Systematic assessment of protein profiles of primary and cultured human cells

Ein wichtiges Ziel der Proteom Forschung ist die Erstellung von Proteinprofilen von biologischen Proben, wodurch neue Erkenntnisse über die Expressionsmuster von Proteinen der untersuchten Zellen oder Geweben gewonnen werden und ein neues Verständnis für biologische Prozesse entsteht, was zur Ermittlung von Biomarkern und bisher unbekannten Angriffspunkten für Medikamente führen kann. Das Ziel dieser Doktorarbeit war die Erstellung von Protein-Referenzlisten von unterschiedlichen Zelltypen, wie von normalen primären Zellen, aber auch von immortalisierten oder transformierten kultivierten Zellen und von Zellen in unterschiedlichen funktionellen Zuständen. Die Analyse der entsprechenden Proteinprofile wurde mit dem Ziel, zelltypspezifische und zellfunktionsassoziierte Proteine zu identifizieren, durchgeführt. Mit Hilfe einer selbstgebauten SQL-Datenbank konnte die Interpretation von Proteinprofilen, die normalerweise mehr als 1000 Proteine umfassen, unterstützt werden. Im Laufe dieser Untersuchungen wurden die folgenden Resultate erzielt. Die zytoplasmatischen Fraktionen von vier sehr unterschiedlichen Zelltypen, nämlich humane dendritische Zellen, Endothelzellen, Fibroblasten und Keratinozyten, wurden verglichen. Proteine, die in allen vier Zelltypen vorkommen, wurden ermittelt und identifiziert. Die Liste dieser ubiquitär exprimierten Proteine kann nun als interner Standard für Proteom Analysen verwendet werden. Proteinprofile von Blutkomponenten, wie Lymphozyten, Monozyten, Neutrophile, Erythrozyten und Blutplättchen, die aus humanen peripheren mononuklearen Blutzellen isoliert worden sind, wurden verglichen, und spezifische Proteine von jeder Komponente wurden erfolgreich identifiziert. Die Zuordnung von Proteinen zu ihrer Herkunftsquelle macht es nun einfacher, die Ergebnisse aus vergleichenden Analysen zu interpretieren. Vergleichende Proteom- und Sekretomanalysen wurden an primeren humanen Hepatozyten und den etablierten Zelllinien HepG2 und Hep3B durchgeführt, um die Differenzierungszustände und funktionellen Zustände dieser Zellen zu bestimmen. In HepG2 konnten mehr hepatozytenspezifische Proteine nachgewiesen werden als in Hep3B. Hep3B exprimieren Proteine, die auf einen epithelial-mesenchymalen Übergang hinweisen. Proteomprofile können demnach dazu beitragen, Differenzierungszustände und funktionelle Zustände umfassend abzuschätzen.An important ambition of proteome research is the establishment of protein profiles of biological samples, providing insight in the protein expression pattern of investigated cells or tissues and thereby supporting the understanding of biological processes and contributing to the identification of biomarkers and novel drug targets. The aim of this PhD-thesis was to generate reference maps of different cell types, such as normal primary cells, but also of immortalized or transformed cultured cells and cells at different functional states. The analysis of the resulting proteome profiles was done in order to identify cell type-specific and cell function-associated proteins. By means of a home-made SQL-database, the interpretation of proteome profiles, typically comprising more than 1000 proteins, was supported. In the course of these investigations, the following results were obtained. The cytoplasmic fractions of four largely differing kinds of cells, namely human dendritic cells, endothelial cells, fibroblasts and keratinocytes were compared and proteins which occurred in each of the four cell types were isolated and identified. The list of these ubiquitously expressed proteins can now be used as internal standard for proteome profiling. Comparative proteome profiling of blood constituents such as lymphocytes, monocytes, neutrophils, erythrocytes and platelets, isolated from human peripheral blood mononuclear cells, was performed and specific proteins of each of the purified constituents were successfully identified. The assignment of proteins to their putative source of origin makes now comparative analyses easier to interpret. Comparative proteome and secretome profiling was performed on primary human hepatocytes and on the well-established cell lines HepG2 and Hep3B, in order to assess the differentiation and functional states of these cells. HepG2 showed more features characteristic for hepatocytes than Hep3B, while Hep3B express proteins indicative for an epithelial-mesenchymal transition. Proteome profiling can thus serve to get a comprehensive assessment of functional cell states and cell differentiation states

Quantification of Cytokines secreted by primary human cells using multiple reaction monitoring: evaluation of analytical parameters

