcsh:Science ,Databases, Protein ,Peptide library ,Multidisciplinary ,030102 biochemistry & molecular biology ,Extramural ,Reproducibility of Results ,General Chemistry ,Non-model organisms ,3. Good health ,030104 developmental biology ,lcsh:Q ,Peptides ,Retention time ,Algorithms ,Chromatography, Liquid ,HeLa Cells - Abstract
Data-independent acquisition approaches typically rely on experiment-specific spectrum libraries, requiring offline fractionation and tens to hundreds of injections. We demonstrate a library generation workflow that leverages fragmentation and retention time prediction to build libraries containing every peptide in a proteome, and then refines those libraries with empirical data. Our method specifically enables rapid, experiment-specific library generation for non-model organisms, which we demonstrate using the malaria parasite Plasmodium falciparum, and non-canonical databases, which we show by detecting missense variants in HeLa., Data-independent acquisition-mass spectrometry (MS) typically requires many preparatory MS runs to produce experiment-specific spectral libraries. Here, the authors show that empirical correction of in silico predicted spectral libraries enables efficient generation of high-quality experiment-specific libraries.
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321. Femtomol sensitivity post-digest 18O labeling for relative quantification of differential protein complex composition
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Marcus Bantscheff, Birgit Dümpelfeld, and Bernhard Kuster
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Chromatography ,Chemistry ,Microchemistry ,Sample (material) ,Absolute quantification ,Organic Chemistry ,Proteins ,Reproducibility of Results ,Computational biology ,Oxygen Isotopes ,Composition (combinatorics) ,Mass spectrometry ,Sensitivity and Specificity ,Analytical Chemistry ,Label-free quantification ,Isotope Labeling ,Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ,Nanotechnology ,Protein identification ,Sensitivity (control systems) ,Spectroscopy ,Chemical labeling - Abstract
Stable isotope labeling (SIL) has emerged as a powerful tool to measure the relative quantitative differences between samples in many differential display-type proteomic applications. However, current SIL procedures tend to suffer from the fact that one needs to decide very early in a biochemical strategy whether or not a sample will be subjected to relative quantification. Typically, the entire strategy has to be adapted to the needs of the particular quantification method chosen which might limit the range of biochemical experiments amenable to quantification. Metabolic labeling approaches, albeit very sensitive, can only be applied to studies using appropriate cell culture systems which might not necessarily be compatible with the biological system under investigation. Chemical labeling of complex protein mixtures by, e.g., isotope-coded affinity tags (ICAT), can offer great simplification of protein mixtures but is restricted by the accessibility of the often few suitable peptides (i.e. cysteine containing peptides) for both protein identification and quantification. Here, we describe a post-digest 18O-labeling method that can circumvent some of the above limitations by separating protein identification from quantification. An aliquot of all samples in a set can be used for rapid protein ID using, e.g., matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOFMS). In a second step, relative quantification is performed using trypsin-catalyzed 18O incorporation into all tryptic peptides. This two-stage procedure introduces significant experimental flexibility because it enables postponement of the decision about which pairs of samples from a given set of experiments are to be compared until after the protein ID stage. In-gel digested protein quantities between 50 fmol and 15 pmol are amenable to this new method, with a dynamic range of 1:10 within one sample. Accuracy for measured relative abundances is similar to those reported for other SIL strategies (errors typically
322. Ammonium containing buffers should be avoided during enzymatic release of glycans from glycoproteins when followed by reducing terminal derivatization
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Bernhard Kuster and Harvey, D. J.
