Podcasts about silac

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2023.08.03.551843v1?rss=1 Authors: Sun, S., Tranchina, D., Gresham, D. Abstract: Cells arrest growth and enter a quiescent state upon nutrient deprivation. However, the molecular processes by which cells respond to different starvation signals to regulate exit from the cell division cycle and initiation of quiescence remains poorly understood. To study the role of protein expression and signaling in quiescence we combined temporal profiling of the proteome and phosphoproteome using stable isotope labeling with amino acids in cell culture (SILAC) in Saccharomyces cerevisiae (budding yeast). We find that carbon and phosphorus starvation signals activate quiescence through largely distinct remodeling of the proteome and phosphoproteome. However, increased expression of mitochondrial proteins is associated with quiescence establishment in response to both starvation signals. Deletion of the putative quiescence regulator RIM15, which encodes a serine-threonine kinase, results in reduced survival of cells starved for phosphorus and nitrogen, but not carbon. However, we identified common protein phosphorylation roles for RIM15 in quiescence that are enriched for RNA metabolism and translation. We also find evidence for RIM15-mediated phosphorylation of some targets, including IGO1, prior to starvation consistent with a functional role for RIM15 in proliferative cells. Finally, our results reveal widespread catabolism of amino acids in response to nitrogen starvation, indicating widespread amino acid recycling via salvage pathways in conditions lacking environmental nitrogen. Our study defines an expanded quiescent proteome and phosphoproteome in yeast, and highlights the multiple coordinated molecular processes at the level of protein expression and phosphorylation that are required for quiescence. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.11.24.517538v1?rss=1 Authors: Sampedro-Castaneda, M., Baltussen, L. L., Lopes, A. T., Qiu, Y., Sirvio, L., Mihaylov, S. R., Claxton, S., Richardson, J. C., Lignani, G., Ultanir, S. K. Abstract: Developmental and epileptic encephalopathies (DEEs) are a group of rare childhood disorders characterized by severe epilepsy and related cognitive deficits. Numerous DEE genes have been discovered thanks to advances in genomic diagnosis, yet putative molecular links between these disorders are not known. CDKL5 deficiency disorder (CDD, DEE2) is one of the most common forms of genetic epilepsy; it is caused by loss-of-function mutations in the brain-enriched kinase CDKL5. To elucidate CDKL5 function, we looked for CDKL5 substrates using a SILAC based phosphoproteomic screen. We identified the voltage-gated Ca2+ channel Cav2.3 (encoded by CACNA1E) as a novel physiological target of CDKL5 in mice and humans. Recombinant channel electrophysiology and interdisciplinary characterization of Cav2.3 phosphomutant mice revealed that the loss of Cav2.3 phosphorylation leads to channel gain-of-function via slower channel inactivation and enhanced acetylcholine-induced stimulation, resulting in increased neuronal excitability. These changes in Cav2.3 closely resemble those described for gain-of-function point-mutations in CACNA1E that cause DEE69, a disorder sharing clinical features with CDD. Our results show that these two single-gene disorders are mechanistically related. We suggest that CDD is partly a channelopathy with Cav2.3 gain-of-function, thus Cav2.3 inhibition could be therapeutic in these DEEs. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Link to bioRxiv paper: http://biorxiv.org/cgi/content/short/2022.11.04.515170v1?rss=1 Authors: Datta, S., Sugatha, J., Priya, A., Raj, P., Jaimon, E., Jose, A. Abstract: Sorting nexins (SNX) are a family of proteins containing the Phox homology domain, which shows a preferential endo-membrane association and regulates cargo sorting processes. Even with the vast amount of information unveiled systematically, the underlying mechanism of sorting remains elusive. Here, we established that SNX32, a SNX-BAR (Bin/Amphiphysin/Rvs) sub-family member, is associated with SNX4 via its BAR domain. We identified A226, Q259, E256, R366 of SNX32, and Y258, S448 of SNX4 at the interface of these two SNX proteins that are important for maintaining the association. Via its PX domain, SNX32 interacts with the Transferrin