TY - GEN A1 - Schwaar, Timm T1 - Entwicklung von multidimensionalen Hochdurchsatzmethoden zur Analyse von partikelbasierten Peptidbibliotheken N2 - Ziel dieser Arbeit war die Entwicklung einer Methode zur Hochdurchsatzanalyse von partikelgebundenen Split-and-Mix-Peptidbibliotheken. Dabei wurde ein System entwickelt, das Partikelbibliotheken in kurzer Zeit auf ihre Bindung zu einem Zielmolekül hin untersucht und im nächsten Schritt eine direkte „Ein-Partikel“-Sequenzierung der Peptide von der festen Phase ermöglicht. Die Analyse der Bindung eines Zielmoleküls an ein selektiv bindendes Peptid wurde dabei in einem Modellsystem simuliert. Dazu wurde FITC-markiertes Neutravidin 3 mit festphasengebundenen biotinylierten und nicht-biotinylierten Peptiden inkubiert. Die Bindung von Neutravidin-FITC an die biotinylierten Peptide konnte erfolgreich mittels eines Fluoreszenzmikroskops nachgewiesen werden, wobei diese Methode für eine Hochdurchsatzanalyse ungeeignet war. So wurde die Analyse des Fluoreszenzassays auf einen Microarrayscanner übertragen (siehe Abbildung 33). Dies gelang erfolgreich wobei die hohe Eigenfluoreszenz des Tentagel-Harzes zu falsch positiven Ergebnissen führte. Mit einem Wechsel zum längerwelligen Fluoreszenzfarbstoff ATTO-633 konnten falsch positive Ergebnisse vermieden werden. Somit wurde die zeitintensive Fluoreszenzanalyse von einem Fluoreszenzmikroskop erfolgreich auf einen Microarrayscanner übertragen, welcher in kurzer Zeit (ca. 5 min pro Objektträger) eine Vielzahl an peptidbeladenen Partikeln gleichzeitig analysieren kann. KW - Peptide KW - OBOC library KW - MALDI-TOF-MS KW - Sequencing KW - Ladder sequencing KW - Solid-phase peptide synthesis KW - SPPS KW - Microarray KW - Fluorescence label KW - Polymer beads KW - Tentagel KW - Merrifield KW - Split and mix technology PY - 2015 SP - 1 EP - 80 PB - Humboldt-Universität zu Berlin CY - Berlin AN - OPUS4-54644 LA - deu AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Eisenreich, F. A1 - Kathan, M. A1 - Dallmann, A. A1 - Ihrig, S. P. A1 - Schwaar, Timm A1 - Schmidt, B. M. A1 - Hecht, S. T1 - A photoswitchable catalyst system for remote-controlled (co)polymerization in situ N2 - The fundamental properties of a polymeric material are ultimately governed by its structure, which mainly relies on monomercomposition and connection, topology, chain length, and polydispersity. Thus far, these structural characteristics are typicallyset ex situ by the specific polymerization procedure, eventually limiting the future design space for the creation of moresophisticated polymers. Herein, we report on a single photoswitchable catalyst system, which enables in situ remote controlover the ring-opening polymerization of l-lactide and further allows regulation of the incorporation of trimethylene carbonateand δ -valerolactone monomers in copolymerizations. By implementing a phenol moiety into a diarylethene-type structure,we exploit light-induced keto–enol tautomerism to switch the hydrogen-bonding-mediated monomer activation reversiblyON and OFF. This general and versatile principle allows for exquisite external modulation of ground-state catalysis of a livingpolymerization process in a closed system by ultraviolet and visible light and should thereby facilitate the generation of newpolymer structures. KW - Polymers PY - 2018 U6 - https://doi.org/10.1038/s41929-018-0091-8 SN - 2520-1158 VL - 1 IS - 7 SP - 516 EP - 522 PB - Nature CY - London AN - OPUS4-45407 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Remmler, Dario A1 - Schwaar, Timm A1 - Pickhardt, M. A1 - Donth, C. A1 - Mandelkow, E. A1 - Weller, Michael G. A1 - Börner, H. T1 - On the way to precision formulation additives: 2D-screening to select solubilizers with tailored host and release capabilities N2 - A 2-dimensional high-throughput screening method is presented to select peptide sequences from large peptide libraries for precision formulation additives, having a high capacity to specifically host a drug of interest and provide tailored drug release properties. The identified sequences are conjugated with poly(ethylene glycol) (PEG) to obtain peptide-PEG conjugates that proved to be valuable as solubilizers for small organic molecule drugs to overcome limitations of poor water-solubility and low bio-availability. The 