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Colorectal cancer (CRC) is one of the top three most common cancers and among the top four causes of cancer-related deaths worldwide (Torre et al., 2015). CRC patients are well characterised on the transcriptome and proteome level, but proteomics data on representative cell lines as model systems for pre-clinical drug sensitivity studies lag behind. Here, label-free quantitative mass spectrometry was used to characterise the kinomes and full proteomes of 65 CRC cell lines, collectively termed the CRC65 cell line panel. This data was integrated with proteomics data on patient samples, as well as public transcriptome and drug sensitivity datasets, which were reanalysed from raw data in order to unify and streamline the data processing. Protein/mRNA ratios were constant across these datasets, enabling linear prediction of protein abundance from mRNA abundance after appropriate adjustment, which was used for mRNA-guided missing value imputation. An exploration of secondary imputation methods prompted the development of a complementary method for minimum-guided missing value imputation. Combining the proteomics datasets on cell lines and patients led to the discovery of integrated proteomic subtypes of CRC and enabled the identification of representative cell lines for each of them. Modelling publicly available dose-response data generated by four large-scale drug sensitivity studies as a function of kinome/full proteome profiles fuelled the prediction of drug sensitivity for cell lines and patients, allowed the identification of drugs differentially effective between the different integrated proteomic subtypes and revealed MERTK as a predictive biomarker for resistance towards MEK1/2 inhibitors. This predictive role of MERTK was subsequently confirmed using in vitro experiments, while immunohistochemistry of TMAs from 1,074 tumours generated as part of the QUASAR2 clinical trial unveiled that MERTK is also a prognostic biomarker in CRC. This dataset will be made available to the scientific community to facilitate the design of prospective clinical studies.

PASEF multiplies the sequencing speed without any loss in sensitivity and is implemented in the timsTOF Pro instrument introduced here. Sequencing speeds above 100 Hz enable single run proteome analysis at a depth of 6000 proteins, making the instrument particularly attractive for rapid and highly sensitive proteomics. Collisional cross sections can be determined with up to 0.1% precision and acquired on a scale of 100,000s, which opens exciting areas for proteomics exploration., Graphical Abstract Highlights Online PASEF achieves greater than 100 MS/MS per second at full sensitivity.Accurate label-free quantification of over 6000 proteins in 2 h.High throughput demonstrated on 50 ng digests measured in 5 min.High-precision determination of 100,000 peptide collisional cross sections., In bottom-up proteomics, peptides are separated by liquid chromatography with elution peak widths in the range of seconds, whereas mass spectra are acquired in about 100 microseconds with time-of-flight (TOF) instruments. This allows adding ion mobility as a third dimension of separation. Among several formats, trapped ion mobility spectrometry (TIMS) is attractive because of its small size, low voltage requirements and high efficiency of ion utilization. We have recently demonstrated a scan mode termed parallel accumulation - serial fragmentation (PASEF), which multiplies the sequencing speed without any loss in sensitivity (Meier et al., PMID: 26538118). Here we introduce the timsTOF Pro instrument, which optimally implements online PASEF. It features an orthogonal ion path into the ion mobility device, limiting the amount of debris entering the instrument and making it very robust in daily operation. We investigate different precursor selection schemes for shotgun proteomics to optimally allocate in excess of 100 fragmentation events per second. More than 600,000 fragmentation spectra in standard 120 min LC runs are achievable, which can be used for near exhaustive precursor selection in complex mixtures or accumulating the signal of weak precursors. In 120 min single runs of HeLa digest, MaxQuant identified more than 6,000 proteins without matching to a library and with high quantitative reproducibility (R > 0.97). Online PASEF achieves a remarkable sensitivity with more than 2,500 proteins identified in 30 min runs of only 10 ng HeLa digest. We also show that highly reproducible collisional cross sections can be acquired on a large scale (R > 0.99). PASEF on the timsTOF Pro is a valuable addition to the technological toolbox in proteomics, with a number of unique operating modes that are only beginning to be explored.