receptor (TfR) and Cation Independent Mannose-6-Phosphate Receptor (CIMPR). We showed that the conserved F131 in its PX domain is important in stabilising the above interactions. Silencing of SNX32 led to a defect in intracellular trafficking of TfR and CIMPR, which could be rescued by overexpressing shRNA-resistant snx32. We also showed that both individual domains play an essential role in trafficking. Our results indicate that SNX4, SNX32 and Rab11 may participate in a common pathway regulating transferrin trafficking; however, the existence of an independent pathway for Rab11 and SNX32 could not be completely ruled out. Further, we established that the PX domain of SNX32 could bind to PI(3)P and PI(4)P, suggesting a possible explanation for its sub-cellular localization. Taken together, our study showed that SNX32 mediate the trafficking of specific cargo molecules along distinct pathway via its PX domain-directed binding to phosphoinositides and its BAR domain-mediated association with other SNX family members. Further, using SILAC-based differential proteomics of the wild type and the mutant SNX32, impaired in cargo binding, we identified Basigin (BSG), an immunoglobulin super family member, as a potential interactor of SNX32 in SH-SY-5Y cells. We then demonstrated that SNX32 binds to BSG through its PX domain and facilitates its trafficking to the cell surface. In Neuro-Glial cell lines, the silencing of SNX32 led to defects in neuronal differentiation. Moreover, abrogation in lactate transport in the SNX32 depleted cells led us to propose that the SNX may contribute to maintaining the neuro-glial coordination via its role in BSG trafficking and the associated Monocarboxylate transporter activity. Copy rights belong to original authors. Visit the link for more info Podcast created by Paper Player, LLC

Episode 5: Dr Alex Makarov In this episode, Chris sits down with Dr Alex Makarov who recently completed a PhD in Eric Schirmer's lab in the Wellcome Centre for Cell Biology at the University of Edinburgh. His research focused on Lamin A proteins, an important structural protein which gives cells their flexibility and shape. Dr Makarov recently published his novel work on the alternative methods to characterise the structure of flexible Lamin A proteins, namely SILAC cross-linking mass spectrometry. Lamin-A function is so important that mutations leading to alterations of this protein's structure are linked to 13 distinct human syndromes ranging from cardiomyopathy to lipodystrophy and progeria.Here is the publication DOI: https://doi.org/10.1038/s41467-019-11063-6Listen until the end to hear our end segment on Veganuary and the future of food.

Tue, 11 Feb 2014 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/18660/ https://edoc.ub.uni-muenchen.de/18660/1/Zeiler_Marlis.pdf Zeiler, Marlis ddc:540, ddc:500, Fakultät für Chemie und Pharmazie

Signaling networks control and regulate outcomes in cells and organisms in both normal physiology and pathophysiological states. Signaling is traditionally represented and studied as a series of stepwise enzymatic events constituting a cascade. However, it is increasingly apparent that such representations limit understanding of signal transduction since these linear cascades function in an interconnected network that includes extensive cross talk among receptors and pathways. Mass spectrometry (MS)-based proteomics is a useful tool that allows a system-wide investigation of signaling events at the levels of post-translational modifications (PTMs), protein-protein interactions and changes in protein expression on a large scale. This technology now allows accurate quantification of thousands of proteins and their modifications in response to any perturbation. This thesis work is dedicated to the optimization and employment of quantitative mass spectrometry to cellular signaling and an application to segregate two lymphoma subtypes at the levels of protein expression and phosphorylation, employing state of the art liquid chromatography (LC)-MS/MS technologies coupled with improved sample preparation techniques and data analysis algorithms. In the first project I investigated the feasibility of a new, high accuracy fragmentation method called higher energy collisional dissociation (HCD) for the analysis of phospho-peptides. Using this method we were able to measure the phospho-proteome of a single