323. Functional organization of a eukaryotic proteome by systematic analysis of protein complexes
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Gavin, A. -C, Krause, R., Bösche, M., Grandi, P., Bauer, A., Schulz, J., Rick, J., Bork, P., Seraphin, B., Neubauer, G., Superti-Furga, G., and Bernhard Kuster
324. Quantification and discovery of sequence determinants of protein per mRNA amount in 29 human tissues
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Thomas Wieland, Fredrik Pontén, Thomas A. Hopf, Hannes Hahne, Bernhard Kuster, Holger Prokisch, Basak Eraslan, Mirjana Gusic, Julien Gagneur, Mathias Uhlén, Dongxue Wang, Björn M. Hallström, and Anna Asplund
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Untranslated region ,0303 health sciences ,Messenger RNA ,RNA ,Computational biology ,Biology ,03 medical and health sciences ,0302 clinical medicine ,Protein sequencing ,Proteome ,Protein biosynthesis ,Human genome ,Gene ,030217 neurology & neurosurgery ,030304 developmental biology - Abstract
Despite their importance in determining protein abundance, a comprehensive catalogue of sequence features controlling protein-to-mRNA (PTR) ratios and a quantification of their effects is still lacking. Here we quantified PTR ratios for 11,575 proteins across 29 human tissues using matched transcriptomes and proteomes. We analyzed the contribution of known sequence determinants of protein synthesis and degradation and 15 novel mRNA and protein sequence motifs that we found by association testing. While the dynamic range of PTR ratios spans more than 2 orders of magnitude, our integrative model predicts PTR ratios at a median precision of 3.2-fold. A reporter assay provided significant functional support for two novel UTR motifs and a proteome-wide competition-binding assay identified motif-specific bound proteins for one motif. Moreover, our direct comparison of protein to RNA levels led to a new metrics of codon optimality. Altogether, this study shows that a large fraction of PTR ratio variance across genes can be predicted from sequence and identified many new candidate post-transcriptional regulatory elements in the human genome.
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325. Plant cytokinesis is orchestrated by the sequential action of the TRAPPII and exocyst tethering complexes
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Eva Facher, Viktor Zarsky, Gerhard Wanner, Staffan Persson, Alexander Steiner, Bernhard Kuster, Ivan Kulich, Lukáš Synek, Susan Klaeger, Farhah F. Assaad, and Katarzyna Rybak
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Models, Molecular ,0106 biological sciences ,Arabidopsis ,Vesicular Transport Proteins ,Exocyst ,Biology ,Microtubules ,01 natural sciences ,Exocytosis ,General Biochemistry, Genetics and Molecular Biology ,03 medical and health sciences ,Microtubule ,Compartment (development) ,Growth Plate ,Molecular Biology ,Cytokinesis ,030304 developmental biology ,0303 health sciences ,Arabidopsis Proteins ,Cytoplasmic Vesicles ,Cell Biology ,Cell plate ,Cell biology ,Membrane protein ,Biogenesis ,010606 plant biology & botany ,Developmental Biology - Abstract
SummaryPlant cytokinesis is initiated in a transient membrane compartment, the cell plate, and completed by a process of maturation during which the cell plate becomes a cross wall. How the transition from juvenile to adult stages occurs is poorly understood. In this study, we monitor the Arabidopsis transport protein particle II (TRAPPII) and exocyst tethering complexes throughout cytokinesis. We show that their appearance is predominantly sequential, with brief overlap at the onset and end of cytokinesis. The TRAPPII complex is required for cell plate biogenesis, and the exocyst is required for cell plate maturation. The TRAPPII complex sorts plasma membrane proteins, including exocyst subunits, at the cell plate throughout cytokinesis. We show that the two tethering complexes physically interact and propose that their coordinated action may orchestrate not only plant but also animal cytokinesis.