Tyrosine kinase inhibitors (TKIs) have become an important therapeutic option for treating several forms of cancer. Gefitinib, an inhibitor of the epidermal growth factor receptor (EGFR), is in clinical use for treating non-small cell lung cancer (NSCLC) harboring activating EGFR mutations. However, despite high initial response rates, many patients develop resistance to gefitinib. The molecular mechanisms of TKI resistance often remain unclear. Here, we describe a chemical proteomic approach comprising kinase affinity purification (kinobeads) and quantitative mass spectrometry for the identification of kinase inhibitor resistance mechanisms in cancer cells. We identified the previously described amplification of MET and found EPHA2 to be more than 10-fold overexpressed (p < 0.001) in gefitinib-resistant HCC827 cells suggesting a potential role in developing resistance. siRNA-mediated EPHA2 knock-down or treating cells with the multikinase inhibitor dasatinib restored sensitivity to gefitinib. Of all dasatinib targets, EPHA2 exhibited the most drastic effect (p < 0.001). In addition, EPHA2 knockdown or ephrin-A1 treatment of resistant cells decreased FAK phosphorylation and cell migration. These findings confirm EPHA2 as an actionable drug target, provide a rational basis for drug combination approaches, and indicate that chemical proteomics is broadly applicable for the discovery of kinase inhibitor resistance.

For the investigation of molecular processes underlying diseases of the bovine mammary gland, primary bovine mammary epithelial cells (pbMEC) are used. They are known to contribute to the innate immune system of the bovine mammary gland. The functionality of pbMEC depends on the maintenance of in vivo characteristics. So far, the optimization of pbMEC culture conditions was intended in a variety of experiments. For this purpose, most of the studies used stable cell lines or primary cells obtained from udder biopsies of slaughtered animals. By contrast, within our study, pbMEC of healthy and first lactating Brown Swiss cows were non-invasively isolated from fresh milk. The non-invasively isolated pbMEC were cultivated on the extracellular matrix-like scaffold Matrigel®. Further, they were challenged with different compositions of proliferation media, containing lactogenic hormones and/or the essential amino acid L-lysine. Changes in expression levels of genes coding for milk proteins and for components of the janus kinase/signal transducers and activators of transcription (JAK-STAT) and mTOR pathways were analyzed by RT-qPCR. The secreted proteins were analyzed by LC-MS/MS measurements. We showed for the first time the establishment of a physiologically functional 3D cell culture model of pbMEC isolated from fresh milk. This represents a primary cell culture model system, based on non-invasive cell collection, that can be used to unravel physiological processes in an unbiased manner.

Phosphorylation is among the most important post-translational modifications of proteins and has numerous regulatory functions across all domains of life. However, phosphorylation is often substoichiometric, requiring selective and sensitive methods to enrich phosphorylated peptides from complex cellular digests. Various methods have been devised for this purpose and we have recently described a Fe-IMAC HPLC column chromatography setup which is capable of comprehensive, reproducible, and selective enrichment of phosphopeptides out of complex peptide mixtures. In contrast to other formats such as StageTips or batch incubations using TiO

An atlas for drug interactions Kinase inhibitors are an important class of drugs that block certain enzymes involved in diseases such as cancer and inflammatory disorders. There are hundreds of kinases within the human body, so knowing the kinase “target” of each drug is essential for developing successful treatment strategies. Sometimes clinical trials can fail because drugs bind more than one target. Yet sometimes off-target effects can be beneficial, and drugs can be repurposed for treatment of additional diseases. Klaeger et al. performed a comprehensive analysis of 243 kinase inhibitors that are either approved for use or in clinical trials. They provide an open-access resource of target summaries that could help researchers develop better drugs, understand how existing drugs work, and design more effective clinical trials. Science , this issue p. eaan4368

Although substantial progress has been made regarding the use of molecularly targeted cancer therapies, resistance almost invariably develops and presents a major clinical challenge. The tumor microenvironment can rescue cancer cells from kinase inhibitors by growth-factor mediated induction of pro-survival pathways. Here we show that epidermal growth factor receptor (EGFR) inhibition by Gefitinib is counteracted by growth factors, notably FGF2, and we assessed the global molecular consequences of this resistance at the proteome and phosphoproteome level in A431 cells. Tandem mass tag peptide labeling and quantitative mass spectrometry allowed the identification and quantification of 22 000 phosphopeptides and 8800 proteins in biological triplicates without missing values. The data show that FGF2 protects the cells from the antiproliferative effect of Gefitinib and largely prevents reprogramming of the proteome and phosphoproteome. Simultaneous EGFR/FGFR or EGFR/GSG2 (Haspin) inhibition overcomes this resistance, and the phosphoproteomic experiments further prioritized the RAS/MEK/ERK as well as the PI3K/mTOR axis for combination treatment. Consequently, the MEK inhibitor Trametinib prevented FGF2-mediated survival of EGFR inhibitor-resistant cells when used in combination with Gefitinib. Surprisingly, the PI3K/mTOR inhibitor Omipalisib reversed resistance mediated by all four growth factors tested, making it an interesting candidate for mitigating the effects of the tumor microenvironment.

