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Aebersold R.,Institute of Molecular Systems Biology | Bader G.D.,University of Toronto | Edwards A.M.,University of Toronto | van Eyk J.,Johns Hopkins University | And 3 more authors.
Proteomics | Year: 2014

At the 12th Annual HUPO World Congress of Proteomics in Japan, the Human Proteome Project (HPP) presented 16 scientific workshop sessions. Here we summarize highlights of ten workshops from the Biology and Disease-driven HPP (B/D-HPP) teams and three from the HPP Resource Pillars. Highlights of the three Chromosome-centric HPP sessions appeared in the many articles of the 2014 C-HPP special issue of the Journal of Proteome Research © 2014 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim. Source

Gillet L.C.,Institute of Molecular Systems Biology
Molecular & cellular proteomics : MCP | Year: 2012

Most proteomic studies use liquid chromatography coupled to tandem mass spectrometry to identify and quantify the peptides generated by the proteolysis of a biological sample. However, with the current methods it remains challenging to rapidly, consistently, reproducibly, accurately, and sensitively detect and quantify large fractions of proteomes across multiple samples. Here we present a new strategy that systematically queries sample sets for the presence and quantity of essentially any protein of interest. It consists of using the information available in fragment ion spectral libraries to mine the complete fragment ion maps generated using a data-independent acquisition method. For this study, the data were acquired on a fast, high resolution quadrupole-quadrupole time-of-flight (TOF) instrument by repeatedly cycling through 32 consecutive 25-Da precursor isolation windows (swaths). This SWATH MS acquisition setup generates, in a single sample injection, time-resolved fragment ion spectra for all the analytes detectable within the 400-1200 m/z precursor range and the user-defined retention time window. We show that suitable combinations of fragment ions extracted from these data sets are sufficiently specific to confidently identify query peptides over a dynamic range of 4 orders of magnitude, even if the precursors of the queried peptides are not detectable in the survey scans. We also show that queried peptides are quantified with a consistency and accuracy comparable with that of selected reaction monitoring, the gold standard proteomic quantification method. Moreover, targeted data extraction enables ad libitum quantification refinement and dynamic extension of protein probing by iterative re-mining of the once-and-forever acquired data sets. This combination of unbiased, broad range precursor ion fragmentation and targeted data extraction alleviates most constraints of present proteomic methods and should be equally applicable to the comprehensive analysis of other classes of analytes, beyond proteomics. Source

Herzig S.,University of Geneva | Raemy E.,University of Geneva | Montessuit S.,University of Geneva | Veuthey J.-L.,University of Geneva | And 4 more authors.
Science | Year: 2012

The transport of pyruvate, the end product of glycolysis, into mitochondria is an essential process that provides the organelle with a major oxidative fuel. Although the existence of a specific mitochondrial pyruvate carrier (MPC) has been anticipated, its molecular identity remained unknown. We report that MPC is a heterocomplex formed by two members of a family of previously uncharacterized membrane proteins that are conserved from yeast to mammals. Members of the MPC family were found in the inner mitochondrial membrane, and yeast mutants lacking MPC proteins showed severe defects in mitochondrial pyruvate uptake. Coexpression of mouse MPC1 and MPC2 in Lactococcus lactis promoted transport of pyruvate across the membrane. These observations firmly establish these proteins as essential components of the MPC. Source

Petrovic A.,Italian National Cancer Institute | Pasqualato S.,Italian National Cancer Institute | Dube P.,Max Planck Institute for Biophysical Chemistry | Krenn V.,Italian National Cancer Institute | And 11 more authors.
Journal of Cell Biology | Year: 2010

Kinetochores are nucleoprotein assemblies responsible for the attachment of chromosomes to spindle microtubules during mitosis. The KMN network, a crucial constituent of the outer kinetochore, creates an interface that connects microtubules to centromeric chromatin. The NDC80, MIS12, and KNL1 complexes form the core of the KMN network. We recently reported the structural organization of the human NDC80 complex. In this study, we extend our analysis to the human MIS12 complex and show that it has an elongated structure with a long axis of ∼22 nm. Through biochemical analysis, crosslinking-based methods, and negative-stain electron microscopy, we investigated the reciprocal organization of the subunits of the MIS12 complex and their contacts with the rest of the KMN network. A highlight of our findings is the identification of the NSL1 subunit as a scaffold supporting interactions of the MIS12 complex with the NDC80 and KNL1 complexes. Our analysis has important implications for understanding kinetochore organization in different organisms. © 2010 Petrovic et al. Source

