670 research outputs found
Accurate Protein Complex Retrieval by Affinity Enrichment Mass Spectrometry (AE-MS) Rather than Affinity Purification Mass Spectrometry (AP-MS)
Protein-protein interactions are fundamental to the understanding of biological processes. Affinity purification coupled to mass spectrometry (AP-MS) is one of the most promising methods for their investigation. Previously, complexes were purified as much as possible, frequently followed by identification of individual gel bands. However, todays mass spectrometers are highly sensitive, and powerful quantitative proteomics strategies are available to distinguish true interactors from background binders. Here we describe a high performance affinity enrichment-mass spectrometry method for investigating protein-protein interactions, in which no attempt at purifying complexes to homogeneity is made. Instead, we developed analysis methods that take advantage of specific enrichment of interactors in the context of a large amount of unspecific background binders. We perform single-step affinity enrichment of endogenously expressed GFP-tagged proteins and their interactors in budding yeast, followed by single-run, intensity-based label-free quantitative LC-MS/MS analysis. Each pull-down contains around 2000 background binders, which are reinterpreted from troubling contaminants to crucial elements in a novel data analysis strategy. First the background serves for accurate normalization. Second, interacting proteins are not identified by comparison to a single untagged control strain, but instead to the other tagged strains. Third, potential interactors are further validated by their intensity profiles across all samples. We demonstrate the power of our AE-MS method using several well-known and challenging yeast complexes of various abundances. AE-MS is not only highly efficient and robust, but also cost effective, broadly applicable, and can be performed in any laboratory with access to high-resolution mass spectrometers
Atmospheric aerosols at the Pierre Auger Observatory and environmental implications
The Pierre Auger Observatory detects the highest energy cosmic rays.
Calorimetric measurements of extensive air showers induced by cosmic rays are
performed with a fluorescence detector. Thus, one of the main challenges is the
atmospheric monitoring, especially for aerosols in suspension in the
atmosphere. Several methods are described which have been developed to measure
the aerosol optical depth profile and aerosol phase function, using lasers and
other light sources as recorded by the fluorescence detector. The origin of
atmospheric aerosols traveling through the Auger site is also presented,
highlighting the effect of surrounding areas to atmospheric properties. In the
aim to extend the Pierre Auger Observatory to an atmospheric research platform,
a discussion about a collaborative project is presented.Comment: Regular Article, 16 pages, 12 figure
Multivalent binding of PWWP2A to H2A.Z regulates mitosis and neural crest differentiation
Replacement of canonical histones with specialized histone variants promotes altering of chromatin structure and function. The essential histone variant H2A.Z affects various DNA-based processes via poorly understood mechanisms. Here, we determine the comprehensive interactome of H2A.Z and identify PWWP2A as a novel H2A.Z-nucleosome binder. PWWP2A is a functionally uncharacterized, vertebrate-specific protein that binds very tightly to chromatin through a concerted multivalent binding mode. Two internal protein regions mediate H2A.Z-specificity and nucleosome interaction, whereas the PWWP domain exhibits direct DNA binding. Genome-wide mapping reveals that PWWP2A binds selectively to H2A.Z-containing nucleosomes with strong preference for promoters of highly transcribed genes. In human cells, its depletion affects gene expression and impairs proliferation via a mitotic delay. While PWWP2A does not influence H2A.Z occupancy, the C-terminal tail of H2A.Z is one important mediator to recruit PWWP2A to chromatin. Knockdown of PWWP2A in Xenopus results in severe cranial facial defects, arising from neural crest cell differentiation and migration problems. Thus, PWWP2A is a novel H2A.Z-specific multivalent chromatin binder providing a surprising link between H2A.Z, chromosome segregation, and organ development
A novel method for the absolute fluorescence yield measurement by AIRFLY
One of the goals of the AIRFLY (AIR FLuorescence Yield) experiment is to
measure the absolute fluorescence yield induced by electrons in air to better
than 10% precision. We introduce a new technique for measurement of the
absolute fluorescence yield of the 337 nm line that has the advantage of
reducing the systematic uncertainty due to the detector calibration. The
principle is to compare the measured fluorescence yield to a well known process
- the Cerenkov emission. Preliminary measurements taken in the BFT (Beam Test
Facility) in Frascati, Italy with 350 MeV electrons are presented. Beam tests
in the Argonne Wakefield Accelerator at the Argonne National Laboratory, USA
with 14 MeV electrons have also shown that this technique can be applied at
lower energies.Comment: presented at the 5th Fluorescence Workshop, El Escorial - Madrid,
Spain, 16 - 20 September 200
Temperature and Humidity Dependence of Air Fluorescence Yield measured by AIRFLY
The fluorescence detection of ultra high energy cosmic rays requires a
detailed knowledge of the fluorescence light emission from nitrogen molecules
over a wide range of atmospheric parameters, corresponding to altitudes typical
of the cosmic ray shower development in the atmosphere. We have studied the
temperature and humidity dependence of the fluorescence light spectrum excited
by MeV electrons in air. Results for the 313.6 nm, 337.1 nm, 353.7 nm and 391.4
nm bands are reported in this paper. We found that the temperature and humidity
dependence of the quenching process changes the fluorescence yield by a
sizeable amount (up to 20%) and its effect must be included for a precise
estimation of the energy of ultra high energy cosmic rays.Comment: presented at the 5th Fluorescence Workshop, El Escorial - Madrid,
Spain, 16 - 20 September 2007, to appear in Nuclear Instruments and Methods
Atmospheric effects on extensive air showers observed with the Surface Detector of the Pierre Auger Observatory
Atmospheric parameters, such as pressure (P), temperature (T) and density,
affect the development of extensive air showers initiated by energetic cosmic
rays. We have studied the impact of atmospheric variations on extensive air
showers by means of the surface detector of the Pierre Auger Observatory. The
rate of events shows a ~10% seasonal modulation and ~2% diurnal one. We find
that the observed behaviour is explained by a model including the effects
associated with the variations of pressure and density. The former affects the
longitudinal development of air showers while the latter influences the Moliere
radius and hence the lateral distribution of the shower particles. The model is
validated with full simulations of extensive air showers using atmospheric
profiles measured at the site of the Pierre Auger Observatory.Comment: 24 pages, 9 figures, accepted for publication in Astroparticle
Physic
Measurement of the Depth of Maximum of Extensive Air Showers above 10^18 eV
We describe the measurement of the depth of maximum, Xmax, of the
longitudinal development of air showers induced by cosmic rays. Almost four
thousand events above 10^18 eV observed by the fluorescence detector of the
Pierre Auger Observatory in coincidence with at least one surface detector
station are selected for the analysis. The average shower maximum was found to
evolve with energy at a rate of (106 +35/-21) g/cm^2/decade below 10^(18.24 +/-
0.05) eV and (24 +/- 3) g/cm^2/decade above this energy. The measured
shower-to-shower fluctuations decrease from about 55 to 26 g/cm^2. The
interpretation of these results in terms of the cosmic ray mass composition is
briefly discussed.Comment: Accepted for publication by PR
The Pierre Auger Observatory III: Other Astrophysical Observations
Astrophysical observations of ultra-high-energy cosmic rays with the Pierre
Auger ObservatoryComment: Contributions to the 32nd International Cosmic Ray Conference,
Beijing, China, August 201
Operations of and Future Plans for the Pierre Auger Observatory
Technical reports on operations and features of the Pierre Auger Observatory,
including ongoing and planned enhancements and the status of the future
northern hemisphere portion of the Observatory. Contributions to the 31st
International Cosmic Ray Conference, Lodz, Poland, July 2009.Comment: Contributions to the 31st ICRC, Lodz, Poland, July 200
- …