170 Fernsehsender, 330 Horfunkprogramme, 1000 Zeitschriften und 350 Tageszeitungen, 100 Millionen Internetseiten, 70 000 neue Bu cher pro Jahr, 3,5 Stunden Fernsehkonsum pro Bundesburger und Tag sowie 40 Minuten Internet: Wie vielfaltig sind die Medien, ihre Moglichkeiten und Grenzen? Und wie vielfaltig zeigt sich Kirche und wird Kirche dargestellt und wahrgenommen in dieser unubersehbar bunten Welt der Medien? Kirche in den Medien – wie bedeutsam und chancenreich ist dieses Miteinander, wie belastend und wie gefahrlich und welche Konsequenzen sollte die Kirche aus dieser Verwobenheit ziehen? Diese Fragen mochte ich mit jeweils drei Einwurfen erortern und zu einer Antwort fuhren. (...)

Es ist auf einer sachlichen Ebene verwunderlich, dass die Gefahr durch Terrorismus die Sicherheitsdebatte dominiert, obwohl im Alltag das Risiko eines Anschlags nahezu bedeutungslos bleibt und eher „gewohnliche“ Kriminalitat eine Rolle spielt. Dennoch hat der „Kampf gegen den Terrorismus“ zu einer intensiveren Debatte uber das Verhaltnis von Sicherheit und Freiheit gefuhrt. Die vermeintliche Sicherheit, nicht von terroristischen Anschlagen oder anderen Angriffen auf die eigene Freiheit getroffen zu werden, wird nicht selten durch die Beschneidung von Freiheiten erkauft. Uberwachung und die Einschrankung von Privatheit, das Sammeln von personenbezogenen Daten, die Durchfuhrung von Kontrollen und die Einschrankung von Bewegungsfreiheiten sind einige Beispiele hierfur. Dies kann einen Abschreckungseffekt zur Folge haben, durch den auch demokratische Grundfreiheiten gefahrdet werden konnen (BVerfGE 65, 1/43 und BVerfGE 69, 315/349). Unter dem Eindruck einer Terrorismusgefahr ist es dabei nicht alternativlos, Freiheiten einzuschranken.

Die Sicherheitsethik sieht sich oft mit dem Problem konfrontiert, dass einem aktuellen, tatsachlichen oder vermeintlichen Sicherheitsproblem mit einer Sicherheitsmasnahme begegnet wird, die dann ethisch bewertet werden soll. Dies ist insofern ein Problem, dass es in diesem ethischen Tagesgeschaft oft keine wirklich befriedigenden Losungen gibt. Die Ethik darf sich zwar nicht aus Bequemlichkeit aus dem ethischen Tagesgeschaft zuruckziehen, doch sie darf sich auch nicht von diesem so unter Druck setzen lassen, dass sie nicht mehr auf eine grosere Perspektive reflektieren kann.

Unter Normalisierung wird die Ausrichtung von Personen an Normalitat verstanden (Brockling 2000, Foucault 2004, Krasmann 2000, Mehrtens 1999, Singelnstein/ Stolle 2006 u. a.). Durch Normalisierung wird die Erwartbarkeit von Handlungen hergestellt. Dies kann durch ein ganzes Spektrum von Steuerungs-Masnahmen geschehen von der Beeinflussung bis zum Zwang. Bis zu einem gewissen Grad ist die Erwartbarkeit von Handlungen notig fur ein Zusammmenleben in Gemeinschaften. Solche Erwartbarkeiten konnen auch zwanglos angestrebt werden, etwa bei der reflektierten Orientierung an moralischen Normen. Der Wechsel von Zwang oder Manipulation zur Ethik bietet hierbei eine Perspektive, auf die am Ende zuruckgekommen werden soll.