Development of capillary based separation techniques for the separation of proteins equilibrated using hexapeptide ligand libraries

Challenges in research areas such as chemistry, medicine, environmental toxicology and biology require the analysis of complex samples. Fast analysis of these samples using separation techniques with spectrometric or spectroscopic detection is common. Most often, chromatographic separation techniques such as gas and liquid chromatography coupled to mass spectrometry are chosen. These techniques, however, often reach their limits when highly charged analytes are investigated. Here, electromigrative separation techniques with their orthogonal separation mechanism are an attractive alternative. A very promising electrophoretic separation technique, which is primarily used during this work, is capillary electrophoresis (CE). One of the greatest challenges using this technique lies in the separation of biological samples, since analytes such as polyamines, peptides and proteins interact with and adsorb on the surface of “bare fused silica”-capillaries which impairs reproducibility. Without efficient suppression of these interactions, separation efficiency and run-to-run reproducibility suffer. A good way to suppress these detrimental interactions of analytes with the capillary surface is to modify the surface using of dynamic, statically adsorbed or covalently bound capillary coatings. In this work, I present approaches for the reproducible separation of polyamines, peptides and proteins: 1) In Chapter 2, the use of poly ethylene oxide as dynamic coating in SDS-CE enables the size-based separation of proteins up to a weight of 100 kDa. Advantages of this technique over classic gelbased SDS-PAGE are separation times of about 20 min and direct quantification via on-line UVdetection without the need of preliminary labeling or subsequent dyeing. Separation times were reduced to 5 min by short-end-injection and modification of the aperture for UV-detection. The presented separation system offers outstanding matrix tolerance: even complex samples such as serum were successfully separated without additional processing. Increased separation performance and efficiency were aspired by the addition of alkanols to the BGE, variation of temperature and the use of enrichment plugs in the capillary. Therein, especially the use of 2- propanol in the BGE proves fruitful regarding separation efficiency in the mass range up to 40 kDa. In Chapter 3 I will interpret my results with an extensive literature search to show, that the observed increase in separation efficiency is linked to a change of the separation mechanism from Reptation- to Ogston-sieving. 2) In Chapter 4, I proudly present, that I achieved CE-separation with MS-hyphenation not only for small polyamines and peptides, but also of large, non-digested proteins. This was possible using a single capillary coating only based on N-acryloylamido ethoxyethanol (AAEE). This highly polar and covalently bound capillary coating offers enjoyably high reproducibly and stability, the latter enabling operating times of 100 h, even when complex samples such as human serum and polyamines in fish eggs were analyzed. In Chapter 5 a novel and parallelized approach for the synthesis of this capillary coating is presented. SEM-measurements of the capillary surface between reaction steps forced me to postulate a novel reaction-mechanism for the formation of the coated surface. Additionally, I present, that pre-conditioning of capillaries with hypercritical water can result in higher reproducibility of capillary-to-capillary performance and reduced synthesis time. In Chapter 6 I will show that the presented separation techniques are excellent for the separation and detection of proteins equilibrated using hexapeptide ligand libraries (HLL). A novel approach for the consecutive equilibration of small sample volumes, which enables a deep insight into the proteome, is critically discussed. Challenges intrinsic to the solid phase extraction of proteins using HLLs are traced back to irreversible binding sites on HLLs. To tackle this issue, different elution and pre-equilibration protocols are designed and investigated. To re-establish binding conditions in consecutive equilibration, which is consecutive equilibration, different protocols for the processing of eluates from HLLs using 10 kDa cut-off filters are presented. Aspects that critically impair yields and recovery rates come to the fore and improved protocols are presented. A further project focused on CE-MS-based pI-value determination of a hardly soluble, cyclic and antibiotic peptide (Chapter 7). Detection of this peptide was not possible using AAEE-coated capillaries. This problem was overcome by using non-coated capillaries and BGEs containing small amounts of citric acid, which functions not only as buffer but also as a dynamic capillary coating. To confirm the determined pI-vaues, a novel and time-saving approach for the sequential injection of amino acid reference substances was developed.Fragestellungen in der Chemie aber auch in vielen anderen Disziplinen wie der