cell line in 24h of measurement time which was a great improvement to previous capabilities. This fragmentation method that was originally thought to be slower and less sensitive than the standard method of low resolution collision induced dissociation (CID) fragmentation. However, our work proves this not to be the case and we showed that HCD outperformed the existing low resolution strategy [1]. In the second project I employed this HCD fragmentation technique on the LTQ-Orbitrap Velos for addressing the clinical question of segregating two subtypes of diffuse B-cell lymphoma (DLBCL). These subtypes are histologically indistinguishable but had been segregated on the basis of a gene expression signature. I employed the recently developed ‘super-SILAC’ approach with a ‘super-SILAC mix’ of multiple labeled cell lines. This heavy reference mix was spiked into several cell lines derived from the two DLBCL subtypes and analyzed LC-MS, resulting in successful segregation based on a distinct proteomic signature [2]. The third project deals with the in-depth analysis of the phospho-proteome of a human cancer cell line on a quadrupole-Orbitrap mass spectrometer using a label-free quantification approach. Our analysis uncovered about 50,000 distinct phosphorylated peptides in a single cell type across a number of cellular conditions allowing assessment of global properties of this large dataset. Strikingly, we found that at least three-quarters of the proteome can be phosphorylated which is much higher than current estimates. We also analyzed phosphotyrosine events using enrichment with anti-phospho-tyrosine antibodies to identify more than 1,500 site specific phosphorylation events. Unexpectedly tyrosine phosphorylated proteins were enriched among higher abundance proteins. The observed difference in phospho-protein abundance correlated with the substrate Km values of tyrosine kinases. For the first time we calculated site specific occupancies using label- free quantification and observed widespread full phosphorylation site occupancy during mitosis. In the final and main project, I applied proteomics and phospho-proteomics to the study of signal transduction in response to transforming growth factor-beta (TGF-β), a multifunctional cytokine. TGF-β signaling regulates many biological outcomes including cell growth, differentiation, morphogenesis, tissue homeostasis and regeneration. The cellular responses to this multifunctional ligand are diverse and can even be opposed to each other, depending on the cell type and the conditions. To shed light on the reasons for the different outcomes, we analyzed the early phospho-proteome and ensuing proteome alterations in response to TGF-β treatment in a keratinocyte cell line. The early SILAC based phospho-proteome analysis uncovered over 20,000 phosphorylation events across five time points (0 to 20 min) of TGF-β treatment. Building on our recent advances in instrumentation, sample preparation, and data analysis algorithms we measured a deep TGF-β responsive proteome at six late time points (6h to 48h) with corresponding controls in only eight days of measurement time. Our label-free approach identified about 8,000 proteins and quantified more than 6,000 of them. This deep proteome covered well established pathways involved in TGF-β signaling, allowing global evaluation at the level of individual pathway members. Combining the TGF-β responsive proteome with an in-silico upstream regulator analysis, we correctly retrieved several known and predicted novel transcription factors driving TGF-β induced cytostasis, de-differentiation and epithelial to mesenchymal transition (EMT). The combined analysis of transcription factor regulation with early phosphorylation changes and proteome changes enabled visualization of the intricate interplay of key transcription factors, kinases and various pathways driving cytostatis, EMT and other processes induced by TGF-β. In summary, my thesis developed a highly efficient phospho-proteomic workflow, which was applied to the measurement of a very deep phospho-proteome of a single cancer cell line allowing analysis of its global features. The main achievement was the first in-depth and combined study of the phospho-proteome and resulting proteome changes following a defined signaling event, in this case leading to a time-resolved view of TGF- β signaling events relevant in cancer.

