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

Core-shell nanoparticle arrays double the strength of steel

Manipulating structure, defects and composition of a material at the atomic scale for enhancing its physical or mechanical properties is referred to as nanostructuring. Here, by combining advanced microscopy techniques, we unveil how formation of highly regular nano-arrays of nanoparticles doubles the strength of an Fe-based alloy, doped with Ti, Mo, and V, from 500 MPa to 1 GPa, upon prolonged heat treatment. The nanoparticles form at moving heterophase interfaces during cooling from the high-temperature face-centered cubic austenite to the body-centered cubic ferrite phase. We observe MoC and TiC nanoparticles at early precipitation stages as well as core-shell nanoparticles with a Ti-C rich core and a Mo-V rich shell at later precipitation stages. The core-shell structure hampers particle coarsening, enhancing the material's strength. Designing such highly organized metallic core-shell nanoparticle arrays provides a new pathway for developing a wide range of stable nano-architectured engineering metallic alloys with drastically enhanced properties. ?The Author(s) 2017.1116Ysciescopu

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

Optical vortex trap for resonant confinement of metal nanoparticles

The confinement and controlled movement of metal nanoparticles and nanorods is an emergent area within optical micromanipulation. In this letter we experimentally realise a novel trapping geometry near the plasmon resonance using an annular light field possessing a helical phasefront that confines the nanoparticle to the vortex core (dark) region. We interpret our data with a theoretical framework based upon the Maxwell stress tensor formulation to elucidate the total forces upon nanometric particles near the particle plasmon resonance. Rotation of the particle due to orbital angular momentum transfer is observed. This geometry may have several advantages for advanced manipulation of metal nanoparticles

Measuring Temperature Gradients over Nanometer Length Scales

When a quantum dot is subjected to a thermal gradient, the temperature of electrons entering the dot can be determined from the dot's thermocurrent if the conductance spectrum and background temperature are known. We demonstrate this technique by measuring the temperature difference across a 15 nm quantum dot embedded in a nanowire. This technique can be used when the dot's energy states are separated by many kT and will enable future quantitative investigations of electron-phonon interaction, nonlinear thermoelectric effects, and the effciency of thermoelectric energy conversion in quantum dots.Comment: 6 pages, 5 figure

The Acidic Tail of the Cdc34 Ubiquitin-conjugating Enzyme Functions in Both Binding to and Catalysis with Ubiquitin Ligase SCFC^(dc4*)

Ubiquitin ligases, together with their cognate ubiquitin-conjugating enzymes, are responsible for the ubiquitylation of proteins, a process that regulates a myriad of eukaryotic cellular functions. The first cullin-RING ligase discovered, yeast SCF^(Cdc4), functions with the conjugating enzyme Cdc34 to regulate the cell cycle. Cdc34 orthologs are notable for their highly acidic C-terminal extension. Here we confirm that the Cdc34 acidic C-terminal tail has a role in Cdc34 binding to SCF^(Cdc4) and makes a major contribution to the submicromolar K_m of Cdc34 for SCF^(Cdc4). Moreover, we demonstrate that a key functional property of the tail is its acidity. Our analysis also uncovers an unexpected new function for the acidic tail in promoting catalysis. We demonstrate that SCF is functional when Cdc34 is fused to the C terminus of Cul1 and that this fusion retains partial function even when the acidic tail has been deleted. The Cdc34-SCF fusion proteins that lack the acidic tail must interact in a fundamentally different manner than unfused SCF and wild type Cdc34, demonstrating that distinct mechanisms of E2 recruitment to E3, as is seen in nature, can sustain substrate ubiquitylation. Finally, a search of the yeast proteome uncovered scores of proteins containing highly acidic stretches of amino acids, hinting that electrostatic interactions may be a common mechanism for facilitating protein assembly

New insights into electron spin dynamics in the presence of correlated noise

The changes of the spin depolarization length in zinc-blende semiconductors when an external component of correlated noise is added to a static driving electric field are analyzed for different values of field strength, noise amplitude and correlation time. Electron dynamics is simulated by a Monte Carlo procedure which keeps into account all the possible scattering phenomena of the hot electrons in the medium and includes the evolution of spin polarization. Spin depolarization is studied by examinating the decay of the initial spin polarization of the conduction electrons through the D'yakonov-Perel process, the only relevant relaxation mechanism in III-V crystals. Our results show that, for electric field amplitude lower than the Gunn field, the dephasing length shortens with the increasing of the noise intensity. Moreover, a nonmonotonic behavior of spin depolarization length with the noise correlation time is found, characterized by a maximum variation for values of noise correlation time comparable with the dephasing time. Instead, in high field conditions, we find that, critically depending on the noise correlation time, external fluctuations can positively affect the relaxation length. The influence of the inclusion of the electron-electron scattering mechanism is also shown and discussed.Comment: Published on "Journal of Physics: Condensed Matter" as "Fast Track Communications", 11 pages, 9 figure

Study of Thermal Properties of Graphene-Based Structures Using the Force Constant Method

The thermal properties of graphene-based materials are theoretically investigated. The fourth-nearest neighbor force constant method for phonon properties is used in conjunction with both the Landauer ballistic and the non-equilibrium Green's function techniques for transport. Ballistic phonon transport is investigated for different structures including graphene, graphene antidot lattices, and graphene nanoribbons. We demonstrate that this particular methodology is suitable for robust and efficient investigation of phonon transport in graphene-based devices. This methodology is especially useful for investigations of thermoelectric and heat transport applications.Comment: 23 pages, 9 figures, 1 tabl

Analysis of and workarounds for element reversal for a finite element-based algorithm for warping triangular and tetrahedral meshes

We consider an algorithm called FEMWARP for warping triangular and tetrahedral finite element meshes that computes the warping using the finite element method itself. The algorithm takes as input a two- or three-dimensional domain defined by a boundary mesh (segments in one dimension or triangles in two dimensions) that has a volume mesh (triangles in two dimensions or tetrahedra in three dimensions) in its interior. It also takes as input a prescribed movement of the boundary mesh. It computes as output updated positions of the vertices of the volume mesh. The first step of the algorithm is to determine from the initial mesh a set of local weights for each interior vertex that describes each interior vertex in terms of the positions of its neighbors. These weights are computed using a finite element stiffness matrix. After a boundary transformation is applied, a linear system of equations based upon the weights is solved to determine the final positions of the interior vertices. The FEMWARP algorithm has been considered in the previous literature (e.g., in a 2001 paper by Baker). FEMWARP has been succesful in computing deformed meshes for certain applications. However, sometimes FEMWARP reverses elements; this is our main concern in this paper. We analyze the causes for this undesirable behavior and propose several techniques to make the method more robust against reversals. The most successful of the proposed methods includes combining FEMWARP with an optimization-based untangler.Comment: Revision of earlier version of paper. Submitted for publication in BIT Numerical Mathematics on 27 April 2010. Accepted for publication on 7 September 2010. Published online on 9 October 2010. The final publication is available at http://www.springerlink.co