Facing Current Quantification Challenges in Protein Microarrays

The proteome is highly variable and differs from cell to cell. The reasons are posttranslational modifications, splice variants, and polymorphisms. Techniques like next-generation sequencing can only give an inadequate picture of the protein status of a cell. Protein microarrays are able to track these changes on the level they occur: the proteomic level. Therefore, protein microarrays are powerful tools for relative protein quantification, to unveil new interaction partners and to track posttranslational modifications. This papers gives an overview on current protein microarray techniques and discusses recent advances in relative protein quantification

Fluorescence-Activated Cell Sorting of EGFP-Labeled Neural Crest Cells From Murine Embryonic Craniofacial Tissue

During the early stages of embryogenesis, pluripotent neural crest cells (NCC) are known to migrate from the neural folds to populate multiple target sites in the embryo where they differentiate into various derivatives, including cartilage, bone, connective tissue, melanocytes, glia, and neurons of the peripheral nervous system. The ability to obtain pure NCC populations is essential to enable molecular analyses of neural crest induction, migration, and/or differentiation. Crossing Wnt1-Cre and Z/EG transgenic mouse lines resulted in offspring in which the Wnt1-Cre transgene activated permanent EGFP expression only in NCC. The present report demonstrates a flow cytometric method to sort and isolate populations of EGFP-labeled NCC. The identity of the sorted neural crest cells was confirmed by assaying expression of known marker genes by TaqMan Quantitative Real-Time Polymerase Chain Reaction (QRT-PCR). The molecular strategy described in this report provides a means to extract intact RNA from a pure population of NCC thus enabling analysis of gene expression in a defined population of embryonic precursor cells critical to development

Implications for the Binding of the Protein G5P to DNA

Microorganisms accumulate molar concentrations of compatible solutes like ectoine to prevent proteins from denaturation. Direct structural or spectroscopic information on the mechanism and about the hydration shell around ectoine are scarce. We combined surface plasmon resonance (SPR), confocal Raman spectroscopy, molecular dynamics simulations, and density functional theory (DFT) calculations to study the local hydration shell around ectoine and its influence on the binding of a gene-S-protein (G5P) to a single-stranded DNA (dT(25)). Due to the very high hygroscopicity of ectoine, it was possible to analyze the highly stable hydration shell by confocal Raman spectroscopy. Corresponding molecular dynamics simulation results revealed a significant change of the water dielectric constant in the presence of a high molar ectoine concentration as compared to pure water. The SPR data showed that the amount of protein bound to DNA decreases in the presence of ectoine, and hence, the protein-DNA dissociation constant increases in a concentration- dependent manner. Concomitantly, the Raman spectra in terms of the amide I region revealed large changes in the protein secondary structure. Our results indicate that ectoine strongly affects the molecular recognition between the protein and the oligonudeotide, which has important consequences for osmotic regulation mechanisms

Beam test performance of a prototype module with Short Strip ASICs for the CMS HL-LHC tracker upgrade

The Short Strip ASIC (SSA) is one of the four front-end chips designed for the upgrade of the CMS Outer Tracker for the High Luminosity LHC. Together with the Macro-Pixel ASIC (MPA) it will instrument modules containing a strip and a macro-pixel sensor stacked on top of each other. The SSA provides both full readout of the strip hit information when triggered, and, together with the MPA, correlated clusters called stubs from the two sensors for use by the CMS Level-1 (L1) trigger system. Results from the first prototype module consisting of a sensor and two SSA chips are presented. The prototype module has been characterized at the Fermilab Test Beam Facility using a 120 GeV proton beam

Comparative evaluation of analogue front-end designs for the CMS Inner Tracker at the High Luminosity LHC

