Applicability of tandem affinity purification MudPIT to pathway proteomics in yeast

A combined multidimensional chromatography-mass spectrometry approach known as "MudPIT" enables rapid identification of proteins that interact with a tagged bait while bypassing some of the problems associated with analysis of polypeptides excised from SDS-polyacrylamide gels. However, the reproducibility, success rate, and applicability of MudPIT to the rapid characterization of dozens of proteins have not been reported. We show here that MudPIT reproducibly identified bona fide partners for budding yeast Gcn5p. Additionally, we successfully applied MudPIT to rapidly screen through a collection of tagged polypeptides to identify new protein interactions. Twenty-five proteins involved in transcription and progression through mitosis were modified with a new tandem affinity purification (TAP) tag. TAP-MudPIT analysis of 22 yeast strains that expressed these tagged proteins uncovered known or likely interacting partners for 21 of the baits, a figure that compares favorably with traditional approaches. The proteins identified here comprised 102 previously known and 279 potential physical interactions. Even for the intensively studied Swi2p/Snf2p, the catalytic subunit of the Swi/Snf chromatin remodeling complex, our analysis uncovered a new interacting protein, Rtt102p. Reciprocal tagging and TAP-MudPIT analysis of Rtt102p revealed subunits of both the Swi/Snf and RSC complexes, identifying Rtt102p as a common interactor with, and possible integral component of, these chromatin remodeling machines. Our experience indicates it is feasible for an investigator working with a single ion trap instrument in a conventional molecular/cellular biology laboratory to carry out proteomic characterization of a pathway, organelle, or process (i.e. "pathway proteomics") by systematic application of TAP-MudPIT

Two‐Dimensional Phase Separation: Co-Adsorption of Hydrogen and Carbon Monoxide on the (111) Surface of Rhodium

The co‐adsorption of CO and H_2 on Rh(111) at low temperature (∼ 100 K) has been studied using thermal desorption mass spectrometry (TDS) and Low‐Energy Electron Diffraction(LEED). The probability of adsorption of CO on rhodium pretreated with hydrogen has been found to decrease slowly with increasing amounts of hydrogen on the surface. In addition, the effect of surface hydrogen on the CO LEED patterns indicates segregation of hydrogen and CO. These results can be explained in terms of a strong repulsive CO–H interaction and a mobile precursor model of CO adsorption

Charting the protein complexome in yeast by mass spectrometry

It has become evident over the past few years that many complex cellular processes, including control of the cell cycle and ubiquitin-dependent proteolysis, are carried out by sophisticated multisubunit protein machines that are dynamic in abundance, post-translational modification state, and composition. To understand better the nature of the macromolecular assemblages that carry out the cell cycle and ubiquitin-dependent proteolysis, we have used mass spectrometry extensively over the past few years to characterize both the composition of various protein complexes and the modification states of their subunits. In this article we review some of our recent efforts, and describe a promising new approach for using mass spectrometry to dissect protein interaction networks

Correlation of plasma levels of digoxin in cardiac patients with dose and measures of renal function

Peer Reviewedhttp://deepblue.lib.umich.edu/bitstream/2027.42/117126/1/cpt1974153291.pd

Bioactive nutrients - Time for tolerable upper intake levels to address safety.

There is increasing interest by consumers, researchers, and regulators into the roles that certain bioactive compounds, derived from plants and other natural sources, can play in health maintenance and promotion, and even prolonging a productive quality of life. Research has rapidly emerged suggesting that a wide range of compounds and mixtures in and from plants (such as fruits and vegetables, tea and cocoa) and animals (such as fish and probiotics) may exert substantial health benefits. There is interest in exploring the possibility of establishing recommended intakes or dietary guidance for certain bioactive substances to help educate consumers. A key aspect of establishing dietary guidance is the assessment of safety/toxicity of these substances. Toxicologists need to be involved in both the development of the safety framework and in the evaluation of the science to establish maximum intake/upper limits

Dynamics of the peroxisomal import cycle of PpPex20p: ubiquitin-dependent localization and regulation

We characterize the peroxin PpPex20p from Pichia pastoris and show its requirement for translocation of PTS2 cargoes into peroxisomes. PpPex20p docks at the peroxisomal membrane and translocates into peroxisomes. Its peroxisomal localization requires the docking peroxin Pex14p but not the peroxins Pex2p, Pex10p, and Pex12p, whose absence causes peroxisomal accumulation of Pex20p. Similarities between Pex5p and Pex20p were noted in their protein interactions and dynamics during import, and both contain a conserved NH2-terminal domain. In the absence of the E2-like Pex4p or the AAA proteins Pex1p and Pex6p, Pex20p is degraded via polyubiquitylation of residue K19, and the K19R mutation causes accumulation of Pex20p in peroxisome remnants. Finally, either interference with K48-branched polyubiquitylation or removal of the conserved NH2-terminal domain causes accumulation of Pex20p in peroxisomes, mimicking a defect in its recycling to the cytosol. Our data are consistent with a model in which Pex20p enters peroxisomes and recycles back to the cytosol in an ubiquitin-dependent manner

Formation and Thermal Stability of sub-10 nm Carbon Templates on Si(100)

We report a lithographic process for creating high-resolution (<10 nm) carbon templates on Si(100). A scanning electron microscope, operating under low vacuum (10E-6 mbar), produces a carbon-containing deposit ("contamination resist") on the silicon surface via electron-stimulated dissociation of ambient hydrocarbons, water and other adsorbed molecules. Subsequent annealing at temperatures up to 1320 K in ultra-high vacuum removes SiO2 and other contaminants, with no observable change in dot shape. The annealed structures are compatible with subsequent growth of semiconductors and complex oxides. Carbon dots with diameter as low as 3.5 nm are obtained with a 200 us electron-beam exposure time.Comment: 13 pages, 4 figure

Development and Performance of the Nanoworkbench: A Four Tip STM for Electrical Conductivity Measurements Down to Sub-micrometer Scales

A multiple-tip ultra-high vacuum (UHV) scanning tunneling microscope (MT-STM) with a scanning electron microscope (SEM) for imaging and molecular-beam epitaxy growth capabilities has been developed. This instrument (nanoworkbench) is used to perform four-point probe conductivity measurements at micrometer spatial dimension. The system is composed of four chambers, the multiple-tip STM/SEM chamber, a surface analysis and preparation chamber, a molecular-beam epitaxy chamber and a load-lock chamber for fast transfer of samples and probes. The four chambers are interconnected by a unique transfer system based on a sample box with integrated heating and temperature-measuring capabilities. We demonstrate the operation and the performance of the nanoworkbench with STM imaging on graphite and with four-point-probe conductivity measurements on a silicon-on-insulator (SOI) crystal. The creation of a local FET, whose dimension and localization are respectively determined by the spacing between the probes and their position on the SOI surface, is demonstrated.Comment: 39 pages, 15 figure