Although protein expression is regulated both temporally and spatially, most proteins have an intrinsic, “typical” range of functionally effective abundance levels. These extend from a few molecules per cell for signaling proteins, to millions of molecules for structural proteins. When addressing fundamental questions related to protein evolution, translation and folding, but also in routine laboratory work, a simple rough estimate of the average wild type abundance of each detectable protein in an organism is often desirable. Here, we introduce a meta-resource dedicated to integrating information on absolute protein abundance levels; we place particular emphasis on deep coverage, consistent post-processing and comparability across different organisms. Publicly available experimental data are mapped onto a common namespace and, in the case of tandem mass spectrometry data, re-processed using a standardized spectral counting pipeline. By aggregating and averaging over the various samples, conditions and cell-types, the resulting integrated data set achieves increased coverage and a high dynamic range. We score and rank each contributing, individual data set by assessing its consistency against externally provided protein-network information, and demonstrate that our weighted integration exhibits more consistency than the data sets individually. The current PaxDb-release 2.1 (at http://pax-db.org/) presents whole-organism data as well as tissue-resolved data, and covers 85,000 proteins in 12 model organisms. All values can be seamlessly compared across organisms via pre-computed orthology relationships.

Comprehensive characterization of a proteome is a fundamental goal in proteomics. To achieve saturation coverage of a proteome or specific subproteome via tandem mass spectrometric identification of tryptic protein sample digests, proteomics data sets are growing dramatically in size and heterogeneity. The trend toward very large integrated data sets poses so far unsolved challenges to control the uncertainty of protein identifications going beyond well established confidence measures for peptide-spectrum matches. We present MAYU, a novel strategy that reliably estimates false discovery rates for protein identifications in large scale data sets. We validated and applied MAYU using various large proteomics data sets. The data show that the size of the data set has an important and previously underestimated impact on the reliability of protein identifications. We particularly found that protein false discovery rates are significantly elevated compared with those of peptide-spectrum matches. The function provided by MAYU is critical to control the quality of proteome data repositories and thereby to enhance any study relying on these data sources. The MAYU software is available as standalone software and also integrated into the Trans-Proteomic

Understanding how proteins and their complex interaction networks convert the genomic information into a dynamic living organism is a fundamental challenge in biological sciences. As an important step towards understanding the systems biology of a complex eukaryote, we cataloged 63% of the predicted Drosophila melanogaster proteome by detecting 9,124 proteins from 498,000 redundant and 72,281 distinct peptide identifications. This unprecedented high proteome coverage for a complex eukaryote was achieved by combining sample diversity, multidimensional biochemical fractionation and analysis-driven experimentation feedback loops, whereby data collection is guided by statistical analysis of prior data. We show that high-quality proteomics data provide crucial information to amend genome annotation and to confirm many predicted gene models. We also present experimentally identified proteotypic peptides matching approximately 50% of D. melanogaster gene models. This library of proteotypic peptides should enable fast, targeted and quantitative proteomic studies to elucidate the systems biology of this model organism.

The nematode Caenorhabditis elegans is a popular model system in genetics, not least because a majority of human disease genes are conserved in C. elegans. To generate a comprehensive inventory of its expressed proteome, we performed extensive shotgun proteomics and identified more than half of all predicted C. elegans proteins. This allowed us to confirm and extend genome annotations, characterize the role of operons in C. elegans, and semiquantitatively infer abundance levels for thousands of proteins. Furthermore, for the first time to our knowledge, we were able to compare two animal proteomes (C. elegans and Drosophila melanogaster). We found that the abundances of orthologous proteins in metazoans correlate remarkably well, better than protein abundance versus transcript abundance within each organism or transcript abundances across organisms; this suggests that changes in transcript abundance may have been partially offset during evolution by opposing changes in protein abundance.

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Neuroserpin is an axonally secreted serine proteinase inhibitor that is expressed in neurons during embryogenesis and in the adult nervous system. To identify target proteinases, we used a eucaryotic expression system based on the mouse myeloma cell line J558L and vectors including a promoter from an Ig--variable region, an Ig-enhancer, and the exon encoding the Igconstant region (C) and produced recombinant neuroserpin as a wild-type protein or as a fusion protein with C. We investigated the capability of recombinant neuroserpin to form SDS-stable complexes with, and to reduce the amidolytic activity of, a variety of serine proteinases in vitro. Consistent with its primary structure at the reactive site, neuroserpin exhibited inhibitory activity against trypsin-like proteinases. Although neuroserpin bound and inactivated plasminogen activators and plasmin, no interaction was observed with thrombin. A reactive site mutant of neuroserpin neither formed complexes with nor inhibited the amidolytic activity of any of the tested proteinases. Kinetic analysis of the inhibitory activity revealed neuroserpin to be a slow binding inhibitor of plasminogen activators and plasmin. Thus, we postulate that neuroserpin could represent a regulatory element of extracellular proteolytic events in the nervous system mediated by plasminogen activators or plasmin.Extracellular proteolysis exerted by serine proteinases has been implicated in a variety of processes in the nervous system during development and in adulthood. Among the serine proteinases recently reported to play a role in neural development and function, there are several well known proteins that had previously been found and characterized in nonneuronal functions, in particular blood coagulation and fibrinolysis. For example, tissue-type plasminogen activator (tPA) 1 and urokinase plasminogen activator (uPA) were found to be expressed in the nervous system (1, 2), and they have been demonstrated to be engaged in developmental processes such as cerebellar granule cell migration (3, 4), Schwann cell migration, and wrapping of axons (5), or neuromuscular synapse elimination (6). In the period of neurite outgrowth, plasminogen activators (PAs) have been found to be secreted at the growth cones of cultured neurons or neuronal cell lines (7,8), and they were demonstrated to modify the molecular composition of the neurites' substrata in vitro (9). In the adult nervous system, tPA is induced in the hippocampus after seizure, kindling, and long term potentiation (LTP) (10) and in the cerebellum after motor learning tasks (11), and mice lacking the gene for tPA (12) show a different form of hippocampal LTP (13,14). Furthermore, tPA has been demonstrated to be involved in excitotoxin-induced neuronal cell death in the murine hippocampus by converting locally secreted plasminogen to active plasmin (15, 16). Thrombin, which has been extensively characterized due to its important function in the blood clotting system, has been reported to be expressed in the nervous system (17). I...

Synaptosomes are isolated synapses produced by subcellular fractionation of brain tissue. They contain the complete presynaptic terminal, including mitochondria and synaptic vesicles, and portions of the postsynaptic side, including the postsynaptic membrane and the postsynaptic density (PSyD). A proteomic characterisation of synaptosomes isolated from mouse brain was performed employing the isotope-coded affinity tag (ICAT) method and tandem mass spectrometry (MS/MS). After isotopic labelling and tryptic digestion, peptides were fractionated by cation exchange chromatography and cysteine-containing peptides were isolated by affinity chromatography. The peptides were identified by microcapillary liquid chromatography-electrospray ionisation MS/MS (muLC-ESI MS/MS). In two experiments, peptides representing a total of 1131 database entries were identified. They are involved in different presynaptic and postsynaptic functions, including synaptic vesicle exocytosis for neurotransmitter release, vesicle endocytosis for synaptic vesicle recycling, as well as postsynaptic receptors and proteins constituting the PSyD. Moreover, a large number of soluble and membrane-bound molecules serving functions in synaptic signal transduction and metabolism were detected. The results provide an inventory of the synaptic proteome and confirm the suitability of the ICAT method for the assessment of synaptic structure, function and plasticity.

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