2D-screening method selects peptide sequences on both (i) high loading capacities and (ii) preferred drug-release capabilities as demonstrated on an experimental Tau-protein aggregation inhibitor/Tau- deaggregator with potentials for an anti-Alzheimer disease drug (BB17). To enable 2D-screening, a one-bead one-compound (OBOC) peptide library was immobilized on a glass slide, allocating individual beads to permanent positions. While the first screening step involved incubation of the supported OBOC library with BB17 to identify beads with high drug binding capacities by fluorescence scanner readouts, the second step reveals release properties of the high capacity binders by incubation with blood plasma protein model solutions. Efficiently peptides with high BB17 capacities and either keeper or medium or fast releaser properties can be identified by direct sequence readouts from the glass slide supported resin beads via matrix-assisted laser desorption/ionization time-of-flight tandem mass spectrometry. Four peptides are synthesized as peptide-PEG solubilizers representing strong, medium, weak releasers and non-binders. Loading capacities reached up to 1:3.4 (mol drug per mol carrier) and release kinetics (fast/medium/slow) are in agreement with the selection process as investigated by fluorescence anisotropy and fluorescence correlation spectroscopy. The ability of BB17/conjugate complexes to inhibit the aggregation of Tau4RDΔK (four repeat Tau ((M)Q244-E372 with deletion of K280), 129 residues) in N2a cells is studied by a Tau-pelleting assay showing the modulation of cellular Tau aggregation. Promising effects such as the reduction of 55% of total Tau load are observed for the strong releaser additive. Studies of in vitro Thioflavin S Tau-aggregation assays show half-maximal inhibitory activities (IC50 values) of BB17/conjugates in the low micro-molar range. KW - Block copolymer KW - Drug transporters KW - Peptide library screening KW - Formulation additives PY - 2018 U6 - https://doi.org/10.1016/j.jconrel.2018.06.032 SN - 0168-3659 SN - 1873-4995 VL - 285 SP - 96 EP - 105 PB - Elsevier CY - Amsterdam AN - OPUS4-45483 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schwaar, Timm A1 - Remmler, Dario A1 - Börner, H. G. A1 - Weller, Michael G. T1 - Multidimensional high throughput methods for the analysis of particle-based peptide libraries N2 - Screening of one-bead-one-peptide libraries is a useful procedure for the identification of protein ligands. The demand for binders with high affinity and high specificity towards various target proteins has surged in the biomedical field in the recent years. The 1 traditional peptide screening procedure involves tedious steps as selection, sequencing, and characterization. Herein, we developed a high-throughput – “all in one chip” system to avoid time-consuming separation steps. T2 - Chemistry and Biology of Peptides Gordon Research Conference CY - Ventura, CA, USA DA - 10.02.2018 KW - Lab-on-a-chip KW - Combinatorial peptide library PY - 2018 AN - OPUS4-44611 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schwaar, Timm A1 - Remmler, Dario A1 - Lettow, Maike A1 - Börner, H. G. A1 - Weller, Michael G. T1 - MALDI-Supported Screening of Large Peptide Libraries for Identification of Protein Binders N2 - Screening of one-bead-one-peptide libraries is a useful procedure for the identification of protein ligands. The demand for binders with high affinity and high specificity towards various target proteins has surged in the biomedical field in the recent years. The traditional peptide screening procedure involves tedious steps as selection, sequencing, and characterization. Herein, we developed a high-throughput – “all in one chip” system to avoid time-consuming separation steps. T2 - European Mass Spectrometry Conference (EMSC) 2018 CY - Saarbrücken, Germany DA - 11.02.2018 KW - Lab-on-a-chip KW - Combinatorial peptide library PY - 2018 AN - OPUS4-44612 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - THES A1 - Schwaar, Timm T1 - Entwicklung von multidimensionalen Hochdurchsatzmethoden zur Analyse von Partikel-basierten Peptidbibliotheken N2 - Gegenwärtig ist das Interesse und der Bedarf von Proteinbindern insbesondere in der Biotechnik und Pharmaforschung sehr