The CMS Inner Tracker, made of silicon pixel modules, will be entirely replaced prior to the start of the High Luminosity LHC period. One of the crucial components of the new Inner Tracker system is the readout chip, being developed by the RD53 Collaboration, and in particular its analogue front-end, which receives the signal from the sensor and digitizes it. Three different analogue front-ends (Synchronous, Linear, and Differential) were designed and implemented in the RD53A demonstrator chip. A dedicated evaluation program was carried out to select the most suitable design to build a radiation tolerant pixel detector able to sustain high particle rates with high efficiency and a small fraction of spurious pixel hits. The test results showed that all three analogue front-ends presented strong points, but also limitations. The Differential front-end demonstrated very low noise, but the threshold tuning became problematic after irradiation. Moreover, a saturation in the preamplifier feedback loop affected the return of the signal to baseline and thus increased the dead time. The Synchronous front-end showed very good timing performance, but also higher noise. For the Linear front-end all of the parameters were within specification, although this design had the largest time walk. This limitation was addressed and mitigated in an improved design. The analysis of the advantages and disadvantages of the three front-ends in the context of the CMS Inner Tracker operation requirements led to the selection of the improved design Linear front-end for integration in the final CMS readout chip

The CMS Phase-1 pixel detector upgrade

The CMS detector at the CERN LHC features a silicon pixel detector as its innermost subdetector. The original CMS pixel detector has been replaced with an upgraded pixel system (CMS Phase-1 pixel detector) in the extended year-end technical stop of the LHC in 2016/2017. The upgraded CMS pixel detector is designed to cope with the higher instantaneous luminosities that have been achieved by the LHC after the upgrades to the accelerator during the first long shutdown in 2013–2014. Compared to the original pixel detector, the upgraded detector has a better tracking performance and lower mass with four barrel layers and three endcap disks on each side to provide hit coverage up to an absolute value of pseudorapidity of 2.5. This paper describes the design and construction of the CMS Phase-1 pixel detector as well as its performance from commissioning to early operation in collision data-taking.Peer reviewe

Test beam performance of a CBC3-based mini-module for the Phase-2 CMS Outer Tracker before and after neutron irradiation

The Large Hadron Collider (LHC) at CERN will undergo major upgrades to increase the instantaneous luminosity up to 5–7.5×10$^{34}$ cm$^{-2}$s$^{-1}$. This High Luminosity upgrade of the LHC (HL-LHC) will deliver a total of 3000–4000 fb-1 of proton-proton collisions at a center-of-mass energy of 13–14 TeV. To cope with these challenging environmental conditions, the strip tracker of the CMS experiment will be upgraded using modules with two closely-spaced silicon sensors to provide information to include tracking in the Level-1 trigger selection. This paper describes the performance, in a test beam experiment, of the first prototype module based on the final version of the CMS Binary Chip front-end ASIC before and after the module was irradiated with neutrons. Results demonstrate that the prototype module satisfies the requirements, providing efficient tracking information, after being irradiated with a total fluence comparable to the one expected through the lifetime of the experiment