Determination of secreted proteins provides highly valuable information about cell functions. While the typical methods for the determination of biologically relevant but low-abundant molecular species still relies on the use of specific antibodies, mass spectrometry-based methods are now gaining sufficient sensitivity to cope with such challenges as well. In the current study we have identified several cytokines and chemokines which were induced in primary human umbilical vein endothelial cells upon inflammatory activation. Based on the high-resolution mass spectrometry data obtained with a Q Exactive orbitrap, we built an MRM method to quantify the most relevant molecules selected from the screening experiment. All experimental data are available via ProteomeXchange, PXD002211/12, and Panorama, www.panoramaweb.org. Using nano-flow Chip-HPLC coupled to a 6490 triple-quadrupole MS for MRM analyses we achieved calibration curves covering a linear range of four orders of magnitude and detection limits in the low attomol per microliter concentration range. Carryover was consistently less than 0.005%, the accuracy was between 80% and 120%, and the median coefficient of variation for LC/MS was only 2.2%. When including the variance of quantification introduced by cell culture and digestion, the coefficient of variation was less than 20% for most peptides. With appropriate marker molecules we monitored typical variations introduced by cell culture caused by differences in cell numbers, proliferative states and cell death. As a result, here, we present a robust and efficient MRM-based assay for the accurate and sensitive determination of cytokines and chemokines representative for functional cell states and including comprehensive quality controls

Membrane disruption, but not metabolic rewiring, is the key mechanism of anticancer-action of FASN-inhibitors: a multi-omics analysis in ovarian cancer.

Fatty-acid(FA)-synthase(FASN) is a druggable lipogenic oncoprotein whose blockade causes metabolic disruption. Whether drug-induced metabolic perturbation is essential for anticancer drug-action, or is just a secondary-maybe even a defence response-is still unclear. To address this, SKOV3 and OVCAR3 ovarian cancer(OC) cell lines with clear cell and serous histology, two main OC subtypes, were exposed to FASN-inhibitor G28UCM. Growth-inhibition was compared with treatment-induced cell-metabolomes, lipidomes, proteomes and kinomes. SKOV3 and OVCAR3 were equally sensitive to low-dose G28UCM, but SKOV3 was more resistant than OVCAR3 to higher concentrations. Metabolite levels generally decreased upon treatment, but individual acylcarnitines, glycerophospholipids, sphingolipids, amino-acids, biogenic amines, and monosaccharides reacted differently. Drug-induced effects on central-carbon-metabolism and oxidative-phosphorylation (OXPHOS) were essentially different in the two cell lines, since drug-naïve SKOV3 are known to prefer glycolysis, while OVCAR3 favour OXPHOS. Moreover, drug-dependent increase of desaturases and polyunsaturated-fatty-acids (PUFAs) were more pronounced in SKOV3 and appear to correlate with G28UCM-tolerance. In contrast, expression and phosphorylation of proteins that control apoptosis, FA synthesis and membrane-related processes (beta-oxidation, membrane-maintenance, transport, translation, signalling and stress-response) were concordantly affected. Overall, membrane-disruption and second-messenger-silencing were crucial for anticancer drug-action, while metabolic-rewiring was only secondary and may support high-dose-FASN-inhibitor-tolerance. These findings may guide future anti-metabolic cancer intervention.The authors would like to thank Kratos/Shimadzu (Manchester, UK) for providing the MALDI-MS instrumentation used in this study and Dr. Steven Pelech (Kinexus Bioinformatics Corp, Vancouver, BC, Canada) for initial instruction in antibody microarray kinomic analysis. This work was financially supported by the Medical Scientific Fund of the Mayor of the City of Vienna, by the ‘Initiative Krebsforschung’ of the Medical University of Vienna, and by the Herzfelder Familienstiftung, Vienna, Austria.S

Comprehensive Assessment of Proteins Regulated by Dexamethasone Reveals Novel Effects in Primary Human Peripheral Blood Mononuclear Cells

Inflammation is a physiological process involved in many diseases. Monitoring proteins involved in regulatory effects may help to improve our understanding of inflammation. We have analyzed proteome alterations induced in peripheral blood mononuclear cells (PBMCs) upon inflammatory activation in great detail using high-resolution mass spectrometry. Moreover, the activated cells were treated with dexamethasone to investigate their response to this antiphlogistic drug. From a total of 6886 identified proteins, 469 proteins were significantly regulated upon inflammatory activation. Data are available via ProteomeXchange with identifiers PXD001415–23. Most of these proteins were counter-regulated by dexamethasone, with some exceptions concerning members of the interferon-induced protein family. To confirm some of these results, we performed targeted MRM analyses of selected peptides. The inflammation-induced upregulation of proteins such as IL-1β, IL-6, CXCL2, and GROα was confirmed, however, with strong quantitative interindividual differences. Furthermore, the inability of dexamethasone to downregulate inflammation-induced proteins such as PTX3 and TSG6 was clearly demonstrated. In conclusion, the relation of cell function as well as drug-induced modulation thereof was successfully mapped to proteomes, suggesting targeted analysis as a novel and powerful drug evaluation method. Although most consequences of dexamethasone were found to be compatible with the expected mode of action, some unexpected but significant observations may be related to adverse effects

Multi-omics Analysis of Serum Samples Demonstrates Reprogramming of Organ Functions Via Systemic Calcium Mobilization and Platelet Activation in Metastatic Melanoma