groß. Kombinatorische, Partikel-basierte (One-Bead-One-Compound) Peptidbibliotheken sind eine Technik, um selektiv bindende Proteine zu identifizieren. Allerdings beinhaltet das Screening dieser Peptidbibliotheken aufwendige Schritte, wie die Separation, Sequenzierung und Charakterisierung von identifizierten Bindern. In dieser Arbeit wurde ein Chip-System entwickelt, auf dem alle Schritte eines Screenings durchgeführt werden können. Dafür wurde ein Glasobjektträger mit einem magnetisch leitenden, doppelseitigen Klebeband versehen. Die Partikel der Bibliothek wurden durch ein Sieb aufgetragen. Dies führte zu einer geordneten Immobilisierung der Partikel auf dem Chip. Über 30.000 Partikel konnten so auf einem Chip immobilisiert werden. Für die Identifizierung von selektiven Protein-bindenden Peptiden wird die immobilisierte Peptidbibliothek mit einem Fluorophor-markierten Protein inkubiert, bindende Partikel mittels Fluoreszenzscan identifiziert und die Peptidsequenz direkt auf dem Chip mittels Matrix-Assisted-Laser-Desorption/Ionization-(MALDI)-Flugzeit-(TOF)-Massenspektroskopie (MS) bestimmt. Die Durchführung einer Abbruchsequenz-Methode erlaubt die eindeutige Bestimmung der Peptidsequenzen mit einer nahezu 100 % Genauigkeit. Die entwickelte Technologie wurde in einem FLAG-Peptid-Modell validiert. Bei dem Screening wurden neue anti-FLAG-Antikörper-bindende Peptide identifiziert. Anschließend wurden in einem Screening von ca. 30.000 Partikeln IgG-bindende Peptide mit mittleren mikromolaren Dissoziationskonstanten identifiziert. Für die Identifizierung stärkerer Binder wurde eine magnetische Anreicherung entwickelt, die dem Chip-Screening vorgeschaltet werden kann. Hiermit wurden aus ca. 1 Million gescreenter Partikel, Peptide mit Dissoziationskonstanten im niedrigen mikromolaren Bereich identifiziert. N2 - The screening of one-bead-one-compound (OBOC) libraries is a well-established technique for the identification of protein-binding ligands. The demand for binders with high affinity and specificity towards various targets has surged in the biomedical and pharmaceutical field in recent years. The combinatoric peptide screening traditionally involves tedious steps such as affinity selection, bead picking, sequencing and characterization. In this thesis, a high-throughput “all-on-one chip” system is presented to avoid slow and technically complex bead picking steps. Beads of a combinatorial peptide library are immobilized on a conventional glass slide equipped with an electrically conductive tape. The beads are applied by using a precision sieve, which allows the spatially ordered immobilization of more than 30,000 beads on one slide. For the target screening, the immobilized library is subsequently incubated with a fluorophore-labeled target protein. In a fluorescence scan followed by matrix-assisted laser desorption/ionization (MALDI)-time of flight (TOF) mass spectrometry (MS), high-affinity binders are directly and unambiguously sequenced directly from the bead. The use of an optimized ladder sequencing approach improved the accuracy of the de-novo sequencing step to 100 %. This new technique was validated by employing a FLAG-based model system. In a first step, new peptide binders for the M2 anti-FLAG monoclonal antibody were identified. Finally, this system was utilized to screen for IgG-binding peptides. The screening of about 30.000 peptides on one chip led to the identification of peptide binders in the mid micromolar range. A magnetic enrichment technique was developed to increase the number of screened beads. By combining the magnetic enrichment strategy with the chip system, 1 million beads were screened and IgG-binders in the low micromolar range were identified. KW - Peptidbibliotheken KW - Hochdurchsatzscreening KW - Peptidsequenzierung KW - Chip-Screening PY - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:b43-511649 SP - 1 EP - 153 PB - Humboldt-Universität CY - Berlin AN - OPUS4-51164 LA - deu AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Wieczorek, S. A1 - Schwaar, Timm A1 - Senge, M. O. A1 - Börner, H.G. T1 - Specific drug formulation additives: Revealing the impact of architecture and block length ratio N2 - Combining poly(ethylene glycol) (PEG) with sequence-defined peptides in PEG–peptide conjugates offers opportunities to