Medizin, Umwelttoxikologie und Biologie, erfordern die Untersuchung von teilweise sehr komplexen Stoffgemischen. Eine schnelle Untersuchung von komplexen Proben ist mittels Trenntechniken, gefolgt von spektroskopischer oder spektrometrischer Detektion möglich. Zu den am weitesten verbreiteten Techniken gehören hierbei chromatographische Methoden wie die Gas- und Flüssigchromatographie, häufig gekoppelt mit Massenspektrometrie. Geraten diese chromatographischen Techniken an ihre Grenzen, beispielsweise bei hoch geladenen Stoffen, so bietet sich der Einsatz von elektromigrativen Trenntechniken an, da diese einen orthogonalen Trennmechanismus besitzen. Die Kapillarelektrophorese (engl.: capillary electrophoresis, CE), mit welcher sich diese Arbeit befasst, zählt zu den elektromigrativen Trenntechniken. Eine große Herausforderung dieser Technik besteht u.a. in der geringen Reproduzierbarkeit bei der Analytik von Biomolekülen, welche sich auf die Verwendung von „bare fused silica“-Kapillaren und deren Wechselwirkung mit den Analyten zurückführen lässt. Wird diese Wechselwirkung nicht effizient unterdrückt, so sinkt die Trenneffizienz innerhalb und die Wiederholbarkeit zwischen Trennungen. Durch die Verwendung von dynamischen, statisch adsorbierten oder kovalent gebundenen Kapillarbeschichtungen kann diese Wechselwirkung effizient unterdrückt werden. In dieser Arbeit werden mehrere Möglichkeiten zur reproduzierbaren Trennung von Polyaminen, Peptiden und Proteinen vorgestellt: 1) In Kapitel 2 wird durch die Verwendung von Polyethylenoxid als dynamische Beschichtung und Siebmatrix in der SDS-CE die größenbasierte Trennung von Proteinen bis zu einer Masse von 100 kDa erreicht. Ein Vorteil gegenüber klassischen gelbasierten Verfahren wie SDS-PAGE ist hierbei, dass Proben innerhalb von 20 min getrennt und durch on-column UV-Detektion quantifiziert werden können, ohne vor- oder nachträgliches Anfärben oder Derivatisieren. Durch eine selbst entworfene Modifikation der Trennapparatur und Injektion vom kurzen Ende der Kapillare konnte diese Trennzeit für ein schnelles Screening auf 5 Minuten reduziert werden. Das vorgestellte Trennsystem zeichnet sich durch eine hohe Matrixtoleranz aus; selbst Proben wie menschliches Serum können ohne Aufarbeitung injiziert werden. Eine Verbesserung der Auflösung und Trenneffizienz wurde durch die Verwendung von verschiedenen Alkoholen im Hintergrundelektrolyten, die Variation der Trenntemperatur und die Etablierung von Anreicherungszonen in der Kapillare angestrebt. Hierbei zeigte vor allem die Verwendung von 2- Propanol im Hintergrundelektrolyten eine erhöhte Trenneffizienz im Massenbereich bis ca. 40 kDa. In Kapitel 3 wird anhand der eigenen Ergebnisse und durch ausführliche Literaturarbeit gezeigt, dass diese Verbesserung der Auflösung durch eine Verschiebung des Trennmechanismus von Reptation- zu Ogstonsieben ermöglicht wird. 2) Durch die Verwendung einer sehr polaren kovalenten Kapillarbeschichtung, dem NAcryloylamido- ethoxyethanol (AAEE), lässt sich die kapillarelektrophoretische Trennung von kleinen Polyaminen und Peptiden, aber auch großen und unverdauten Proteinen, bei gleichzeitiger massenspektrometrischer Detektion erreichen. Dies wird in Kapitel 4 vorgestellt. Erfreulich hohe Einsatzzeiten von ca. 100 h und gute Wiederholbarkeiten werden sogar bei der Untersuchung von menschlichem Serum oder Polyaminen in Fischeiern beobachtet. In Kapitel 5 werden ein neuer und ABSTRACT (DEUTSCH) 4 parallelisierter Ansatz zur Kapillarsynthese sowie ein koexistenter und durch SEM-Aufnahmen postulierter Reaktionsmechanismus vorgestellt. Zusätzlich wird ein Ansatz zur Vorbehandlung der Kapillaren durch die Verwendung von überkritischem Wasser vorgeschlagen, welcher ersten Versuchen nach zu einer höheren Reproduzierbarkeit der Beschichtung und einer beschleunigten Synthese führen kann. In Kapitel 6 werden die vorgestellten Analysetechniken werden für die Bestimmung von Proteinen in mittels Hexapeptidligandenbibliotheken angereicherten Proben verwendet. Ein Ansatz zur mehrfachen Anreicherung, der einen tiefen Blick ins Proteom erlauben soll, wird kritisch bewertet. Herausforderungen in der Festphasenanreicherung werden auf irreversible Bindungsstellen zurückgeführt. In diesem Zusammenhang werden verschiedene Elutions- und PreÄquilibrierungsprotokolle untersucht. Für die erfolgreiche Wiederbeladung, welche einen tieferen Blick ins Proteom erlaubt, werden verschiedene Protokolle zur Aufarbeitung des Eluats mittels 10 kDa-Cut-off-Filtern vorgestellt. Kritische Aspekte, welche die Ausbeuten beeinträchtigen, werden beleuchtet und Lösungswege aufgezeigt. In Kapitel 7 wird die MS-basierte pI-Wertbestimmung eines schwerlöslichen zyklischen Peptidantibiotikums vorgestellt. Dieses konnte mit AAEE-beschichteten Kapillaren nicht erfasst werden. Diese Herausforderung konnte durch die Verwendung unbeschichteter Kapillaren und geringen Mengen Zitronensäure im Hintergrundelektrolyt, welche als dynamische Beschichtung fungiert, umgangen werden. Zur Absicherung der bestimmten pI-Werte mit Aminosäurestandards wurde ein neuer und zeitsparender Ansatz der sequentiellen Injektion entwickelt