326. The emerging landscape of single-molecule protein sequencing technologies
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Patroklos Samaras, Cecil J Howard, Aleksei Aksimentiev, Amit Meller, Derek Stein, Mike Filius, Chan Cao, Rienk Eelkema, Georges Bedran, Adam Pomorski, David R. Goodlett, Zvi Kelman, Nicholas Drachman, Stuart Lindsay, Meni Wanunu, Sonja Schmid, Chirlmin Joo, Etienne Coyaud, Peggy R. Bohländer, Neil L. Kelleher, Giovanni Maglia, Ryan T. Kelly, Michael Mayer, Umesh Kalathiya, Edward M. Marcotte, Eric V. Anslyn, Mingjie Dai, Shilo Ohayon, Kumar Sarthak, Peng Yin, Sebastien Hentz, Sung Hyun Kim, Mauro Chinappi, Javier A. Alfaro, Gunnar Dittmar, John P. Marino, Christophe Masselon, Bernhard Kuster, David Rodriguez-Larrea, Mathias Wilhelm, Xander F. van Kooten, Cees Dekker, Lusia Sepiashvili, Swiss National Science Foundation, National Institutes of Health (US), Welch Foundation, US Army Research Office, Erisyon, National Institute of General Medical Sciences (US), Wyss Institute of Biologically Inspired Engineering, Harvard Medical School, Peter and Traudl Engelhorn Foundation, Netherlands Organization for Scientific Research, Azrieli Foundation, Paul G. Allen Family Foundation, National Science Foundation (US), Human Frontier Science Program, Polish National Agency for Academic Exchange, European Commission, European Research Council, Fonds National de la Recherche Luxembourg, Adolphe Merkle Foundation, Michael J. Fox Foundation for Parkinson's Research, Swiss National Supercomputing Centre, Foundation for Polish Science, Genome British Columbia, Genome Canada, Alfaro, Javier Antonio, Dai, Mingjie, Filius, Mike, Pomorski, Adam, Schmid, Sonja, Aksimentiev, Aleksei, Anslyn, Eric V., Cao, Chan, Hentz, Sébastien, Kalathiya, Umesh, Kelleher, Neil L., Kuster, Bernhard, Rodríguez-Larrea, David, Maglia, Giovanni, Marino, John P., Masselon, Christophe, Samaras, Patroklos, Stein, Derek, Wilhelm, Mathias, Yin, Peng, Meller, Amit, Joo, Chirlmin, University of Gdańsk (UG), Delft University of Technology (TU Delft), Harvard University, Harvard Medical School [Boston] (HMS), University of Texas at Austin [Austin], Technion - Israel Institute of Technology [Haifa], Wageningen University and Research [Wageningen] (WUR), University of Illinois at Urbana-Champaign [Urbana], University of Illinois System, Ecole Polytechnique Fédérale de Lausanne (EPFL), Università degli Studi di Roma Tor Vergata [Roma], CHU Lille, Protéomique, Réponse Inflammatoire, Spectrométrie de Masse (PRISM) - U 1192 (PRISM), Institut National de la Santé et de la Recherche Médicale (INSERM)-Université de Lille-Centre Hospitalier Régional Universitaire [Lille] (CHRU Lille), Luxembourg Institute of Health (LIH), University of Luxembourg [Luxembourg], Brown University, University of Victoria [Canada] (UVIC), Commissariat à l'énergie atomique et aux énergies alternatives - Laboratoire d'Electronique et de Technologie de l'Information (CEA-LETI), Direction de Recherche Technologique (CEA) (DRT (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA), Université Grenoble Alpes (UGA), Northwestern University [Evanston], Brigham Young University (BYU), Institute for Bioscience and Biotechnology Research [Rockville, MD, États-Unis] (IBBR), University of Maryland [College Park], University of Maryland System-University of Maryland System, Technische Universität Munchen - Université Technique de Munich [Munich, Allemagne] (TUM), Universidad del Pais Vasco / Euskal Herriko Unibertsitatea [Espagne] (UPV/EHU), Arizona State University [Tempe] (ASU), University of Groningen [Groningen], Laboratoire de Biologie à Grande Échelle (BGE - UMR S1038), Institut National de la Santé et de la Recherche Médicale (INSERM)-Institut de Recherche Interdisciplinaire de Grenoble (IRIG), Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Direction de Recherche Fondamentale (CEA) (DRF (CEA)), Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Commissariat à l'énergie atomique et aux énergies alternatives (CEA)-Université Grenoble Alpes (UGA), Université de Fribourg = University of Fribourg (UNIFR), University of Toronto, Northeastern University [Boston], Harvard University [Cambridge], University of Fribourg, Bruley, Christophe, INSERM, Université de Lille, Medical University of Gdańsk, Delft University of Technology [TU Delft], Wageningen University and Research [Wageningen] [WUR], Ecole Polytechnique Fédérale de Lausanne [EPFL], Protéomique, Réponse Inflammatoire, Spectrométrie de Masse (PRISM) - U1192, Technische Universität Munchen - Université Technique de Munich [Munich, Allemagne] [TUM], and Chemical Biology 1