Relative Protein Quantification Using Reverse Phase Protein Microarrays

Tabellenverzeichnis i Abbildungsverzeichnis ii Abkürzungsverzeichnis iv 1 Einleitung 1 1.1 Methodik 4 1.2 Microarrayformate 9 1.2.1 Antikörper- / Aptamerarrays 11 1.2.2 Peptidmicroarrays 12 1.2.3 Reverse Phase Protein Microarrays 14 1.3 Validierung der Antikörper und Detektion 16 2 Material und Methoden 18 2.1 Material 18 2.1.1 Chemikalien und Lösungsmittel 18 2.1.2 Zellkulturlinien 19 2.1.3 Bakterienstamm 19 2.1.4 Verbrauchsmaterialien und Geräte 20 2.1.5 Software 21 2.1.6 Antikörper 21 2.1.7 Puffer 22 2.2 Methoden 25 2.2.1 Zellkultur 25 2.2.2 Zelllyse 25 2.2.3 Antikörpervalidierung 25 2.2.4 Natriumdodecylsulfat-Polyacrylamidgelelektrophorese (SDS PAGE) 28 2.2.5 Western Blot 29 2.2.6 Immunopräzipitation 31 2.2.7 Assay mit Bicinchoninsäure (BCA) 31 2.2.8 Bradford 31 2.2.9 Electrophoretic Mobility Shift Assay (EMSA) 32 2.2.10 Überexpression und Aufreinigung von CREB 32 2.2.11 in vitro Phosphorylierung von CREB 33 2.2.12 Benzonaseverdau der Zelllysate 33 2.2.13 Herstellung der Reverse Phase Protein Microarrays 33 2.2.14 Herstellung der Epoxy beschichteten Slides 35 2.2.15 Handhabung der Epoxy beschichteten Slides 35 2.2.16 Handhabung der Nitrozellulose beschichteten Slides 36 2.2.17 Epicocconone Färbung 37 2.2.18 Datenanalyse und Normierung der Daten 37 3 Ergebnisse 39 3.1 Zellkultur, Signaltransduktion, Zellaufschluss und Quantifizierung der Lysate 39 3.1.1 Zellkultur 39 3.1.2 Untersuchung der Lyseeffizienz beim Zellaufschluss 40 3.1.3 Signaltransduktion 44 3.2 Antikörpervalidierung und Herstellung der Kontrollen 46 3.2.1 Positivkontrollen 46 3.2.2 Immunopräzipitation zur Antikörpervalidierung unter nativen Bedingungen 48 3.2.3 EMSA Experimente zur Konformationsuntersuchung der Proteine 49 3.2.4 Western Blots zur Antikörpervalidierung 52 3.3 Herstellen von Reverse Phase Microarrays 53 3.3.1 Spotten von Zellextrakt 55 3.3.2 Benzonaseverdau der Lysate 55 3.3.3 Auswahl der Slides und Optimierung der Inkubationsbedingungen 56 3.4 Auswerten der Microarraydaten 69 3.4.1 Donutstrukturen auf RPMAs 70 3.4.2 Normalisierung der Proteinkonzentrationen mittels Epicocconone-Färbung 71 3.4.3 Autofluoreszenz der Lysate auf dem RPMA 72 3.4.4 Reproduzierbarkeit der Positivkontrollen auf dem RPMA 74 3.5 Signaltransduktion auf RPMAs 76 4 Diskussion 79 4.1 Zellkultur, Signaltransduktion, Zellaufschluss und Quantifizierung der Lysate 79 4.2 Antikörpervalidierung und Herstellung der Kontrollen 80 4.3 Herstellen von Reverse Phase Microarrays 81 4.4 Auswerten der Microarraydaten 85 4.4.1 Massentransport 86 4.4.2 Normalisierung der Microarraydaten 87 4.5 Signaltransduktion auf RPMAs 87 5 Zusammenfassung in Deutsch 88 6 Zusammenfassung in Englisch 89 7 Literaturverzeichnis 90 8 Verzeichnis der erfolgten Publikationen 95 9 Lebenslauf 107 10 Anhang 109Im Gegensatz zum Genom ist das Proteom hochvariabel. Es variiert nicht nur von Organismus zu Organismus, sondern auch zwischen verschiedenen Geweben und innerhalb eines Gewebes von Zelle zu Zelle. Grund hierfür sind unter anderem unterschiedliche Splicevarianten und posttranslationale Modifikationen (PTMs). Diese sind zeitlich begrenzt, sehr dynamisch und abhängig vom Zellzyklus oder externen Stimuli und beeinflussen die Proteinzusammensetzung einer Zelle. Nur ein Bruchteil dieser Modifikationen spielt bei regulatorischen Prozessen eine Rolle. Eine der wichtigsten PTMs ist die Phosphorylierung von Proteinen, denn diese spielt eine entscheidende Rolle bei der Signaltransduktion und damit einhergehend, der Veränderung des Proteoms. Zum Verständnis von Signaltransduktionsevents gehört die Analyse der zeitlichen und räumlichen Dynamik von posttranslationalen Modifikationen. Deswegen ist es von besonderem Interesse diese Änderung der Verhältnisse möglichst genau zu untersuchen. Proteinbasierte Nachweismethoden, wie die Analyse mittels Reverse Phase Protein Microarrays, sind in der Lage Änderungen des Proteoms direkt nachweisen zu können. Ziel dieser Arbeit war die relative Quantifizierung von Proteinen mittels RPMAs anhand der Stimulierung des PKA Signaltransduktionsweges. Um dieses Ziel zu erreichen, wurde der gesamte Vorgang vom Herstellen der Microarrays, über den Assay auf den Slides und der Auswertung bis zur Stimulierung der Zellen optimiert. In diesem Zusammenhang wurden verschiedene Zelllinien und Stimuli getestet und die Lysebedingungen an die Anforderungen des Piezo-Spotter angepasst. Bei der Herstellung der RPMAs wurde auf eine möglichst kurze Verweildauer der Lysate und eine schnelle, automatisierte