Posttranslational modifications (PTMs) of proteins by covalent attachment of functional groups (like phosphorylation, acetylation, methylation, glycosylation, etc.) are of key importance for the cell as they regulate various aspects of protein behavior after its synthesis, e.g., dictate protein interaction properties, change catalytic activity of enzymes, induce conformational changes, guide subcellular localization and determine protein stability. A special class of protein PTMs is the conjugation of small proteins of the ubiquitin family to typically acceptor lysine residues of the substrates. The reversible nature of this PTM and the presence of dedicated domains that specifically recognize modified substrates make this type of protein modification instrumental for the regulation of numerous biological pathways. For ubiquitylation, strong substrate selectivity due to the presence of highly diversified conjugation machinery is characteristic and well studied, especially in case of ubiquitin’s proteolytic role. On the contrary, much less is known about the principles of substrate specificity and mechanisms of PTM action in the ubiquitin-like protein SUMO modification system. Despite the fact that SUMOylation specifically targets hundreds of substrates and major conjugation steps are identical with ubiquitin system, strikingly only a handful of enzymes operate in the SUMO pathway, suggesting that other principles of substrate selectivity must apply and perhaps distinct mechanisms of PTM action exist in the SUMO pathway. Moreover, the recognition of SUMO modification is surprisingly simple and relies mainly on a short hydrophobic sequence known as SUMO-interacting motif (SIM), in striking contrast to the ubiquitin system, where numerous ubiquitin-binding domains exist with different interaction specificities. All these, together with the observations that SUMO conjugation machinery seems rather promiscuous in vitro, that typically only a small fraction of a protein is being SUMOylated at a given time, and that specific SUMOylation-defective mutants often exhibit no obvious phenotypes, whereas SUMO pathway mutants do, emphasize the question of substrate specificity in the SUMO system and suggest other principles of SUMO action on its substrates. Here, we address the question of SUMOylation specificity and function using DNA double-strand break (DSB) repair pathway via homologous recombination (HR) as a case study because of its strong ties to the SUMO system. First, using SILAC-based proteomic approach we show that proteins acting in the same DNA repair pathway become collectively SUMOylated upon a specific stimulus (HR factors – upon DSB induction; nucleotide excision repair factors – upon exposure to UV light), suggesting that SUMO machinery often targets protein groups within the same pathway. Then, focusing on the DSB repair we find that DNA-bound SUMO ligase Siz2 catalyzes collective multisite SUMOylation of a whole set of HR factors. Repair proteins are loaded onto resected single-stranded DNA (ssDNA) in the vicinity of the ligase, thus making exposure of ssDNA a precise trigger for modification. Protein group SUMOylation fosters physical interactions between the HR proteins engaged in DNA repair, because not only that they become collectively modified at multiple SUMO-acceptor sites, but they also possess multiple SIMs, which promote SUMO-SIM mediated complex formation. Only wholesale elimination of SUMOylation of the core HR proteins significantly affects the HR pathway by slowing down DNA repair, suggesting that SUMO acts synergistically on several proteins. Thus, we show that SUMOylation collectively targets functionally engaged protein group rather than individual proteins, whereas localization of modification enzymes and specific triggers ensure substrate specificity.

Aneuploidy is a change in number or structure of one or more chromosomes that are not a multiple of the whole chromosome set. One of the best known pathological aneuploidies is trisomy 21 (Down syndrome), with chromosome 21 present in three instead of two copies. Patients with Down syndrome display severe mental retardation and growth defects. In fact, most abnormal aneuploid karyotypes lead to spontaneous abortions during embryogenesis, indicating that aneuploidy is not well tolerated in humans. Aneuploidy was also shown to be a common hallmark of cancer tissues; however, the debate is ongoing whether aneuploidy is rather a by-product or a trigger of tumorigenesis. Even though aneuploid karyotypes were already identified more than 100 years ago little is understood about cellular physiology of aneuploidy cells, especially in humans. To uncover the consequences of numerical aneuploidy in human cells, I generated aneuploid cell lines derived from the human cell lines HCT116 and RPE-1 hTERT. First, we showed that aneuploid cells proliferate slower compared to their disomic counterparts. A detailed cell cycle analysis revealed that this delay was due to a prolonged G1 and S phase, whereas G2 and M phase remained unperturbed. Furthermore, we conducted an in depth genome wide comparison of DNA, mRNA and protein levels in aneuploid cells. Using CGH, mRNA array and SILAC technology, we quantified the changes in DNA, mRNA and protein abundance. We revealed that extra chromosomes are actively transcribed and translated. However, the abundance of some proteins, particularly subunits of protein complexes and protein kinases, are adjusted towards disomic levels. Additionally, we asked how the cellular physiology is affected by the addition of a specific chromosome. Two scenarios are possible: either the cellular response depends on the additional chromosomes or all aneuploid cells show the same changes of cellular physiology. Indeed, we found that all aneuploid cell lines show similar physiological responses, irrespective of the type of additional chromosome. All aneuploid cell lines down-regulate DNA and RNA metabolism and up-regulate among others energy metabolism, lysosome function and membrane biosynthesis pathways. Lysosomes which are involved in autophagy are besides the ubiquitin-proteasome system important for cellular protein turn over. We found p62-dependent selective autophagy increased in all analyzed cell lines with extra chromosomes suggesting a role of p62-dependent selective autophagy in maintenance of protein homeostasis upon expression of extra protein in these cell lines.

