Development and application of high-performance liquid chromatography- and mass spectrometry-based two-dimensional methods for proteome analysis

The availability of robust, sensitive and reliable analytic methods is a highly relevant requirement for proteome analysis. In this work two gel-free two-dimensional high-performance liquid chromatography-matrix assisted laser desorption/ionization mass spectrometry (2D HPLC-MALDI-MS) platforms suitable for proteome analysis were developed and compared. To provide an alternative approach to the classical bottom-up method combining strong cation-exchange- and ion-pair reversed phase HPLC (SCX x IP-RP-HPLC), in which peptides are separated in both dimensions, a semi top-down method was elaborated. This new approach employed separation of proteins in the first and of peptides in the second dimension, respectively. Both separation dimensions of the new method were operated in the ion-pair reversed phase (IP-RP) chromatographic mode using poly-(styrene-divinylbenzene) (PS-DVB)-based monolithic columns and hydro-organic eluents at a pH value of 2.1. Both methods were validated using the same 10-protein mixture. The elaborated semi top-down- and the classical bottom-up approach were applied to the proteome analysis of a Glioblastoma multiforme tissue protein extract including the comparison of the two approaches. Fewer proteins were identified with the semi top-down approach. However, the sequence coverage of the proteins identified was slightly higher. The major advantage of this new method is the elution of proteins in less fractions of the first separation dimension than for the bottom-up approach. Thus, only the fractions containing the target proteins have to be digested in further experiments. Five of 13 potential biomarkers previously identified applying the SEREX (serological identification of antigens by recombinant expression screening) approach, could be confirmed on molecular level by combining the results of both chromatographic approaches.Robuste, sensitive und zuverlässige Analysenmethoden sind eine enorm wichtige Voraussetzung für die Proteomanalyse. In dieser Arbeit wurden zwei zwei-dimensionale gelfreie Plattformen basierend auf Hochleistungsflüssig-chromatographie und matrixunterstützter Laser Desorptions-/Ionisations Massen-spektrometrie (2D HPLC-MALDI-MS), die off-line gekoppelt wurden, entwickelt und miteinander verglichen. Eine Alternativmethode zur klassischen bottom-up Methode, welche starken Kationenaustausch- mit Ionenpaar-Umkehrphasenchromatographie verbindet (SCX x IP-RP-HPLC) und bei der in beiden Dimensionen Peptide getrennt werden, wurde erarbeitet. Diese alternative semi top-down Methode beinhaltete die Trennung von Proteinen in der ersten und von Peptiden in der zweiten Dimension. In beiden Dimensionen der neuen Methode wurde der chromatographische Ionen-Umkehrphasen Modus (IP-RP-HPLC) in Verbindung mit Polystyrol-Divenylbenzol (PS-DVB) basierten Monolithen und hydro-organischen Eluenten bei einem pH Wert von 2.1 verwendet. Beide Methoden wurden mit einem 10-Proteinstandard validiert. Die erarbeitete semi top-down und die klassische bottom-up Methode wurden auf die Proteomanalyse eines Glioblastoma multiforme Gewebe Proteinextraktes angewandt und verglichen. Mit der semi top-down Methode wurden zwar weniger Proteine identifiziert, aber deren Sequenzabdeckung war höher. Der Vorteil dieser Methode ist die Elution der Proteine in weniger Fraktionen der ersten Dimension der Trennung. Nur die Fraktionen, die die Targetproteine enthalten, müssen in weiteren Experimenten verdaut werden. Fünf von 13 Biomarkern, die vorher mit der SEREX (serological identification of antigens by recombinant expression screening) - Methode identifiziert wurden, konnten auf der molekularen Ebene unter Verbindung der Ergebnisse beider chromatographischer Methoden, nachgewiesen werden

Selective Attenuation of Norepinephrine Release and Stress-Induced Heart Rate Increase by Partial Adenosine A1 Agonism

The release of the neurotransmitter norepinephrine (NE) is modulated by presynaptic adenosine receptors. In the present study we investigated the effect of a partial activation of this feedback mechanism. We hypothesized that partial agonism would have differential effects on NE release in isolated hearts as well as on heart rate in vivo depending on the genetic background and baseline sympathetic activity. In isolated perfused hearts of Wistar and Spontaneously Hypertensive Rats (SHR), NE release was induced by electrical stimulation under control conditions (S1), and with capadenoson 6 · 10−8 M (30 µg/l), 6 · 10−7 M (300 µg/l) or 2-chloro-N6-cyclopentyladenosine (CCPA) 10−6 M (S2). Under control conditions (S1), NE release was significantly higher in SHR hearts compared to Wistar (766+/−87 pmol/g vs. 173+/−18 pmol/g, p<0.01). Capadenoson led to a concentration-dependent decrease of the stimulation–induced NE release in SHR (S2/S1 = 0.90±0.08 with capadenoson 6 · 10−8 M, 0.54±0.02 with 6 · 10−7 M), but not in Wistar hearts (S2/S1 = 1.05±0.12 with 6 · 10−8 M, 1.03±0.09 with 6 · 10−7 M). CCPA reduced NE release to a similar degree in hearts from both strains. In vivo capadenoson did not alter resting heart rate in Wistar rats or SHR. Restraint stress induced a significantly greater increase of heart rate in SHR than in Wistar rats. Capadenoson blunted this stress-induced tachycardia by 45% in SHR, but not in Wistar rats. Using a [35S]GTPγS assay we demonstrated that capadenoson is a partial agonist compared to the full agonist CCPA (74+/−2% A1-receptor stimulation). These results suggest that partial adenosine A1-agonism dampens stress-induced tachycardia selectively in rats susceptible to strong increases in sympathetic activity, most likely due to a presynaptic attenuation of NE release