Herstellungsprozedur geachtet. Um die zeitlich unterschiedlich stimulierten Lysate miteinander vergleichen zu können wurde eine Normalisierung gegen den Gesamtproteingehalt am Ende des Assays durchgeführt. Eine Änderung der Signalintensität ist somit auf die Stimulierung und nicht auf einen unterschiedlichen Proteingehalt nach dem Spotten auf den Arrays zurückzuführen. Die Aktivierung des PKA Signaltransduktionsweges konnte in relativen Verhältnissen dargestellt werden. Es konnte durch Stimulierung eine Aktivierung der PKA nach wenigen Minuten gemessen werden. Wie die Ergebnisse zeigen, sind Phosphorylierungen im Zelllysat nur über einen begrenzten Zeitraum stabil und die Analysedauer ab Probennahme sollte möglichst kurz gehalten werden. Dabei traten biologische Varianzen auf, welche nicht durch die Optimierung des Protokolls oder eine verbesserte Auswertung zu korrigieren waren. Faktoren wie der Grad der Konfluenz oder der Druck des Zellkulturmediums haben bereits im Vorfeld einen Einfluss auf die Signaltransduktion und konnten, auch durch ein vorangegangenes Aushungern der Zellen und eine optimierte Herstellung der RPMAs selbst, nicht minimiert werden. Es konnte gezeigt werden, dass die relative Quantifizierung mittels Reverse Phase Protein Microarrays möglich, jedoch von vielen Faktoren abhängig ist. Um die Vergleichbarkeit der Versuche, auch zwischen verschiedenen Laboren, zu gewährleisten ist eine strikte Einhaltung einheitlicher Protokolle von der Stimulierung der Zellen über die Prozessierung und Lagerung der Lysate unabdingbar.In contrast to the genome, the proteome is highly variable. It not only differs from organism to organism but also between different tissues. Even cells within one tissue type may have different protein content. Reasons for this diversity are various splice variants and post translational modifications. These PTMs are time limited, highly dynamic and depend on external stimuli or cell cycle. However, only a fraction is involved in regulatory processes like cell signaling, transcription and translation. One modification that plays a vital role in signal transduction and therefore also in gene regulation, is the phosphorylation of proteins. In order to understand signal transduction events the investigation of phosphorylation time frames upon stimulation is crucial. Protein based methods like Reverse Phase Protein Microarrays are able to track these changes on the level they occur – the proteomic level. Using the PKA signal transduction network as a model system, the aim of this study was the relative quantification of proteins using the RPMA technique. Goal of this thesis was the relative quantification of proteins using reverse phase protein microarrays. The PKA signal transduction pathway was chosen as a model system to optimize various steps along the way from cell culture over the actual production of the array to the final data acquisition and evaluation. Diverse stimuli and cell lines were tested and optimized for parameters like PKA activation, protein content and lysis conditions. To ensure automated high quality microarray production, fabrication times could be reduced to a minimum. On-array normalization was established to guarantee suitable estimation of all changes to the number of proteins immobilized on the array. The activation of the PKA signal transduction pathway could be monitored in relative amounts. Upon stimulation of the cells, PKA activity was measured after a couple of minutes and was depleted after 45 minutes. As the results show, phosphorylations are only stable for a limited amount of time. As a direct consequence the time between the stimulation experiment and the actual assay on the RPMA have to be kept as short as possible. Parameters like confluence of the cells or changes in pressure on top of the cells influence the phosphorylation pattern in signal transduction cascades before / during and after starvation and stimulation. Such parameters can neither be normalized by total protein staining nor be corrected by an improved fabrication protocol. Results showed that relative quantification of proteins using reverse phase protein microarray technology is possible. The outcome is greatly influenced by various factors along the production chain. Factors like sample processing and storage have a high impact on the stability and amount of PTMs measured. In order to be able to compare the results, not only from day to day, but also with different laboratories, the strict adherence to standard operation procedures is mandatory