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Proteomics ,Computer science ,[SDV]Life Sciences [q-bio] ,BIOCONJUGATION ,Biophysics ,Settore ING-IND/06 ,Computational biology ,NATIVE PROTEINS ,Biochemistry ,Genome ,Article ,Mass Spectrometry ,03 medical and health sciences ,Protein sequencing ,Single-molecule biophysics ,Sequence Analysis, Protein ,Nanotechnology ,Life Science ,SELECTIVE MODIFICATION ,Molecular Biology ,030304 developmental biology ,Profiling (computer programming) ,0303 health sciences ,IDENTIFICATION ,Sequence Analysis, RNA ,Protein ,RECOGNITION ,Proteins ,PEPTIDES ,Cell Biology ,MASS-SPECTROMETRY ,Single Molecule Imaging ,TRANSLOCATION ,[SDV] Life Sciences [q-bio] ,Biofysica ,Cellular heterogeneity ,DISCRIMINATION ,NANOPORE ,Proteome ,RNA ,Identification (biology) ,Single-Cell Analysis ,Sequence Analysis ,Biotechnology - Abstract
Single-cell profiling methods have had a profound impact on the understanding of cellular heterogeneity. While genomes and transcriptomes can be explored at the single-cell level, single-cell profiling of proteomes is not yet established. Here we describe new single-molecule protein sequencing and identification technologies alongside innovations in mass spectrometry that will eventually enable broad sequence coverage in single-cell profiling. These technologies will in turn facilitate biological discovery and open new avenues for ultrasensitive disease diagnostics., S.S. acknowledges Postdoc Mobility fellowship no. P400PB 180889 from the Swiss National Science Foundation. E.M.M. and E.V.A. acknowledge funding from the NIH (R35 GM122480 and R01 DK110520 to E.M.M.), Welch Foundation (F1515 to E.M.M. and F-0046 to E.V.A.), Army Research Office grant W911NF-12-1-0390 and Erisyon. E.M.M. and E.V.A. are co-founders and shareholders of Erisyon. R.T.K. acknowledges funding from NIGMS (R01 GM138931). P.Y. acknowledges funding from an NIH Director’s New Innovator Award (1DP2OD007292), an NIH Transformative Research Award (1R01EB018659), an NIH Pioneer Award (1DP1GM133052), and the Molecular Robotics Initiative fund at the Wyss Institute for Biologically Inspired Engineering. M.D. acknowledges funding from a Systems Biology Department Fellowship from Harvard Medical School and a Technology Development Fellowship from Wyss Institute for Biologically Inspired Engineering. C.C. acknowledges the Peter and Traudl Engelhorn Foundation. C.D. acknowledges the ERC Advanced Grant Looping DNA (no. 883684) and the NWO programs NanoFront and Basyc., S.O. acknowledges the support of the Azrieli fellowship foundation. N.L.K. acknowledges funding from the Paul G. Allen Frontiers Program (11715), the NIH HuBMAP program (UH3 CA246635) and NIGMS (P41 GM108569). J.P.M. and Z.K. acknowledge internal funding from NIST and are co-inventors on patents relevant to this work. M. Wanunu acknowledges funding from the NIH (HG009186). K.S. and A.A. acknowledge funding from the NSF (PHY-1430124). C.J., C.D. and R.E. acknowledge funding from NWO-I (SMPS). C.J. acknowledges funding from HFSP (RGP0026/2019). A.P. acknowledges Bekker fellowship no. PPN/BEK/2018/1/00296 from the Polish National Agency for Academic Exchange. C.M. and S.H. acknowledge funding from the European Research Council (ERC ‘Enlightened’, GA 616251) and the CEA Transverse Program ‘Instrumentation and Detection’ (PTC-ID VIRIONEMS). Support from the Proteomics French Infrastructure (PROFI) is also gratefully acknowledged. G.D. acknowledges funding from FNR (C17/BM/11642138). M.M. acknowledges funding from the Adolphe Merkle Foundation, the Michael J. Fox Foundation for Parkinson’s Research (grant 17924) and the Swiss National Science Foundation (grant no. 200021-169304). A.M. acknowledges funding from the European Union’s Horizon 2020 research and innovation programme under grant agreement no. 833399-ERC NanoProt-ID and ISF award 3485/19. M.C. acknowledges computational resources from CINECA (NATWE project) and the Swiss National Super-Computing Centre (CSCS), under projects sm11 and s865. E.C. acknowledges funding from I-Site Lille, Région Hauts-de-France, and the European Union’s Horizon 2020 Marie Skłodowska-Curie no. 843052. The study was supported by the project ‘International Centre for Cancer Vaccine Science’ that is carried out within the International Agendas Programme of the Foundation for Polish Science co-financed by the European Union under the European Regional Development Fund. D.G. thanks Genome Canada and Genome British Columbia for financial support for Genomics Technology Platforms (GTP) funding for operations and technology development (264PRO).