Modelling human choices: MADeM and decision‑making

Research supported by FAPESP 2015/50122-0 and DFG-GRTK 1740/2. RP and AR are also part of the Research, Innovation and Dissemination Center for Neuromathematics FAPESP grant (2013/07699-0). RP is supported by a FAPESP scholarship (2013/25667-8). ACR is partially supported by a CNPq fellowship (grant 306251/2014-0)

Entwicklung und Anwendung von zweidimensionalen Methoden für die Proteomanalyse basierend auf Hochleistungsflüssigchromatographie und Massenspektrometrie

The availability of robust, sensitive and reliable analytic methods is a highly relevant requirement for proteome analysis. In this work two gel-free two-dimensional high-performance liquid chromatography-matrix assisted laser desorption/ionization mass spectrometry (2D HPLC-MALDI-MS) platforms suitable for proteome analysis were developed and compared. To provide an alternative approach to the classical bottom-up method combining strong cation-exchange- and ion-pair reversed phase HPLC (SCX x IP-RP-HPLC), in which peptides are separated in both dimensions, a semi top-down method was elaborated. This new approach employed separation of proteins in the first and of peptides in the second dimension, respectively. Both separation dimensions of the new method were operated in the ion-pair reversed phase (IP-RP) chromatographic mode using poly-(styrene-divinylbenzene) (PS-DVB)-based monolithic columns and hydro-organic eluents at a pH value of 2.1. Both methods were validated using the same 10-protein mixture. The elaborated semi top-down- and the classical bottom-up approach were applied to the proteome analysis of a Glioblastoma multiforme tissue protein extract including the comparison of the two approaches. Fewer proteins were identified with the semi top-down approach. However, the sequence coverage of the proteins identified was slightly higher. The major advantage of this new method is the elution of proteins in less fractions of the first separation dimension than for the bottom-up approach. Thus, only the fractions containing the target proteins have to be digested in further experiments. Five of 13 potential biomarkers previously identified applying the SEREX (serological identification of antigens by recombinant expression screening) approach, could be confirmed on molecular level by combining the results of both chromatographic approaches.Robuste, sensitive und zuverlässige Analysenmethoden sind eine enorm wichtige Voraussetzung für die Proteomanalyse. In dieser Arbeit wurden zwei zwei-dimensionale gelfreie Plattformen basierend auf Hochleistungsflüssig-chromatographie und matrixunterstützter Laser Desorptions-/Ionisations Massen-spektrometrie (2D HPLC-MALDI-MS), die off-line gekoppelt wurden, entwickelt und miteinander verglichen. Eine Alternativmethode zur klassischen bottom-up Methode, welche starken Kationenaustausch- mit Ionenpaar-Umkehrphasenchromatographie verbindet (SCX x IP-RP-HPLC) und bei der in beiden Dimensionen Peptide getrennt werden, wurde erarbeitet. Diese alternative semi top-down Methode beinhaltete die Trennung von Proteinen in der ersten und von Peptiden in der zweiten Dimension. In beiden Dimensionen der neuen Methode wurde der chromatographische Ionen-Umkehrphasen Modus (IP-RP-HPLC) in Verbindung mit Polystyrol-Divenylbenzol (PS-DVB) basierten Monolithen und hydro-organischen Eluenten bei einem pH Wert von 2.1 verwendet. Beide Methoden wurden mit einem 10-Proteinstandard validiert. Die erarbeitete semi top-down und die klassische bottom-up Methode wurden auf die Proteomanalyse eines Glioblastoma multiforme Gewebe Proteinextraktes angewandt und verglichen. Mit der semi top-down Methode wurden zwar weniger Proteine identifiziert, aber deren Sequenzabdeckung war höher. Der Vorteil dieser Methode ist die Elution der Proteine in weniger Fraktionen der ersten Dimension der Trennung. Nur die Fraktionen, die die Targetproteine enthalten, müssen in weiteren Experimenten verdaut werden. Fünf von 13 Biomarkern, die vorher mit der SEREX (serological identification of antigens by recombinant expression screening) - Methode identifiziert wurden, konnten auf der molekularen Ebene unter Verbindung der Ergebnisse beider chromatographischer Methoden, nachgewiesen werden

Diffuse Glioneuronal tumour with Oligodendroglioma‐like features and Nuclear Clusters (DGONC) – a molecularly‐defined glioneuronal CNS tumour class displaying recurrent monosomy 14

International audienc