Release of amino-terminal fragments from amyloid precursor protein reporter and mutated derivatives in cultured cells.

Abnormal proteolytic processing of amyloid precursor protein (APP) is thought to be central to the formation and deposition of beta amyloid peptide in Alzheimer's disease. A putative "secretase" activity normally releases an amino-terminal APP fragment by cleaving APP at residues within the beta amyloid peptide thereby precluding amyloidogenesis. In order to better understand the requirements for APP cleavage by secretase, we have expressed a modified cDNA construct representing the 751-amino acid isoform of APP (APP-REP) and mutated APP-REP proteins in cultured cells. Here, we show that: (a) APP-REP is predominantly associated with membranes; (b) intracellular turnover and processing of APP-REP is similar to that reported for the intact APP protein; (c) secretion appears unaltered by introduction of the glutamate to glutamine mutation found in the APP gene of patients suffering from hereditary cerebral hemorrhage with amyloidosis of Dutch origin; (d) a mutation in which the 18 juxtamembranous amino acids encompassing the secretase site are deleted also allows release of an amino-terminal fragment into the conditioned medium; and (e) kinetics of cleavage of APP-REP and its mutated derivatives are similar. These results indicate that the secretory cleavage of the extracellular amino-terminal fragments of APP-REP can occur in the presence of different novel juxtamembranous amino acid sequences.     

SuperHirn - a novel tool for high resolution LC-MS-based peptide/protein profiling

Label-free quantification of high mass resolution LC-MS data has emerged as a promising technology for proteome analysis. Computational methods are required for the accurate extraction of peptide signals from LC-MS data and the tracking of these features across the measurements of different samples. We present here an open source software tool, SuperHirn, that comprises a set of modules to process LC-MS data acquired on a high resolution mass spectrometer. The program includes newly developed functionalities to analyze LC-MS data such as feature extraction and quantification, LC-MS similarity analysis, LC-MS alignment of multiple datasets, and intensity normalization. These program routines extract profiles of measured features and comprise tools for clustering and classification analysis of the profiles. SuperHirn was applied in an MS1-based profiling approach to a benchmark LC-MS dataset of complex protein mixtures with defined concentration changes. We show that the program automatically detects profiling trends in an unsupervised manner and is able to associate proteins to their correct theoretical dilution profile.     

Reproducible and Consistent Quantification of the Saccharomyces cerevisiae Proteome by SWATH-mass spectrometry

Targeted mass spectrometry by selected reaction monitoring (S/MRM) has proven to be a suitable technique for the consistent and reproducible quantification of proteins across multiple biological samples and a wide dynamic range. This performance profile is an important prerequisite for systems biology and biomedical research. However, the method is limited to the measurements of a few hundred peptides per LC-MS analysis. Recently, we introduced SWATH-MS, a combination of data independent acquisition and targeted data analysis that vastly extends the number of peptides/proteins quantified per sample, while maintaining the favorable performance profile of S/MRM. Here we applied the SWATH-MS technique to quantify changes over time in a large fraction of the proteome expressed in Saccharomyces cerevisiae in response to osmotic stress.We sampled cell cultures in biological triplicates at six time points following the application of osmotic stress and acquired single injection data independent acquisition data sets on a high-resolution 5600 tripleTOF instrument operated in SWATH mode. Proteins were quantified by the targeted extraction and integration of transition signal groups from the SWATH-MS datasets for peptides that are proteotypic for specific yeast proteins. We consistently identified and quantified more than 15,000 peptides and 2500 proteins across the 18 samples. We demonstrate high reproducibility between technical and biological replicates across all time points and protein abundances. In addition, we show that the abundance of hundreds of proteins was significantly regulated upon osmotic shock, and pathway enrichment analysis revealed that the proteins reacting to osmotic shock are mainly involved in the carbohydrate and amino acid metabolism. Overall, this study demonstrates the ability of SWATH-MS to efficiently generate reproducible, consistent, and quantitatively accurate measurements of a large fraction of a proteome across multiple samples.In systems biology and biomedical studies targeted mass spectrometry via selected reaction monitoring (SRM)¹ (also known as multiple reaction monitoring, MRM) has emerged as a powerful technique for the consistent and reproducible quantification of proteins across numerous complex samples (1–6). Optimal sets of precursor/fragment ion pairs, called transitions, uniquely represent a specific peptide. They constitute a definitive mass spectrometric assay for the detection of targeted peptides, and thus the proteins from which they derive, in the complex matrix of trypsinized biological samples (1, 7). Protein quantification is then performed by relating the intensity of the acquired transition signals to suitable reference signals. Most quantification strategies commonly used in proteomics are compatible with this method (8). Recently, the high-throughput development of S/MRM assays has been achieved via the generation of MS/MS spectral libraries from the measurements of thousands of synthetic peptides representing proteotypic peptides (9). Moreover, many experimental and bioinformatics workflows have been developed for assay generation, assay optimization, data evaluation, and the dissemination of optimized S/MRM assays (10–16). In combination, these developments have supported the creation of mass-spectrometric maps of entire proteomes of selected species including Streptococcus pyogenes, Mycobacterium tuberculosis, and Saccharomyces cerevisae (5, 17–19) and the robust use of these resources to quantify specific protein sets across multiple biological samples.Currently, targeted proteomics by S/MRM can be multiplexed to a maximum set of ∼100 proteins that can be measured in a single LC-S/MRM run at optimal quantitative accuracy, limit of detection and dynamic range. The quantification of higher numbers of proteins per run compromises some of the performance parameters of the method because of well understood tradeoffs (8). Attempts have been made to further increase the degree of multiplexing of S/MRM, either by automated adjustment of the scheduled detection windows (20) or by acquiring, in a data-dependent manner, the complete set of precursor-fragment ion pairs of a given assay (21). Alternatively, parallel reaction monitoring (PRM) approach operated on quadrupole-orbitrap mass spectrometer has shown detection and quantification performances similar or better than those obtained in SRM, because of the increased selectivity of the mass analyzer (22–24). These approaches are promising, but their application relies on prior knowledge of the precursor ions that need to be targeted during the data acquisition, and they still are subject of the above-mentioned tradeoffs.Recently, we developed a novel MS strategy that combines data independent acquisition (DIA) of trypsinized protein samples with S/MRM-like, in silico targeted analysis of the acquired complete fragment ion maps (25). We termed the method SWATH-MS, and applied the sequential isolation window acquisition principle (26) to repeatedly cycle, in a single injection, through 32 consecutive 25-Da precursor isolation windows (swaths). The process acquires fragment ion spectra of all precursors in a space defined by the 400–1200 m/z precursor range and a user-specified retention time window. We used the prior information in MS/MS spectral libraries to extract groups of signals that uniquely identify a specific peptide, and to demonstrate that peptides could be identified and quantified over a dynamic range of four orders of magnitude, even when the precursors were not detectable in a survey MS scan. For the 45 proteins involved in the central carbon metabolism of yeast, we demonstrated that the accuracy of quantification was equivalent to that of S/MRM (25). However, because of the lack of adequate software tools at that time, the extensive high-throughput targeted data analysis of the SWATH-MS maps could not be fully demonstrated in that first study.Here we demonstrate the multiplexing capabilities of SWATH-MS for the detection and quantification of significantly larger fractions of a proteome as compared with S/MRM, without compromising reproducibility, consistency, and quantitative accuracy. We describe the large scale deployment of fragment ion spectral libraries and the use of S/MRM-like analysis tools specifically adapted to SWATH-MS data for the detection and quantification of temporal changes of the S. cerevisae proteome in response to osmotic stress.     

Climate and geographic trends in hatch delay of the treehole mosquito,Aedes triseriatus Say (Diptera: Culicidae)

Eggs of Aedes triseriatus mosquitoes are stimulated to hatch when inundated with water, but only a small fraction of eggs from the same batch will hatch for any given stimulus. Similar hatching or germination patterns are observed in desert plants, copepods, rotifers, insects, and many other species. Bet hedging theory suggests that parents stagger offspring emergence into vulnerable life history stages in order to avoid catastrophic reproductive failures. For Ae. triseriatus, a treehole breeding mosquito, immediate hatching of an entire clutch leaves all of the parent's progeny vulnerable to extinction in the event of a severe drought. Natural selection has likely favored parents that pursued a bet hedging strategy where the risk of reproductive failure is distributed over time. Considering treehole mosquitoes, bet hedging theory could be used to predict that hatch delay would be positively correlated with the likelihood of drought. To test this prediction, we collected Ae. triseriatus from habitats that varied widely in mean annual precipitation and exposed them to several hatch stimuli in the laboratory. Here we report that, as predicted, Ae. triseriatus eggs from high precipitation regions showed less hatch delay than areas of low precipitation. This strategy probably allows Ae. triseriatus to cope with the wide variety of climatic conditions that it faces in its extensive geographical range.     

Are the taxonomic concepts of agamospermous genera useful for autogamous groups — A critical discussion using the example ofEuphrasia (Scrophulariaceae)

Problems and difficulties in the taxonomy ofEuphrasia (Scrophulariaceae) are discussed. They are partly due to autogamous breeding systems, but also due to patterns of ecological convergences. The use of taxonomic approaches to agamospermous complexes within autogamous groups in the genusEuphrasia is discussed and rejected because of producing unnecessary taxa for ephemeral variants. Also for the taxonomic treatment of agamospermous complexes in general the existence of a broad set of populations with stable morphological, ecological and/or geographical integrities should be the inevitable basis for describing of new taxa.     

Cdc25A localisation and shuttling: characterisation of sequences mediating nuclear export and import

The Cdc25 phosphatases play crucial roles in cell cycle progression by removing inhibitory phosphates from tyrosine and threonine residues of cyclin-dependent kinases. Cdc25A is an important regulator of the G1/S transition but functions also in the mitotic phase of the human cell cycle. In this paper, we investigate the sub-cellular localisation of exogenously expressed Cdc25A. We show that YFP-Cdc25A is localised both in the nucleus and the cytoplasm of HeLa cells and untransformed fibroblasts. Cell fusion assays and fluorescence loss in photobleaching (FLIP) assays reveal that the localisation is dynamic and the protein shuttles between the nucleus and the cytoplasm. We demonstrate that nuclear export of Cdc25A is partly mediated by an N-terminal nuclear export sequence (NES), in a manner not sensitive to the Exportin 1-inhibitor leptomycin B. A nuclear localisation signal (NLS) is also characterised, mutation of which leads to cytoplasmic localisation of Cdc25A. Our results imply that the Cdc25A phosphatase may interact with substrates and regulators both in the nucleus and the cytoplasm.