News Article
Site: http://www.chromatographytechniques.com/rss-feeds/all/rss.xml/all

Biologists at ETH Zurich have developed a method that, for the first time, makes it possible to measure concentration changes of several hundred metabolic products simultaneously, and almost in real-time. The technique could inspire basic biological research and the search for new pharmaceutical agents. Genomics, proteomics, metabolomics. Scientists who work in a field that ends with the suffix -omics analyse the totality of something. In the case of metabolomics, it is the totality of all metabolites of a cell or organism. The research groups of Uwe Sauer, professor of Systems Biology at ETH Zurich, and Nicola Zamboni, group leader at the Institute of Molecular Systems Biology, are among the leaders in this field. They have now developed a method by which they can identify the concentration of hundreds of metabolites simultaneously and almost in real time. The analysis of all metabolites in one go is not particularly easy since metabolites are a very diverse class of biological substances. “Various sugars, fats, messenger materials and amino acids belong to this group – thus, completely different molecules. Their only similarity is that they are small, at least compared with proteins and RNA molecules that occur on a mass scale in cells,” explains Sauer. For a long time, the simultaneous measurement of hundreds of metabolites in a fluid – for instance, urine or blood – or in cells was very time consuming. Most biologists used methods in which the substance mixture was first separated by chromatography and then the separated ingredients were identified in a mass spectrometer. A few years ago Sauer, Zamboni and their colleagues developed a method that made chromatographic separation unnecessary. “We can now analyse a sample directly in a mass spectrometer and filter out information about the ingredients from a huge amount of data using a software that we developed,” says Sauer. Identification of 300 to 800 different metabolites in a sample takes only a minute, which means that analysis of thousands of samples in one day – previously only a dream – has now become a reality. “The success of this high-throughput measurement method brought us to the idea of real-time measurements,” says Sauer. This is helpful because metabolism responds very quickly to stimulus changes: “If, for example, you shine a light on a plant in the dark, the concentrations of its metabolites change in just a few seconds.” The precise timing of a concentration change in response to new stimuli is important and meaningful information in biology. The ETH scientists implemented their real-time measurement idea by using different cells in a culture: two bacterial species, a yeast species and mice cells. The researchers let the cells grow in a growth medium directly next to a measuring instrument. An automatic pump system extracted a tiny amount from the cell culture every 10 seconds in order to analyse it in the instrument. The researchers not only managed to prove that, in principle, such on-line measurements are possible with all types of cell cultures; thanks to their technology, they also gained new insights into how E. coli bacteria switch from a ‘stand-by’ mode into a growth phase. They let the bacteria starve for two hours by keeping them in the growth medium without sugar. As a consequence the bacteria switch to the ‘stand-by’ programme by stopping production of most metabolites and breaking down the existing ones in order to gain energy for survival. Following this starvation phase, the scientists again provided the bacteria with sugar. Within one minute, the cells resumed production of metabolites in order to grow and divide. However, the scientists were baffled by the behaviour of 10 of the nearly 300 metabolites studied, which behaved differently from the majority: their concentration increased during the starvation phase and decreased during the optimal supply phase. The researchers believe that these are key metabolites that influence the extremely fast switch of the overall metabolism between the two phases. These 10 metabolites are eight specific amino acids – the building blocks of proteins – and two molecules, from which the cells produce DNA and RNA building blocks. And they have one thing in common: the cells have to expend a large amount of energy to produce them. “We assume that the cells do not break down such valuable building blocks during the starvation phase, but instead save them to have the best possible starting conditions for the subsequent growth phase,” says Sauer. Using a systems biological computer model, the scientists were able to show how the regulation works: the 10 metabolites saved during the starvation phase prevent the cells from producing more of them at the beginning of the growth phase by means of a feedback mechanism. As a result, the cells do not waste energy in the expensive construction of the 10 metabolites, but put their resources entirely into the synthesis of the other molecules. Helpful in the development of medication Sauer is currently making the new real-time method known to the scientific community. “It is a very useful method to get a first overview of how cells react to an external stimulus. This makes it suitable for the analysis of all metabolic processes that take place over a period of time of half an hour to several hours,” he says. He sees possible applications not only in basic biological research, but also, for example, in the screening of potential new pharmaceutical agents. This would make it possible to discover how a drug alters metabolism – a method that Sauer’s group now uses for such investigations.

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