Upon emerging from the ribosomal exit tunnel, folding of the polypeptide chain is necessary to form the fully functional protein. In E. coli, correct and efficient protein folding is mainly secured by an organized and complex chaperone system which includes two main principles: The first principle consists of the nascent binding chaperones including trigger factor (TF) and the DnaK/DnaJ system, while the second principle is represented by the downstream GroEL/ES chaperonin system. The identification of ~250 natural GroEL substrates demonstrated that GroEL/ES specifically folds a small group of proteins with complex domain topologies (Kerner et al., 2005) which include some essential proteins. Although the structural, functional and mechanistic aspects of DnaK, the E. coli Hsp70 chaperone, have been extensively studied, a systematic profiling of the natural DnaK substrates is still missing. Moreover, the cooperation between the two main chaperone systems remains to be elucidated. Here we analyzed the central role of DnaK in the bacterial chaperone network and its cooperation with the ribosome-associated chaperone TF and the downstream chaperonin GroEL/GroES using SILAC-based proteomics of DnaK-pulldowns. In parallel, we also analyzed the changes at the global proteome level under conditions of single or combined chaperone deletion. Our measurements show that DnaK normally interacts with at least ~700 newly-synthesized and pre-existent proteins (~30 % of all cytosolic proteins), including ~200 aggregation-prone substrates. Individual deletion of TF or depletion of GroEL/ES at 30 oC-37 oC leads to limited but highly specific changes in the DnaK interactome and in global proteome composition. Specifically, loss of TF results in increased interaction of DnaK with ribosomal and other small, basic proteins, and in a specific defect in the biogenesis of outer membrane -barrel proteins. While deletion of DnaK/DnaJ leads to the degradation or aggregation of ~150 highly DnaK-dependent proteins of large size, massive proteostasis collapse is only observed upon combined deletion of the DnaK system and TF, and is accompanied by extensive aggregation of GroEL substrates and ribosomal proteins. We conclude that DnaK is a central hub in the cytosolic E. coli chaperone network, interfacing with the upstream TF and the downstream chaperonin. These three major chaperone machineries have partially overlapping and non-redundant functions.