Spermidine, but not spermine, is essential for pigment pattern formation in zebrafish

Polyamines are small poly-cations essential for all cellular life. The main polyamines present in metazoans are putrescine, spermidine and spermine. Their exact functions are still largely unclear; however, they are involved in a wide variety of processes affecting cell growth, proliferation, apoptosis and aging. Here we identify idefix, a mutation in the zebrafish gene encoding the enzyme spermidine synthase, leading to a severe reduction in spermidine levels as shown by capillary electrophoresis-mass spectrometry. We show that spermidine, but not spermine, is essential for early development, organogenesis and colour pattern formation. Whereas in other vertebrates spermidine deficiency leads to very early embryonic lethality, maternally provided spermidine synthase in zebrafish is sufficient to rescue the early developmental defects. This allows us to uncouple them from events occurring later during colour patterning. Factors involved in the cellular interactions essential for colour patterning, likely targets for spermidine, are the gap junction components Cx41.8, Cx39.4, and Kir7.1, an inwardly rectifying potassium channel, all known to be regulated by polyamines. Thus, zebrafish provide a vertebrate model to study the in vivo effects of polyamines

Is the pi-particle responsible for the 41 meV peak in YBa_2Cu_3O_7?

It is argued that there is no low-energy resonance associated with the pi-operators introduced by Demler and Zhang. This implies that the Hubbard model does not possess an approximate SO(5) symmetry generated by these operators. Recent finite-size studies are re-interpreted accordingly.Comment: 3 pages, latex (revtex

Uniformity in association schemes and coherent configurations: cometric Q-antipodal schemes and linked systems

Inspired by some intriguing examples, we study uniform association schemes and uniform coherent configurations, including cometric Q-antipodal association schemes. After a review of imprimitivity, we show that an imprimitive association scheme is uniform if and only if it is dismantlable, and we cast these schemes in the broader context of certain --- uniform --- coherent configurations. We also give a third characterization of uniform schemes in terms of the Krein parameters, and derive information on the primitive idempotents of such a scheme. In the second half of the paper, we apply these results to cometric association schemes. We show that each such scheme is uniform if and only if it is Q-antipodal, and derive results on the parameters of the subschemes and dismantled schemes of cometric Q-antipodal schemes. We revisit the correspondence between uniform indecomposable three-class schemes and linked systems of symmetric designs, and show that these are cometric Q-antipodal. We obtain a characterization of cometric Q-antipodal four-class schemes in terms of only a few parameters, and show that any strongly regular graph with a ("non-exceptional") strongly regular decomposition gives rise to such a scheme. Hemisystems in generalized quadrangles provide interesting examples of such decompositions. We finish with a short discussion of five-class schemes as well as a list of all feasible parameter sets for cometric Q-antipodal four-class schemes with at most six fibres and fibre size at most 2000, and describe the known examples. Most of these examples are related to groups, codes, and geometries.Comment: 42 pages, 1 figure, 1 table. Published version, minor revisions, April 201

The tails in the Helix Nebula NGC 7293

We have examined a stream-source model for the production of the cometary tails observed in the Helix Nebula NGC 7293 in which a transonic or moderately supersonic stream of ionized gas overruns a source of ionized gas. Hydrodynamic calculations reveal velocity structures which are in good agreement with the observational data on tail velocities and are consistent with observations of the nebular structure. The results also are indicative of a stellar atmosphere origin for the cometary globules. Tail remnants persist for timescales long enough for their identification with faint striations visible in the nebula gas to be plausible.Comment: 7 pages, 6 figures, accepted for publication in A&

Time--delay autosynchronization of the spatio-temporal dynamics in resonant tunneling diodes

The double barrier resonant tunneling diode exhibits complex spatio-temporal patterns including low-dimensional chaos when operated in an active external circuit. We demonstrate how autosynchronization by time--delayed feedback control can be used to select and stabilize specific current density patterns in a noninvasive way. We compare the efficiency of different control schemes involving feedback in either local spatial or global degrees of freedom. The numerically obtained Floquet exponents are explained by analytical results from linear stability analysis.Comment: 10 pages, 16 figure

The mass-loss return from evolved stars to the Large Magellanic Cloud III. Dust properties for carbon-rich asymptotic giant branch stars

