Development of an X-band Photoinjector at SLAC

As part of a National Cancer Institute contract to develop a compact source of monoenergetic X-rays via Compton backscattering, we have completed the design and construction of a 5.5 cell Photoinjector operating at 11.424 GHz. Successful completion of this project will result in the capability of generating a monoenergetic X-ray beam, continuously tunable from 20 - 85 KeV. The immediate goal is the development of a Photoinjector producing 7 MeV, 0.5 nC, sub-picosecond electron bunches with normalized RMS emittances of approximately 1 pi-mm-mR at repetition rates up to 60 Hz. This beam will then be further accelerated to 60 MeV using a 1.05 m accelerating structure. This Photoinjector is somewhat different than the traditional 1.5 cell design both because of the number of cells and the symmetrically fed input coupler cell. Its operating frequency is also unique. Since the cathode is non-removable, cold-test tuning was somewhat more difficult than in other designs. We will present results of "bead-drop" measurements used in tuning this structure. Initial beam measurements are currently in progress and results will be presented as well as results of RF conditioning to high gradients at X-band. Details of the RF system, emittance-compensating solenoid, and cathode laser system as well as PARMELA simulations will also be presented.Comment: 3 pages, 6 figures, 1 Table, LINAC 200

Identification of protein complexes with quantitative proteomics in S. cerevisiae

Lipids are the building blocks of cellular membranes that function as barriers and in compartmentalization of cellular processes, and recently, as important intracellular signalling molecules. However, unlike proteins, lipids are small hydrophobic molecules that traffic primarily by poorly described nonvesicular routes, which are hypothesized to occur at membrane contact sites (MCSs). MCSs are regions where the endoplasmic reticulum (ER) makes direct physical contact with a partnering organelle, e.g., plasma membrane (PM). The ER portion of ER-PM MCSs is enriched in lipid-synthesizing enzymes, suggesting that lipid synthesis is directed to these sites and implying that MCSs are important for lipid traffic. Yeast is an ideal model to study ER-PM MCSs because of their abundance, with over 1000 contacts per cell, and their conserved nature in all eukaryotes. Uncovering the proteins that constitute MCSs is critical to understanding how lipids traffic is accomplished in cells, and how they act as signaling molecules. We have found that an ER called Scs2p localize to ER-PM MCSs and is important for their formation. We are focused on uncovering the molecular partners of Scs2p. Identification of protein complexes traditionally relies on first resolving purified protein samples by gel electrophoresis, followed by in-gel digestion of protein bands and analysis of peptides by mass spectrometry. This often limits the study to a small subset of proteins. Also, protein complexes are exposed to denaturing or non-physiological conditions during the procedure. To circumvent these problems, we have implemented a large-scale quantitative proteomics technique to extract unbiased and quantified data. We use stable isotope labeling with amino acids in cell culture (SILAC) to incorporate staple isotope nuclei in proteins in an untagged control strain. Equal volumes of tagged culture and untagged, SILAC-labeled culture are mixed together and lysed by grinding in liquid nitrogen. We then carry out an affinity purification procedure to pull down protein complexes. Finally, we precipitate the protein sample, which is ready for analysis by high-performance liquid chromatography/ tandem mass spectrometry. Most importantly, proteins in the control strain are labeled by the heavy isotope and will produce a mass/ charge shift that can be quantified against the unlabeled proteins in the bait strain. Therefore, contaminants, or unspecific binding can be easily eliminated. By using this approach, we have identified several novel proteins that localize to ER-PM MCSs. Here we present a detailed description of our approach