Mon, 25 Oct 2010 12:00:00 +0100 https://edoc.ub.uni-muenchen.de/15536/ https://edoc.ub.uni-muenchen.de/15536/1/Dulla_Kalyana_Chakravarthi.pdf Dulla, Kalyana Chakravarthi ddc:570, ddc:500, Fakultät für Biologi

Ein lebender Organismus ist unter anderem durch seine Fähigkeit zum präzisen Auf- und Zusammenbau höherer molekularer Strukturen charakterisiert, wobei die Faltung und Assemblierung von Proteinen eine bedeutende Rolle spielt. Die Proteinfaltung wird durch molekulare Chaperone unterstützt und optimiert, bis ein Protein seine native, biologisch funktionelle Struktur eingenommen hat. Durch exogene Einflüsse oder endogene Veränderungen eines Proteins, z.B. bei neurodegenerativen Erkrankungen wie M. Alzheimer, M. Parkinson oder Chorea Huntington, oder des gesamten Proteinnetzwerkes, kann Proteinfehlfaltung, Aggregation und die Ausbildung amyloider Strukturen, verbunden mit Zytotoxizität, auftreten. Die zur Fehlfaltung und Bildung ähnlicher amyloider Aggregate führenden strukturellen Determinanten der Zytotoxizität, verursacht durch Proteine unterschiedlicher Primärstruktur und Länge, sind nur unzureichend erforscht. Eine Hypothese besagt, dass lösliche intermediäre Oligomere der aggregierenden Proteine die toxische Spezies in einem wahrscheinlich multifunktionellen pathogenen Geschehen darstellen. Es gibt Hinweise, dass eine zusammenbrechende Proteostase verbunden mit einer zu geringeren Kapazität molekularer Chaperone zu den deletären Effekten führt. Auch ist nicht abschließend geklärt, ob und zu welchem Anteil die Toxizität durch Aggregation des Proteins und damit verbundener erhöhter Pathogenität bedingt ist, oder inwieweit durch einen Funktionsverlust des fehlgefalteten Proteins selbst. Um zytotoxische Effekte in humanen Zellen zu analysieren, wurden de novo generierte beta-Faltblattproteine untersucht, welche durch Aggregation in der Zelle keine Autofunktionsstörung auslösen sollten. Es wurde gezeigt, dass diese artifiziellen Proteine in HEK293T-Zellen amyloide Aggregate bildeten und zytotoxisch wirkten, im Vergleich zu de novo generierten alpha-helikalen Proteinen, welche löslich und homogen in der Zelle verteilt vorlagen und nahezu keine Zytotoxizität aufwiesen. Drei aus einer kombinatorischen Bibliothek ausgewählte de novo amyloide Proteine, beta4, beta17 und beta23, waren zytotoxisch mit der Gradierung beta4 < beta17 < beta23, sie induzierten Apoptose und veränderten die Zellmorphologie. Die Zytotoxizität korrelierte mit vorhandenen präfibrillären, intermediären Oligomeren. Die Proteine beeinträchtigten die Rückfaltung von GFP-Luciferase in gleicher Abstufung, ebenso eine Induktion der Stressantwort und die Proteinbiogenese. Die Aggregate colokalisierten mit GFP-Luciferase, jedoch nicht mit GFP. Eine massenspektrometrische Untersuchung der Interaktionspartner der drei de novo amyloiden Proteine in Kombination mit SILAC und Co-IP wies Interaktionen mit metastabilen Proteinen essentieller zellulärer Funktionen nach, dabei wurde Hsp110 als stark angereichertes Chaperon unter den Interaktoren identifiziert. Eine Überexpression von Hsp110 verminderte die Zytotoxizität der de novo Proteine beta4 und beta17, jedoch nicht beta23. Hsp110 war ebenfalls in der Lage, Aggregate teilweise zu solubilisieren und eine normalisierte Zellmorphologie wieder herzustellen. Um einen beta-Strang verkürzte oder verlängerte Mutanten der semitoxischen beta-Faltblattproteine beta4 und beta17 wiesen eine erhöhte Zytotoxizität auf, so dass wahrscheinlich generell beta-Faltblattproteine mit einer ungeraden Anzahl an beta-Strängen toxischer sind als ihre Derivate mit gerader Anzahl an beta-Strängen, da ungepaarte reaktive beta-Stränge vorliegen dürften. Zusammenfassend stellen die de novo beta-Faltblattproteine ein attraktives Modell dar, um aggregierende, amyloide Proteine ohne biologische Funktion in vivo zu untersuchen. Inkubation humaner Zellen mit dem Prolin-Analogon Azetidin-2-carbonsäure führte in Anwesenheit eines proteasomalen Inhibitors zur Verstärkung der Zytotoxizität, es entstanden amyloide Aggregate und präfibrilläre Intermediate, so dass die Hypothese der Verstärkung von Funktion und Pathogenität durch Aggregation in diesem System weiter untermauert wurde. Expression von Huntingtin mit expandierter PolyQ-Sequenz und einem angefügten hydrophoben CL1-Degron führte zu einer Erhöhung der Löslichkeit, zu verstärkter Inhibition des Ubiquitin-Proteasom-Systems und zu erhöhter Zytotoxitzität im Vergleich zu expandiertem Huntingtin ohne CL1-Degron. Die Zytotoxizität des mit Degron versehenen Huntingtins konnte mittels Überexpression von expandiertem Huntingtin ohne Degron durch Coaggregation verringert werden. Die Ergebnisse sprechen für die Hypothesen, dass präfibrilläre Intermediate die maßgeblichen zytotoxischen Spezies darstellen, während große Aggregate eine protektive Funktion einnehmen können. Eine Überexpression fehlfaltender Proteine kann in multifaktorieller Weise zur Interaktion mit essentiellen zellulären Proteinen führen und die Funktion metastabiler Proteine beeinträchtigen, was u.a. im Falle der de novo amyloiden Proteine zur Inhibition der Proteinbiogenese und der HSR führt. Akkumulation endogener fehlgefalteter Proteine durch proteasomale Inhibition legt den Mechanismus einer Verstärkung der Zytotoxizität durch amyloide, aggregierende Proteine per se nahe.