We present a 2Dust model for the dust shell around a LMC long-period variable (LPV) previously studied as part of the OGLE survey. OGLE LMC LPV 28579 (SAGE J051306.40-690946.3) is a carbon-rich asymptotic giant branch (AGB) star for which we have photometry and spectra from the Spitzer SAGE and SAGE-Spec programs along with UBVIJHK_s photometry. By modeling this source, we obtain a baseline set of dust properties to be used in the construction of a grid of models for carbon stars. We reproduce its spectral energy distribution using a mixture of AmC and SiC (15% by mass). The grain sizes are distributed according to the KMH model. The best-fit model has an optical depth of 0.28 for the shell at the peak of the SiC feature, with R_in~1430 R_sun or 4.4 R_star. The temperature at this inner radius is 1310 K. Assuming an expansion velocity of 10 km s^-1, we obtain a dust mass-loss rate of 2.5x10^-9 M_sun yr-1. We calculate a 15% variation in this rate by testing the fit sensitivity against variation in input parameters. We also present a simple model for the molecular gas in the extended atmosphere that could give rise to the 13.7 \mu m feature seen in the spectrum. We find that a combination of CO and C_2H_2 gas at an excitation temperature of about 1000 K and column densities of 3x10^21 cm^-2 and 10^19 cm^-2 respectively are able to reproduce the observations. Given that the excitation temperature is close to T_dust(R_in), most of the molecular contribution probably arises from the inner shell region. The luminosity corresponding to the first epoch of SAGE observations is 6580 L_sun. For an effective temperature of about 3000 K, this implies a stellar mass of 1.5-2 M_sun and an age of 1-2.5 Gyr. For a gas:dust ratio of 200, we obtain a gas mass-loss rate of 5.0x10^-7 M_sun yr^-1, consistent with the gas mass-loss rates estimated from the period, color and 8 \mu m flux of the source.Comment: 14 pages, 5 figures, accepted for publication in Astronomy & Astrophysic

Geochemistry and paleogeographic implications of Permo-Triassic metasedimentary cover from the Tauern Window (Eastern Alps)

The chemical composition of metasediments is a valuable source of paleogeographic information about the protolith's sedimentary environment. Here, we compile major- and trace-element whole-rock data, including B contents, and 10/11 B-isotope ratios from the Permo-Triassic metasedimentary cover of the Pfitsch–Mörchner basin, overlying the Variscan basement in the western Tauern Window, Eastern Alps (Austria and Italy). The basement consists of orthogneiss (“Zentralgneis”, metamorphosed Variscan granitoids with intrusion ages between 305 and 280 Ma), and the roof pendant consists of granites (amphibolites, paragneiss, and minor serpentinites). The Zentralgneis is partly hydrothermally altered into pyrite quartzite with high Al–S contents, low Na–Sr–Ca–Mg contents, and very strong depletion of the light rare earth elements. Comparison with published detailed mapping of this and other time-equivalent basins in the western Tauern Window, with radiometric age data in the literature, and with unmetamorphosed basins in the South Alpine realm yields a late Permian to Early Triassic age of sedimentation. Although during Alpine metamorphism all rocks were strongly deformed, the whole-rock chemical compositions of the metasediments were not pervasively changed during deformation. We show that the sediments were deposited in a small, probably lacustrine–fluviatile, intramontane basin, under arid to semi-arid climatic conditions. The sequence starts with metaconglomerates, which can be interpreted as a mixture of the different basement rocks, based on a combination of major-element ratios Na2O /  (Na2O + K2O) and MgO / (MgO + Fe2O3) with concentrations of trace elements Cr, V, and Ni. The sequence is overlain by a fining-upwards sequence of clastic sediments, in which the behavior of K, Rb, and Sr allows the reconstruction of intense diagenetic K–B metasomatism, which raised the K2O contents up to ∼ 10 wt %. The average B content of 218 µg g−1 is well above the B content of common sediments, and the B-isotope composition reaches extremely low values of down to −33 ‰ δ11B. The top of the sequence is a lazulite quartzite, interpreted as a former conglomeratic phosphatic sandstone, which marks the transition from a closed Permian basin to an open Triassic basin. Within the clastic sequence, the presence of hydrothermal tourmalinite veins documents a hydrothermal event after deposition but before the onset of Alpine metamorphism. A metamorphosed mafic dike swarm in the orthogneiss indicates a post-Variscan event of basaltic magmatism, and this event is tentatively correlated with increased heat flow in the Triassic basin and hydrothermal activity. A consistent conceptual model of this basin and its diagenetic modifications, based on a combination of geochemical data with petrographical and field information, provides the geodynamic context of the European margin at the onset of the Alpine orogeny.DFG, 414044773, Open Access Publizieren 2021 - 2022 / Technische Universität Berli