327. Proteome activity landscapes of tumor cell lines determine drug responses
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Martin Frejno, Benjamin Ruprecht, Patroklos Samaras, Wilko Weichert, Chen Meng, Jana Zecha, Sebastian Scheich, Hans-Michael Kvasnicka, Julia Mergner, Karin Kleigrewe, Stephanie Heinzlmeir, Dominic Helm, Bernhard Kuster, Thomas Oellerich, Enken Drecoll, Alexander Hogrebe, Julia Dorn, Hubert Serve, and Mathias Wilhelm
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Proteomics ,0301 basic medicine ,Cellular signalling networks ,Proteome ,Science ,Predictive medicine ,General Physics and Astronomy ,Adenylate kinase ,Antineoplastic Agents ,Breast Neoplasms ,02 engineering and technology ,Computational biology ,Drug action ,Biology ,Proteome informatics ,Article ,General Biochemistry, Genetics and Molecular Biology ,Transcriptome ,03 medical and health sciences ,Drug Development ,Cell Line, Tumor ,Neoplasms ,Progesterone receptor ,Animals ,Humans ,Computer Simulation ,Cancer models ,lcsh:Science ,Multidisciplinary ,Adenylate Kinase ,Cytarabine ,Computational Biology ,Myeloid leukemia ,Genomics ,General Chemistry ,021001 nanoscience & nanotechnology ,3. Good health ,ddc ,Leukemia, Myeloid, Acute ,030104 developmental biology ,Drug development ,Raloxifene Hydrochloride ,lcsh:Q ,Receptors, Progesterone ,0210 nano-technology ,Signal Transduction - Abstract
Integrated analysis of genomes, transcriptomes, proteomes and drug responses of cancer cell lines (CCLs) is an emerging approach to uncover molecular mechanisms of drug action. We extend this paradigm to measuring proteome activity landscapes by acquiring and integrating quantitative data for 10,000 proteins and 55,000 phosphorylation sites (p-sites) from 125 CCLs. These data are used to contextualize proteins and p-sites and predict drug sensitivity. For example, we find that Progesterone Receptor (PGR) phosphorylation is associated with sensitivity to drugs modulating estrogen signaling such as Raloxifene. We also demonstrate that Adenylate kinase isoenzyme 1 (AK1) inactivates antimetabolites like Cytarabine. Consequently, high AK1 levels correlate with poor survival of Cytarabine-treated acute myeloid leukemia patients, qualifying AK1 as a patient stratification marker and possibly as a drug target. We provide an interactive web application termed ATLANTiC (http://atlantic.proteomics.wzw.tum.de), which enables the community to explore the thousands of novel functional associations generated by this work., Proteome activity has a major role in cancer progression and response to drugs. Here, the authors use comprehensive proteomic and phosphoproteomic data, in conjunction with drug-sensitivity screens, to generate a community resource consisting of landscapes of pathway and kinase activity across different cell lines
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328. Glycosylation analysis of gel-separated proteins
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Bernhard Kuster, Krogh, T. N., Mortz, E., and Harvey, D. J.