The aerobic, haloalkaliphilic archaeon Natronomonas pharaonis is able to survive in salt-saturated lakes of pH 11. With genome-wide shotgun proteomics, 886 soluble proteins (929 proteins in total) of the theoretical Natronomonas pharaonis soluble proteome consisting of 2187 proteins have been confidentially identified by MS/MS. By comparing the identified proteins of Natronomonas pharaonis with homologues of other organisms, both extreme diversity between halophiles and occasional extraordinary sequence conservation to proteins from unrelated species were observed, substantiating genetic exchange between organisms that are evolutionary nearly unrelated to cope with several extreme conditions. Alternative and largely overlapping open reading frames (called overprinting) could not be identified in the genome of neither Natronomonas pharaonis nor Halobacterium salinarum, leading to the conclusion that in halophiles, not more than one protein can be produced from the same genomic sequence stretch. In the second part of this work, analyses on both the transcriptional and translational level have been performed on the halophilic archaeon Halobacterium salinarum, to gain insights into its lifestyle changes leading to cell response when challenged by heat shock. Thereby, quantitative proteomic data obtained from two different approaches regarding the labeling method (ICPL; SILAC), the fractionation of the protein or peptide mixtures (2DE; 1DE-LC), the mass spectrometric analysis (MALDI-TOF/TOF; ESI Q-TOF), and the choice of the growth medium (complex; synthetic) were integrated with data from whole-genome DNA microarrays, real-time quantitative PCR (RTqPCR), and Northern analyses. A number of genes congruently displayed substantial induction after heat shock on both the transcript and protein level as in the case of the thermosome, two AAA-type ATPases, a Dps-like ferritin protein (DpsA), a hsp5-type molecular chaperone, and the transcription initiation factor tfbB. In contrast, the dnaK operon (hsp70) did not exhibit any significant upregulation in either of the approaches. Some genes encoding enzymes of the TCA cycle, of pathways flowing into the latter, and of pathways leading to pyrimidine synthesis, were only translationally induced. Finally, differential transcriptional induction of the transcription initiation factors tfbB and tfbA, determined by RTqPCR, led to the conclusion that they may regulate genes by reciprocal action. The multiplicity of proteomics and transcriptomics methods are complementing one another, covering a bigger area on the one hand, but also confirming some unexpected findings.