realize next-generation drug formulation additives for overcoming undesired pharmacological profiles of difficult small molecule drugs. The tailored peptide segments provide sequence-specific, noncovalent drug binding, and the hydrophilic PEG block renders the complexes water soluble. On the basis of a peptide sequence known to bind the photosensitizer m-tetra(hydroxyphenyl)chlorin (m-THPC) for photodynamic cancer therapy, a set of different conjugate architectures is synthesized and studied. Variations in PEG block length and amplification of the peptidic binding domain of PEG–peptide conjugates are used to fine tune critical parameters for hosting m-THPC, such as drug payload capacities, aggregation sizes, and drug release and activation kinetics. PY - 2015 U6 - https://doi.org/10.1021/acs.biomac.5b00961 SN - 1525-7797 VL - 16 IS - 10 SP - 3308 EP - 3312 PB - ACS Publ. CY - Washington, DC, USA AN - OPUS4-35037 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schwaar, Timm A1 - Weller, Michael G. A1 - Remmler, Dario A1 - Börner, H.-G. T1 - MALDI-Imaging of large peptide libraries for pharmaceutical applications T2 - Tag der Chemie CY - FREIEN UNIVERSITÄT BERLIN DA - 2015-06-18 PY - 2015 AN - OPUS4-34460 LA - deu AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schwaar, Timm A1 - Weller, Michael G. A1 - Remmler, Dario A1 - Börner, H.-G. T1 - Development of multidimensional high-throughput methods for the analysis of particle-based peptide libraries T2 - PhD Students Day 2015 CY - Berlin DA - 2015-07-14 PY - 2015 AN - OPUS4-34461 LA - deu AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Tscheuschner, Georg A1 - Schwaar, Timm A1 - Weller, Michael G. T1 - Fast Confirmation of Antibody Identity by MALDI-TOF MS Fingerprints N2 - Thousands of antibodies for diagnostic and other analytical purposes are on the market. However, it is often difficult to identify duplicates, reagent changes, and to assign the correct original publications to an antibody. This slows down scientific progress and might even be a cause of irreproducible research and a waste of resources. Recently, activities were started to suggest the sole use of recombinant antibodies in combination with the open communication of their sequence. In this case, such uncertainties should be eliminated. Unfortunately, this approach seems to be rather a long-term vision since the development and manufacturing of recombinant antibodies remain quite expensive in the foreseeable future. Nearly all commercial antibody suppliers also may be reluctant to publish the sequence of their antibodies, since they fear counterfeiting. De novo sequencing of antibodies is also not feasible today for a reagent user without access to the hybridoma clone. Nevertheless, it seems to be crucial for any scientist to have the opportunity to identify an antibody undoubtedly to guarantee the traceability of any research activity using antibodies from a third party as a tool. For this purpose, we developed a method for the identification of antibodies based on a MALDI-TOF MS fingerprint. To circumvent lengthy denaturation, reduction, alkylation, and enzymatic digestion steps, the fragmentation was performed with a simple formic acid hydrolysis step. Eighty-nine unknown monoclonal antibodies were used for this study to examine the feasibility of this approach. Although the molecular assignment of peaks was rarely possible, antibodies could be easily recognized in a blinded test, simply from their mass-spectral fingerprint. A general protocol is given, which could be used without any optimization to generate fingerprints for a database. We want to propose that, in most scientific projects relying critically on antibody reagents, such a fingerprint should be established to prove and document the identity of the used antibodies, as well as to assign a specific reagent to a datasheet of a commercial supplier, public database record, or antibody ID. KW - Reproducibility KW - Quality Control KW - Traceability KW - Diagnostics KW - Hybridoma KW - Monoclonal Antibody KW - Recombinant Antibody PY - 2020 U6 - http://nbn-resolving.de/urn/resolver.pl?urn:nbn:de:kobv:b43-506611 SN - 2310-287X VL - 9 IS - 2 SP - 1 EP - 17 PB - MDPI CY - Basel AN - OPUS4-50661 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER -