Linking genome and proteome by mass spectrometry: Large-scale identification of yeast proteins from two dimensional gels

The function of many of the uncharacterized open reading frames discovered by genomic sequencing can be determined at the level of expressed gene products, the proteome. However, identifying the cognate gene from minute amounts of protein has been one of the major problems in molecular biology. Using yeast as an example, we demonstrate here that mass spectrometric protein identification is a general solution to this problem given a completely sequenced genome. As a first screen, our strategy uses automated laser desorption ionization mass spectrometry of the peptide mixtures produced by in-gel tryptic digestion of a protein. Up to 90% of proteins are identified by searching sequence data bases by lists of peptide masses obtained with high accuracy. The remaining proteins are identified by partially sequencing several peptides of the unseparated mixture by nanoelectrospray tandem mass spectrometry followed by data base searching with multiple peptide sequence tags. In blind trials, the method led to unambiguous identification in all cases. In the largest individual protein identification project to date, a total of 150 gel spots—many of them at subpicomole amounts—were successfully analyzed, greatly enlarging a yeast two-dimensional gel data base. More than 32 proteins were novel and matched to previously uncharacterized open reading frames in the yeast genome. This study establishes that mass spectrometry provides the required throughput, the certainty of identification, and the general applicability to serve as the method of choice to connect genome and proteome.

RANK is the intrinsic hematopoietic cell surface receptor that controls osteoclastogenesis and regulation of bone mass and calcium metabolism

We have generated RANK (receptor activator of NF-κB) nullizygous mice to determine the molecular genetic interactions between osteoprotegerin, osteoprotegerin ligand, and RANK during bone resorption and remodeling processes. RANK−/− mice lack osteoclasts and have a profound defect in bone resorption and remodeling and in the development of the cartilaginous growth plates of endochondral bone. The osteopetrosis observed in these mice can be reversed by transplantation of bone marrow from rag1−/− (recombinase activating gene 1) mice, indicating that RANK−/− mice have an intrinsic defect in osteoclast function. Calciotropic hormones and proresorptive cytokines that are known to induce bone resorption in mice and human were administered to RANK−/− mice without inducing hypercalcemia, although tumor necrosis factor α treatment leads to the rare appearance of osteoclast-like cells near the site of injection. Osteoclastogenesis can be initiated in RANK−/− mice by transfer of the RANK cDNA back into hematopoietic precursors, suggesting a means to critically evaluate RANK structural features required for bone resorption. Together these data indicate that RANK is the intrinsic cell surface determinant that mediates osteoprotegerin ligand effects on bone resorption and remodeling as well as the physiological and pathological effects of calciotropic hormones and proresorptive cytokines.

Mutation R120G in αB-crystallin, which is linked to a desmin-related myopathy, results in an irregular structure and defective chaperone-like function

αB-crystallin, a member of the small heat shock protein family, possesses chaperone-like function. Recently, it has been shown that a missense mutation in αB-crystallin, R120G, is genetically linked to a desmin-related myopathy as well as to cataracts [Vicart, P., Caron, A., Guicheney, P., Li, A., Prevost, M.-C., Faure, A., Chateau, D., Chapon, F., Tome, F., Dupret, J.-M., et al. (1998) Nat. Genet. 20, 92–95]. By using α-lactalbumin, alcohol dehydrogenase, and insulin as target proteins, in vitro assays indicated that R120G αB-crystallin had reduced or completely lost chaperone-like function. The addition of R120G αB-crystallin to unfolding α-lactalbumin enhanced the kinetics and extent of its aggregation. R120G αB-crystallin became entangled with unfolding α-lactalbumin and was a major portion of the resulting insoluble pellet. Similarly, incubation of R120G αB-crystallin with alcohol dehydrogenase and insulin also resulted in the presence of R120G αB-crystallin in the insoluble pellets. Far and near UV CD indicate that R120G αB-crystallin has decreased β-sheet secondary structure and an altered aromatic residue environment compared with wild-type αB-crystallin. The apparent molecular mass of R120G αB-crystallin, as determined by gel filtration chromatography, is 1.4 MDa, which is more than twice the molecular mass of wild-type αB-crystallin (650 kDa). Images obtained from cryoelectron microscopy indicate that R120G αB-crystallin possesses an irregular quaternary structure with an absence of a clear central cavity. The results of this study show, through biochemical analysis, that an altered structure and defective chaperone-like function of αB-crystallin are associated with a point mutation that leads to a desmin-related myopathy and cataracts.

Direct genetic analysis by matrix-assisted laser desorption/ionization mass spectrometry

An approach to analyzing single-nucleotide polymorphisms (SNPs) found in the human genome has been developed that couples a recently developed invasive cleavage assay for nucleic acids with detection by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). The invasive cleavage assay is a signal amplification method that enables the analysis of SNPs by MALDI-TOF MS directly from human genomic DNA without the need for initial target amplification by PCR. The results presented here show the successful genotyping by this approach of twelve SNPs located randomly throughout the human genome. Conventional Sanger sequencing of these SNP positions confirmed the accuracy of the MALDI-TOF MS analysis results. The ability to unambiguously detect both homozygous and heterozygous genotypes is clearly demonstrated. The elimination of the need for target amplification by PCR, combined with the inherently rapid and accurate nature of detection by MALDI-TOF MS, gives this approach unique and significant advantages in the high-throughput genotyping of large numbers of SNPs, useful for locating, identifying, and characterizing the function of specific genes.

Viral mediated expression of insulin-like growth factor I blocks the aging-related loss of skeletal muscle function

During the aging process, mammals lose up to a third of their skeletal muscle mass and strength. Although the mechanisms underlying this loss are not entirely understood, we attempted to moderate the loss by increasing the regenerative capacity of muscle. This involved the injection of a recombinant adeno-associated virus directing overexpression of insulin-like growth factor I (IGF-I) in differentiated muscle fibers. We demonstrate that the IGF-I expression promotes an average increase of 15% in muscle mass and a 14% increase in strength in young adult mice, and remarkably, prevents aging-related muscle changes in old adult mice, resulting in a 27% increase in strength as compared with uninjected old muscles. Muscle mass and fiber type distributions were maintained at levels similar to those in young adults. We propose that these effects are primarily due to stimulation of muscle regeneration via the activation of satellite cells by IGF-I. This supports the hypothesis that the primary cause of aging-related impairment of muscle function is a cumulative failure to repair damage sustained during muscle utilization. Our results suggest that gene transfer of IGF-I into muscle could form the basis of a human gene therapy for preventing the loss of muscle function associated with aging and may be of benefit in diseases where the rate of damage to skeletal muscle is accelerated.

DNA-bound transcription factor complexes analysed by mass-spectrometry: binding of novel proteins to the human c-fos SRE and related sequences

Transcription factors control eukaryotic polymerase II function by influencing the recruitment of multiprotein complexes to promoters and their subsequent integrated function. The complexity of the functional ‘transcriptosome’ has necessitated biochemical fractionation and subsequent protein sequencing on a grand scale to identify individual components. As a consequence, much is now known of the basal transcription complex. In contrast, less is known about the complexes formed at distal promoter elements. The c-fos SRE, for example, is known to bind Serum Response Factor (SRF) and ternary complex factors such as Elk-1. Their interaction with other factors at the SRE is implied but, to date, none have been identified. Here we describe the use of mass-spectrometric sequencing to identify six proteins, SRF, Elk-1 and four novel proteins, captured on SRE duplexes linked to magnetic beads. This approach is generally applicable to the characterisation of nucleic acid-bound protein complexes and the post-translational modification of their components.

Electrospray ionization mass spectrometry analysis of changes in phospholipids in RBL-2H3 mastocytoma cells during degranulation

Biological membranes contain an extraordinary diversity of lipids. Phospholipids function as major structural elements of cellular membranes, and analysis of changes in the highly heterogeneous mixtures of lipids found in eukaryotic cells is central to understanding the complex functions in which lipids participate. Phospholipase-catalyzed hydrolysis of phospholipids often follows cell surface receptor activation. Recently, we demonstrated that granule fusion is initiated by addition of exogenous, nonmammalian phospholipases to permeabilized mast cells. To pursue this finding, we use positive and negative mode Fourier-transform ion cyclotron resonance mass spectrometry (FTICR-MS) to measure changes in the glycerophospholipid composition of total lipid extracts of intact and permeabilized RBL-2H3 (mucosal mast cell line) cells. The low energy of the electrospray ionization results in efficient production of molecular ions of phospholipids uncomplicated by further fragmentation, and changes were observed that eluded conventional detection methods. From these analyses we have spectrally resolved more than 130 glycerophospholipids and determined changes initiated by introduction of exogenous phospholipase C, phospholipase D, or phospholipase A2. These exogenous phospholipases have a preference for phosphatidylcholine with long polyunsaturated alkyl chains as substrates and, when added to permeabilized mast cells, produce multiple species of mono- and polyunsaturated diacylglycerols, phosphatidic acids, and lysophosphatidylcholines, respectively. The patterns of changes of these lipids provide an extraordinarily rich source of data for evaluating the effects of specific lipid species generated during cellular processes, such as exocytosis.

The Lateral Trigger Probability function for the Ultra-High Energy Cosmic Ray showers detected by the Pierre Auger Observatory

In this paper we introduce the concept of Lateral Trigger Probability (LTP) function, i.e., the probability for an Extensive Air Shower (EAS) to trigger an individual detector of a ground based array as a function of distance to the shower axis, taking into account energy, mass and direction of the primary cosmic ray. We apply this concept to the surface array of the Pierre Auger Observatory consisting of a 1.5 km spaced grid of about 1600 water Cherenkov stations. Using Monte Carlo simulations of ultra-high energy showers the LTP functions are derived for energies in the range between 10(17) and 10(19) eV and zenith angles up to 65 degrees. A parametrization combining a step function with an exponential is found to reproduce them very well in the considered range of energies and zenith angles. The LTP functions can also be obtained from data using events simultaneously observed by the fluorescence and the surface detector of the Pierre Auger Observatory (hybrid events). We validate the Monte Carlo results showing how LTP functions from data are in good agreement with simulations.

On the steric and mass-induced contributions to the annual sea level variations in the Mediterranean Sea

The sea level variation (SLVtotal) is the sum of two major contributions: steric and mass-induced. The steric SLVsteric is that resulting from the thermal and salinity changes in a given water column. It only involves volume change, hence has no gravitational effect. The mass-induced SLVmass, on the other hand, arises from adding or subtracting water mass to or from the water column and has direct gravitational signature. We examine the closure of the seasonal SLV budget and estimate the relative importance of the two contributions in the Mediterranean Sea as a function of time. We use ocean altimetry data (from TOPEX/Poseidon, Jason 1, ERS, and ENVISAT missions) to estimate SLVtotal, temperature, and salinity data (from the Estimating the Circulation and Climate of the Ocean ocean model) to estimate SLVsteric, and time variable gravity data (from Gravity Recovery and Climate Experiment (GRACE) Project, April 2002 to July 2004) to estimate SLVmass. We find that the annual cycle of SLVtotal in the Mediterranean is mainly driven by SLVsteric but moderately offset by SLVmass. The agreement between the seasonal SLVmass estimations from SLVtotal – SLVsteric and from GRACE is quite remarkable; the annual cycle reaches the maximum value in mid-February, almost half a cycle later than SLVtotal or SLVsteric, which peak by mid-October and mid-September, respectively. Thus, when sea level is rising (falling), the Mediterranean Sea is actually losing (gaining) mass. Furthermore, as SLVmass is balanced by vertical (precipitation minus evaporation, P–E) and horizontal (exchange of water with the Atlantic, Black Sea, and river runoff) mass fluxes, we compared it with the P–E determined from meteorological data to estimate the annual cycle of the horizontal flux.

Self-heating function of carbon nanofiber cement pastes

The viability of carbon nanofiber (CNF) composites in cement matrices as a self-heating material is reported in this paper. This functional application would allow the use of CNF cement composites as a heating element in buildings, or for deicing pavements of civil engineering transport infrastructures, such as highways or airport runways. Cement pastes with the addition of different CNF dosages (from 0 to 5% by cement mass) have been prepared. Afterwards, tests were run at different fixed voltages (50, 100 and 150V), and the temperature of the specimens was registered. Also the possibility of using a casting method like shotcrete, instead of just pouring the fresh mix into the mild (with no system’s efficiency loss expected) was studied. Temperatures up to 138 °C were registered during shotcrete-5% CNF cement paste tests (showing initial 10 °C/min heating rates). However a minimum voltage was required in order to achieve a proper system functioning.

Analysis of minerals from Arctic Ocean sediments

The Arctic Ocean System is a key player regarding the climatic changes of Earth. Its highly sensitive ice Cover, the exchange of surface and deep water masses with the global ocean and the coupling with the atmosphere interact directly with global climatic changes. The output of cold, polar water and sea ice influences the production of deep water in the North Atlantic and controls the global ocean circulation ("the conveyor belt"). The Arctic Ocean is surrounded by the large Northern Hemisphere ice sheets which not only affect the sedimentation in the Arctic Ocean but also are supposed to induce the Course of glacials and interglacials. Terrigenous sediment delivered from the ice sheets by icebergs and meltwater as well as through sea ice are major components of Arctic Ocean sediments. Hence, the terrigenous content of Arctic Ocean sediments is an outstanding archive to investigate changes in the paleoenvironment. Glazigenic sediments of the Canadian Arctic Archipelago and surface samples of the Arctic Ocean and the Siberian shelf regions were investigated by means of x-ray diffraction of the bulk fraction. The source regions of distinct mineral compositions were to be deciphered. Regarding the complex circumpolar geology stable christalline shield rocks, active and ancient fold belts including magmatic and metamorphic rocks, sedimentary rocks and wide periglacial lowlands with permafrost provide a complete range of possible mineral combinations. Non- glaciated shelf regions mix the local input from a possible point source of a particular mineral combination with the whole shelf material and function as a sampler of the entire region draining to the shelf. To take this into account, a literature research was performed. Descriptions of outcropping lithologies and Arctic Ocean sediments were scanned for their mineral association. The analyses of glazigenic and shelf sediments yielded a close relationship between their mineral composition and the adjacent source region. The most striking difference between the circumpolar source regions is the extensive outcrop of carbonate rocks in the vicinity of the Canadian Arctic Archipelago and in N Greenland while siliciclastic sediments dominate the Siberian shelves. In the Siberian shelf region the eastern Kara Sea and the western Laptev Sea form a destinct region defined by high smectite, (clino-) pyroxene and plagioclase input. The source of this signal are the extensive outcrops of the Siberian trap basalt in the Putorana Plateau which is drained by the tributaries of the Yenissei and Khatanga. The eastern Laptev Sea and the East Siberian Sea can also be treated as one source region containing a feldspar, quartz, illite, mica, and chlorite asscciation combined with the trace minerals hornblende and epidote. Franz Josef Land provides a mineral composition rich in quartz and kaolinite. The diverse rock suite of the Svalbard archipelago distributes specific mineral compositions of highly metamorphic christalline rocks, dolomite-rich carbonate rocks and sedimentary rocks with a higher diagenetic potential manifested in stable newly built diagenetic minerals and high organic maturity. To reconstruct the last 30,000 years as an example of the transition between glacial and interglacial conditions a profile of sediment cores, recovered during the RV Polarstern" expedition ARK-VIIIl3 (ARCTIC '91), and additional sediment cores around Svalbard were investigated. Besides the mineralogy of different grain size fractions several additional sedimentological and organo-geochemical Parameterswere used. A detailed stratigraphic framework was achieved. By exploiting this data set changes in the mineral composition of the Eurasian Basin sediments can be related to climatic changes. Certain mineral compositions can even be associated with particular transport processes, e.g. the smectitel pyroxene association with sea ice transport from the eastern Kara Sea and the western Laptev Sea. Hence, it is possible to decipher the complex interplay between the influx of warm Atlantic waters into the Southwest of the Eurasian Basin, the waxing and waning of the Svalbard1Barents- Sea- and Kara-Sea-Ice-Sheets, the flooding of the Siberian shelf regions and the surface and deep water circulation. Until now the Arctic Ocean was assumed to be a rather stable System during the last 30,000 years which only switched from a completely ice covered situation during the glacial to seasonally Open waters during the interglacial. But this work using mineral assemblages of sediment cores in the vicinity of Svalbard revealed fast changes in the inflow of warm Atlantic water with the Westspitsbergen Current (< 1000 years), short periods of advances and retreats of the marine based Eurasian ice sheets (1000-3000 years), and short melting phases (400 years?). Deglaciation of the marine-based Eurasian and the land-based north American and Greenland ice sheets are not simultaneous. This thesis postulates that the Kara Sea Ice Sheet released an early meltwater signal prior to 15,000 14C years leading the Barents Sea Ice Sheet while the western land-based ice sheets are following later than 13,500 14C years. The northern Eurasian Basin records the shift between iceberg and sea-ice material derived from the Canadian Arctic Archipelago and N-Greenland and material transported by sea-ice and surface currents from the Siberian shelf region. The phasing of the deglaciation becomes very obvious using the dolomite and quartd phyllosilicate record. It is also supposed that the flooding of the Laptev Sea during the Holocene is manifested in a stepwise increase of sediment input at the Lomonosov Ridge between the Eurasian and Amerasian Basin. Depending on the strength of meltwater pulses from the adjacent ice sheets the Transpolar Drift can probably be relocated. These movements are traceable by the distribution of indicator minerals. Based on the outcome of this work the feasibility of bulk mineral determination can be qualified as excellent tool for paleoenvironmental reconstructions in the Arctic Ocean. The easy preparation and objective determination of bulk mineralogy provided by the QUAX software bears the potential to use this analyses as basic measuring method preceding more time consuming and highly specialised mineralogical investigations (e.g. clay mineralogy, heavy mineral determination).

Fibroblast growth factor 23 and markers of mineral metabolism in individuals with preserved renal function

Fibroblast growth factor 23 (FGF23) is a bone-derived hormone that regulates phosphate homeostasis. Circulating FGF23 is elevated in chronic kidney disease (CKD) and independently associated with poor renal and cardiovascular outcomes and mortality. Because the study of FGF23 in individuals with normal renal function has received little attention, we examined in a large, population based study of 1128 participants the associations of FGF23 with markers of mineral metabolism and renal function. The median estimated glomerular filtration rate (eGFR) of the cohort was 105 ml/min per 1.73 m2, and the median plasma FGF23 was 78.5 RU/ml. FGF23 increased and plasma 1,25-dihydroxyvitamin D3 decreased significantly below an eGFR threshold of 102 and 99 ml/min per 1.73 m2, respectively. In contrast, plasma parathyroid hormone increased continuously with decreasing eGFR and was first significantly elevated at an eGFR of 126 ml/min per 1.73 m2. On multivariable analysis adjusting for sex, age, body mass index, and GFR, FGF23 was negatively associated with 1,25-dihydroxyvitamin D3, and urinary absolute and fractional calcium excretion but not with serum calcium or parathyroid hormone. We found a positive association of FGF23 with plasma phosphate, but no association with urinary absolute or fractional phosphate excretion and, unexpectedly, a positive association with tubular maximum phosphate reabsorption/GFR. Thus, in the absence of CKD, parathyroid hormone increases earlier than FGF23 when the eGFR decreases. The increase in FGF23 occurs at a higher eGFR threshold than previously reported and is closely associated with a decrease in 1,25-dihydroxyvitamin D3. We speculate that the main demonstrable effect of FGF23 in the setting of preserved renal function is suppression of 1,25-dihydroxyvitamin D3 rather than stimulation of renal phosphate excretion.

The zeros and maxima of the Airy function and its first derivative to 25 significant figures.

Mode of access: Internet.

ATCA HI observations of the peculiar galaxy IC 2554

ATCA H I and radio continuum observations of the peculiar southern galaxy IC 2554 and its surroundings reveal typical signatures of an interacting galaxy group. We detected a large H I cloud between IC 2554 and the elliptical galaxy NGC 3136B. The gas dynamics in IC 2554 itself, which is sometimes described as a colliding pair, are surprisingly regular, whereas NGC 3136B was not detected. The H I cloud, which emerges from IC 2554 as a large arc-shaped plume, has a size of similar to30 kpc, larger than that of IC 2554. The total H I mass of the IC 2554 system is similar to2 x 10(9) M., one-third of which resides in the H I cloud. It is possible that tidal interaction between IC 2554 and NGC 3136B caused this spectacular H I cloud, but the possibility of IC 2554 being a merger remnant is also discussed. We also detected H I gas in the nearby galaxies ESO 092-G009 and RKK 1959 and an associated H I cloud, ATCA J1006-6710. Together they have an H I mass of similar to4.6 x 10(8) M-.. Another new H I source, ATCA J1007-6659, with an H I mass of only similar to2.2 x 10(7) M. was detected roughly between IC 2554 and ESO 092-G009 and corresponds to a face-on low surface brightness dwarf galaxy. Star formation is evident only in the galaxy IC 2554 with a rate of similar to4 M. yr(-1).