Mass spectrometry and ribosome profiling, a perfect combination towards a more comprehensive identification strategy of true in vivo protein forms

An increasing number of studies involve integrative analysis of gene and protein expression data, taking advantage of new technologies such as next-generation transcriptome sequencing (RNA-Seq) and highly sensitive mass spectrometry (MS). Recently, a strategy, termed ribosome profiling, based on deep sequencing of ribosome-protected mRNA fragments, indirectly monitoring protein synthesis, has been described. In contrast to routinely employed protein databases in proteomics searches, RIBO-seq derived data gives a more representative expression state and accounts for sequence variation information and alternative translation initiation. To verify the potential of ribosome profiling in providing us with a true snapshot of the translational landscape, we devised a proteogenomic approach generating a database of translation products based on ribosome profiling experiments. The raw and untreated RIBO-seq data is analyzed for both splice isoforms and single nucleotide polymorphisms, as such taking into account transcriptional variation. Next to that, RIBO-seq data for translation start site discovery (treated with harringtonine, lactomidomycin or puromycin) is used to obtain a genome wide blueprint of all possible translation initiation sites and as such taking into account translation variation. By adding protein-DB annotation to the genomic RIBO-seq derived data and after in silico translation a protein database is constructed reflecting the full complexity of the proteome. Using a first version of our proteogenomic approach on an undifferentiated mouse embryonic stem cell line (E14) we could demonstrate an increase of the overall protein identification rate with 2.5% as compared to only searching UniProtKB-SwissProt. Furthermore, identification of N-terminal COFRADIC data resulted in detection of 16 alternative start sites giving rise to N-terminally extended protein variants besides the identification of four translated uORFs

Bis(μ-diisopropyl­phosphanido-κ2 P:P)bis­[hydrido(triisopropyl­phosphane-κP)platinum(II)]

In the centrosymmetric mol­ecular structure of the title compound [Pt2(C6H14P)2H2(C9H21P)2], each PtII atom is bound on one side to a phosphane ligand (PiPr3) and a hydrido ligand. On the other side, it is bound to two phosphanide ligands (μ-PiPr2), which engage a bridging position between the two PtII atoms, forming a distorted square-planar structure motif. The Pt⋯Pt distance is 3.6755 (2) Å. A comparable mol­ecular structure was observed for bis­(μ-di-tert-butyl­phosphanido)bis­[hydrido(triethyl­phosphane)platinum(II)] [Itazaki et al. (2004 ▶). Organometallics, 23, 1610–1621]

Balancing Performance and Energy for Lightweight Data Compression Algorithms

Energy consumption becomes more and more a critical design factor, whereby performance is still an important requirement. Thus, a balance between performance and energy has to be established. To tackle that issue for database systems, we proposed the concept of work-energy profiles. However, generating such profiles requires extensive benchmarking. To overcome that, we propose to approximate work-energy-profiles for complex operations based on the profiles of low-level operations in this paper. To show the feasibility of our approach, we use lightweight data compression algorithms as complex operations, since compression as well as decompression are heavily used in in-memory database systems, where data is always managed in a compressed representation. Furthermore, we evaluate our approach on a concrete hardware system

PROTEOFORMER: deep proteome coverage through ribosome profiling and MS integration

An increasing amount of studies integrate mRNA sequencing data into MS-based proteomics to complement the translation product search space. However, several factors, including extensive regulation of mRNA translation and the need for three- or six-frame-translation, impede the use of mRNA-seq data for the construction of a protein sequence search database. With that in mind, we developed the PROTEOFORMER tool that automatically processes data of the recently developed ribosome profiling method (sequencing of ribosome-protected mRNA fragments), resulting in genome-wide visualization of ribosome occupancy. Our tool also includes a translation initiation site calling algorithm allowing the delineation of the open reading frames (ORFs) of all translation products. A complete protein synthesis-based sequence database can thus be compiled for mass spectrometry-based identification. This approach increases the overall protein identification rates with 3% and 11% (improved and new identifications) for human and mouse, respectively, and enables proteome-wide detection of 5'-extended proteoforms, upstream ORF translation and near-cognate translation start sites. The PROTEOFORMER tool is available as a stand-alone pipeline and has been implemented in the galaxy framework for ease of use

MorphStore — In-Memory Query Processing based on Morphing Compressed Intermediates LIVE

In this demo, we present MorphStore, an in-memory column store with a novel compression-aware query processing concept. Basically, compression using lightweight integer compression algorithms already plays an important role in existing in-memory column stores, but mainly for base data. The continuous handling of compression from the base data to the intermediate results during query processing has already been discussed, but not investigated in detail since the computational effort for compression as well as decompression is often assumed to exceed the benefits of a reduced transfer cost between CPU and main memory. However, this argument increasingly loses its validity as we are going to show in our demo. Generally, our novel compression-aware query processing concept is characterized by the fact that we are able to speed up the query execution by morphing compressed intermediate results from one scheme to another scheme to dynamically adapt to the changing data characteristics during query processing. Our morphing decisions are made using a cost-based approach

Integrated approaches to restore gullies in land prone to soil piping : innovations from the drylands of northern Ethiopia

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High temporal resolution reveals simultaneous plasma membrane recruitment of TPLATE complex subunits

The TPLATE complex (TPC) is a key endocytic adaptor protein complex in plants. TPC in Arabidopsis (Arabidopsis thaliana) contains six evolutionarily conserved subunits and two plant-specific subunits, AtEH1/Pan1 and AtEH2/Pan1, although cytoplasmic proteins are not associated with the hexameric subcomplex in the cytoplasm. To investigate the dynamic assembly of the octameric TPC at the plasma membrane (PM), we performed state-of-the-art dual-color live cell imaging at physiological and lowered temperatures. Lowering the temperature slowed down endocytosis, thereby enhancing the temporal resolution of the differential recruitment of endocytic components. Under both normal and lowered temperature conditions, the core TPC subunit TPLATE and the AtEH/Pan1 proteins exhibited simultaneous recruitment at the PM. These results, together with co-localization analysis of different TPC subunits, allow us to conclude that TPC in plant cells is not recruited to the PM sequentially but as an octameric complex

(1R,2R)-N,N′-Dimethyl­cyclo­hexane-1,2-diamine

The molecule of the title compound, C8H18N2, possesses C 2 symmetry. Owing to its stereochemistry, it is used in the synthesis of chiral ligands and metal complexes for asymmetric synthesis. The cyclo­hexane ring shows a chair conformation with the amino groups in equatorial positions. Contrary to the literature, the title compound is not a liquid, but a crystalline solid at room temperature (293 K). The absolute configuration is assigned from the synthesis