This thesis applies quantitative mass spectrometry to research topics in relation to cancer. Proteome-wide quantification at the protein expression level and phosphorylation level were achieved. The technologies developed and used here cover the latest improvements in instrumentation in mass spectrometry, strategies in phosphopeptide enrichment in large scale, algorithms in data analysis and their streamlined implementation, and data mining in downstream bioinformatics. For each of the projects described in this thesis, proteome mapping routinely resulted in identification and quantitation of around 4,000 proteins and phosphoproteome mapping often lead to quantitation of more than 5,000 phosphorylation sites. This ‘systems-wide’ quantitation of the proteome and phosphoproteome is a completely novel development, which has not been used in cancer related topics before. Three major biology topics are studied in this thesis. In the first project, the phosphoproteome of a mouse liver cancer cell line Hepa1-6 was analyzed in-depth, by using phosphatase inhibitors (calyculin A, deltamethrin, and Na-pervanadate) to boost phosphorylation. The characterization of the phosphoproteome revealed a broad spectrum of cellular compartmentalization and biological functions. Quantitation of phosphatase inhibitor treatment using the Stable Isotope Labeling by Amino Acids in Cell culture (SILAC) method revealed the quantitative effects of these inhibitor compounds on the whole phosphoproteome. To our surprise, these three broadband phosphatase inhibitors displayed very different efficiency, with tyrosine phosphorylation significantly boosted but serine/threonine phosphorylation much less affected. Additionally, a method to estimate an upper bound of the stoichiometry of phosphorylation was introduced by comparing phosphorylation in three SILAC conditions: non-treated cells, stimulated cells (e.g. with insulin), and only phosphatase inhibitor treated cells. The methods developed here can be used directly in development of drugs directed against kinases and phosphatases, key regulators in cancer and other diseases. The second project continues with the application of phosphoproteomics techniques. Kinase inhibitors influence cellular signal transduction processes and therefore are of great potential in rescuing aberrant cellular signaling in tumors. In fact they constitute a significant portion of drug developing programs in pharmaceutical industry. With the aim of quantifying the effect of kinase inhibitors over the entire signaling network, the second project first set out to study two very commonly used kinase inhibitor compounds for MAPKs: U0126 and SB202190. Their effect on epidermal growth factor (EGF) signal transduction was quantified and compared using the HeLa cell system. The study confirmed that the MAPK cascades are the predominant signaling branches for propagating the EGF signaling at early time points of stimulation. These large scale examinations also suggest that U0126 and SB202190 are quite specific inhibitors for MAPKs as the majority of regulated phosphopeptides appears to belong to the MAPK pathways. In the second part of the project, the effect on phosphoproteome changes of the chemical compound dasatinib, which was demonstrated to effectively inhibit the constitutively activated fusion protein BCR-ABL and was recently approved for chronic myelogenous leukemia (CML) therapy, was quantified in the human CML cell line K562. Bioinformatic analysis revealed that the most influenced signal transduction branch was the Erk1/2 cascade. Overall more than 500 phosphorylation sites were found to be regulated by dasatinib, the vast majority not described in the literature yet. The third project compared the proteomes of mouse hepatoma cell line Hepa1-6 with the non-transformed mouse primary hepatocytes. This was performed by combining the SILAC heavy labeled form of Hepa1-6 with the primary hepatocytes. To characterize the features of these two proteomes, quantitation information (i.e. protein ratios between the two cell types) was used to divide all proteins into five quantiles. Each quantile was clustered according to the Gene Ontology and KEGG pathway databases to assess their enriched functional groups and signaling pathways. To integrate this information at a higher level, hierarchical clustering based on the p-value from the first Gene Ontology and KEGG clustering was performed. Using this improved bioinformatic algorithm for data mining, the proteomic phenotypes of the primary cells and transformed cells are immediately apparent. Primary hepatocytes are enriched in mitochondrial functions such as metabolic regulation and detoxification, as well as liver functions with tissue context such as secretion of plasma and low-density lipoprotein (LDL). In contrast, the transformed cancer cell line Hepa1-6 is enriched in cell cycle and growth functions. Interestingly, several aspects of the molecular basis of the “Warburg effect” described in many cancer cells became apparent in Hepa1-6, such as increased expression of glycolysis markers and decreased expression of markers for tricarboxylic acid (TCA) cycle. Studies in this thesis only provide examples of the application of mass spectrometry-based quantitative proteomics and phosphoproteomics in cancer research. The connection to clinical research, especially the assessment of drug effects on a proteome wide scale, is a specific feature of this thesis. Although this development is only in its infancy, it reflects a trend in the quantitative mass spectrometry field. We believe that more and more clinical related topics can and will be studied by these powerful methods.