Pathophysiologies of cancer-associated syndromes such as cachexia are poorly understood and no routine biomarkers have been established, yet. Using shotgun proteomics, known marker molecules including PMEL, CRP, SAA, and CSPG4 were found deregulated in patients with metastatic melanoma. Targeted analysis of 58 selected proteins with multiple reaction monitoring was applied for independent data verification. In three patients, two of which suffered from cachexia, a tissue damage signature was determined, consisting of nine proteins, PLTP, CD14, TIMP1, S10A8, S10A9, GP1BA, PTPRJ, CD44, and C4A, as well as increased levels of glycine and asparagine, and decreased levels of polyunsaturated phosphatidylcholine concentrations, as determined by targeted metabolomics. Remarkably, these molecules are known to be involved in key processes of cancer cachexia. Based on these results, we propose a model how metastatic melanoma may lead to reprogramming of organ functions via formation of platelet activating factors from long-chain polyunsaturated phosphatidylcholines under oxidative conditions and via systemic induction of intracellular calcium mobilization. Calcium mobilization in platelets was demonstrated to alter levels of several of these marker molecules. Additionally, platelets from melanoma patients proved to be in a rather exhausted state, and platelet-derived eicosanoids implicated in tumor growth were found massively increased in blood from three melanoma patients. Platelets were thus identified as important source of serum protein and lipid alterations in late stage melanoma patients. As a result, the proposed model describes the crosstalk between lipolysis of fat tissue and muscle wasting mediated by oxidative stress, resulting in the metabolic deregulations characteristic for cachexia

Proteome profiling in IL-1β and VEGF-activated human umbilical vein endothelial cells delineates the interlink between inflammation and angiogenesis

<div><p>Endothelial cells represent major effectors in inflammation and angiogenesis, processes that drive a multitude of pathological states such as atherosclerosis and cancer. Both inflammation and angiogenesis are interconnected with each other in the sense that many pro-inflammatory proteins possess proangiogenic properties and vice versa. To elucidate this interplay further, we present a comparative proteome study of inflammatory and angiogenic activated endothelial cells. HUVEC were stimulated with interleukin 1-β and VEGF, respectively. Cultured primary cells were fractionated into secreted, cytoplasmic and nuclear protein fractions and processed for subsequent LC-MS/MS analysis. Obtained protein profiles were filtered for fraction-specific proteins to address potential cross fractional contamination, subjected to comparative computational biology analysis (GO-Term enrichment analysis, weighted gene co-expression analysis) and compared to published mRNA profiles of IL-1β respectively VEGF stimulated HUVEC. GO Term enrichment analysis and comparative pathway analysis revealed features such as NOD and NfkB signaling for inflammatory activated HUVEC and VEGF and ErB signaling for VEGF-activated HUVEC with potential crosstalk via map kinases MAP2K2. Weighted protein co-expression network analysis revealed several potential hub genes so far not associated with driver function in inflammation or angiogenesis such as HSPG2, ANXA3, and GPI. “Classical” inflammation or angiogenesis markers such as IL6, CXCL8 or CST1 were found in a less central position within the co-expression networks. In conclusion, this study reports a framework for the computational biology based analysis of proteomics data applied to cytoplasmic, nucleic and extracellular fractions of quiescent, inflammatory and angiogenic activated HUVEC. Novel potential hub genes relevant for these processes were successfully identified.</p></div

Multi-omics analysis of serum samples demonstrates reprogramming of organ functions via systemic calcium mobilization and platelet activation in metastatic melanoma

Pathophysiologies of cancer-associated syndroms such as cachexia are poorly understood and no routine biomarkers have been established, yet. Using shotgun proteomics, known marker molecules including PMEL, CRP, SAA and CSPG4 were found deregulated in patients with metastatic melanoma. Targeted analysis of 58 selected proteins with multiple reaction monitoring was applied for independent data verification. In three patients, two of which suffered from cachexia, a tissue damage signature was determined, consisting of nine proteins, PLTP, CD14, TIMP1, S10A8, S10A9, GP1BA, PTPRJ, CD44 and C4A, as well as increased levels of glycine and asparagine, and decreased levels of polyunsaturated phosphatidylcholine concentrations, as determined by targeted metabolomics. Remarkably, these molecules are known to be involved in key processes of cancer cachexia. Based on these results, we propose a model how metastatic melanoma may lead to reprogramming of organ functions via formation of platelet activating factors from long-chain polyunsaturated phosphatidylcholines under oxidative conditions and via systemic induction of intracellular calcium mobilization. Calcium mobilization in platelets was demonstrated to alterate levels of several of these marker molecules. Additionally, platelets from melanoma patients proved to be in a rather exhausted state, and platelet-derived eicosanoids implicated in tumor growth were found massively increased in blood from three melanoma patients. Platelets were thus identified as important source of serum protein and lipid alterations in late stage melanoma patients. As a result, the proposed model describes the crosstalk between lipolysis of fat tissue and muscle wasting mediated by oxidative stress, resulting in the metabolic deregulations characteristic for cachexia