329. Functional analysis of the human CDC5L complex and identification of its components by mass spectrometry
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Paul Ajuh, Kostya I. Panov, Bernhard Kuster, Matthias Mann, Angus I. Lamond, and Joost C. B. M. Zomerdijk
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Spliceosome ,Multiprotein complex ,DNA, Complementary ,Databases, Factual ,Protein subunit ,RNA Splicing ,Blotting, Western ,Molecular Sequence Data ,Cell Cycle Proteins ,Biology ,Models, Biological ,General Biochemistry, Genetics and Molecular Biology ,Catalysis ,Chromatography, Affinity ,Adenosine Triphosphate ,Schizosaccharomyces ,medicine ,Humans ,Amino Acid Sequence ,RNA, Messenger ,Cloning, Molecular ,Molecular Biology ,Peptide sequence ,Cell Nucleus ,General Immunology and Microbiology ,General Neuroscience ,Articles ,biology.organism_classification ,Molecular biology ,Precipitin Tests ,Recombinant Proteins ,Cell nucleus ,medicine.anatomical_structure ,Biochemistry ,Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization ,RNA splicing ,Schizosaccharomyces pombe ,Spliceosomes ,Electrophoresis, Polyacrylamide Gel ,Schizosaccharomyces pombe Proteins ,Ribosomes ,HeLa Cells - Abstract
Recently, we identified proteins that co-purify with the human spliceosome using mass spectrometry. One of the identified proteins, CDC5L, corresponds to the human homologue of the Schizosaccharomyces pombe CDC5(+) gene product. Here we show that CDC5L is part of a larger multiprotein complex in HeLa nuclear extract that incorporates into the spliceosome in an ATP-dependent step. We also show that this complex is required for the second catalytic step of pre-mRNA splicing. Immunodepletion of the CDC5L complex from HeLa nuclear extract inhibits the formation of pre-mRNA splicing products in vitro but does not prevent spliceosome assembly. The first catalytic step of pre-mRNA splicing is less affected by immunodepleting the complex. The purified CDC5L complex in HeLa nuclear extract restores pre-mRNA splicing activity when added to extracts that have been immunodepleted using anti-CDC5L antibodies. Using mass spectrometry and database searches, the major protein components of the CDC5L complex have been identified. This work reports a first purification and characterization of a functional, human non-snRNA spliceosome subunit containing CDC5L and at least five additional protein factors.
330. MALDI imaging mass spectrometry for in situ proteomic analysis of preneoplastic lesions in pancreatic cancer.
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Barbara M Grüner, Hannes Hahne, Pawel K Mazur, Marija Trajkovic-Arsic, Stefan Maier, Irene Esposito, Evdokia Kalideris, Christoph W Michalski, Jörg Kleeff, Sandra Rauser, Roland M Schmid, Bernhard Küster, Axel Walch, and Jens T Siveke
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Medicine ,Science - Abstract
The identification of new biomarkers for preneoplastic pancreatic lesions (PanINs, IPMNs) and early pancreatic ductal adenocarcinoma (PDAC) is crucial due to the diseases high mortality rate upon late detection. To address this task we used the novel technique of matrix-assisted laser desorption/ionization (MALDI) imaging mass spectrometry (IMS) on genetically engineered mouse models (GEM) of pancreatic cancer. Various GEM were analyzed with MALDI IMS to investigate the peptide/protein-expression pattern of precursor lesions in comparison to normal pancreas and PDAC with cellular resolution. Statistical analysis revealed several discriminative m/z-species between normal and diseased tissue. Intraepithelial neoplasia (PanIN) and intraductal papillary mucinous neoplasm (IPMN) could be distinguished from normal pancreatic tissue and PDAC by 26 significant m/z-species. Among these m/z-species, we identified Albumin and Thymosin-beta 4 by liquid chromatography and tandem mass spectrometry (LC-MS/MS), which were further validated by immunohistochemistry, western blot, quantitative RT-PCR and ELISA in both murine and human tissue. Thymosin-beta 4 was found significantly increased in sera of mice with PanIN lesions. Upregulated PanIN expression of Albumin was accompanied by increased expression of liver-restricted genes suggesting a hepatic transdifferentiation program of preneoplastic cells. In conclusion we show that GEM of endogenous PDAC are a suitable model system for MALDI-IMS and subsequent LC-MS/MS analysis, allowing in situ analysis of small precursor lesions and identification of differentially expressed peptides and proteins.
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- 2012
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