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Proteomics Rates August 1st 2008

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Submitter: brettsp	Released: Mon, 04-Aug-2008

Download Now Rates August 1st 2008	Version: 0 Downloads93 File Size: 136.92 KB Platform: None Home Page: Not Specified Rating: (0 Votes)

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Label-Free Relative Quantitation of Developmentally Expressed Soluble Proteins in the Ripening Fruit

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Submitter: brettsp	Released: Thu, 12-Jun-2008

Download Now The pathways involved in the processes of fruit development and ripening are unique to plants and vary between species. Developmental, physiological, anatomical, biochemical and structural differences contribute to the operation of unique pathways, genes and proteins. Many of these have been characterized in tomato, a climacteric-ripening fruit; however non-climacteric ripening such as that in citrus is poorly understood. Improved understanding of non-climacteric fruit maturation may yield benefits both for public health and agricultural economics.This study aims to identify and quantify developmentally regulated proteins in two stages of "Washington naval" oranges (Citrus sinensis) fruit development; at early stage II, a still-green, smaller, acidic orange; and at stage III, a larger, sweeter, juicier, less acidic orange. Accurate LC-MS/MS data were acquired with a Finnigan LTQ-FT for the differentially expressed proteins in the two developmental stages, then label-free techniques were employed to quantify differences between proteins of the two subsets: Both label-free spectral counting and comparison of peak intensities using the Sieve program. Statistical analyses were conducted with Spotfire software. A citrus genome-wide ESTs database and the NCBI-nr (green plants) database were used to identify proteins, which were subsequently classified according to their putative and assigned functions to known biosynthetic pathways. A number of proteins were found to differ between the two stages of orange, suggesting significant shifts in some pathways; these may suggest mechanisms for the metabolic changes affecting the quality of the fruit and resistance to disease. These studies may also reflect some “synergism” between different pathways during maturation of the fruit	Version: 0 Downloads147 File Size: 0 bytes Platform: None Home Page: Not Specified Rating: (0 Votes)

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Quantitation at the Proteomics Core Facility

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Submitter: brettsp	Released: Thu, 12-Jun-2008

Download Now Quantitation Services MRM Based Quantitation Using our new Triple Quadrupole Mass Spectrometer we can target peptides for quantitation across multiple samples using a technique called Multiple Reaction Monitoring (MRM). This can be done with or without he use of isotopically labeled peptides (AQUA). CV’s are typically between 8-20%. The peptide must be known or sequence hypothesized before the experiment Label Free Quantitation One of the most accurate and sensitive ways to determine protein expression differences. This experiment can be accomplished “blind” without prior knowledge of what to expect. You can also multiple conditions (A vs. B vs. C) Stable Isotope Labeling in Cell Culture Can be used similar to the Label free approach to do expression profiling. Generally considered to be the best way to do quantitation and expression profiling	Version: 0 Downloads93 File Size: 0 bytes Platform: None Home Page: Not Specified Rating: (0 Votes)

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Proteomics Core Facility

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Submitter: brettsp	Released: Thu, 12-Jun-2008

Download Now General info on the facility	Version: 0 Downloads99 File Size: 0 bytes Platform: None Home Page: Not Specified Rating: (0 Votes)

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Novel Method for High-Throughput Cross-Linking Site Mapping

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Submitter: brettsp	Released: Thu, 12-Jun-2008

Download Now Overview We developed a novel tool for cross-linking analysis directly analyzing LC-MS/MS data set: Shotgun cross-linking analysis. Application to cytochrome c identified a total of 31 cross-links, 21 inter- and 10 intra-peptide cross-links in 257 MS/MS spectra; 100% Lys coverage as either of the cross-links; 31% Lys pair coverage with β carbons within 20Å. Application to Dnm1G385D dimer band resulted in 46 cross-links, 38 inter- and 8 intra-peptide cross-links, in 99 MS/MS spectra, the most cross-links identified so far in a single data set. Our program, X!Link, has very low false discovery rate of 1%.	Version: 0 Downloads86 File Size: 0 bytes Platform: None Home Page: Not Specified Rating: (0 Votes)

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Cross-linking Site Mapping of Sindbis Virus Structural Proteins

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Submitter: brettsp	Released: Thu, 12-Jun-2008

Download Now Overview High-throughput approach (nanoLC-MS/MS and “X-link” search) was used for the cross-linking site mapping of Sindbis virus structural proteins. Computer program (“X-link”) was developed to find matching sequences from thousands of MS/MS spectra. Identified E1/E2, E1/E1, and E2/E2 cross-linked peptides using BS3 and DSS cross-linkers. Also found four intra-peptide links. Introduction Sindbis virus (SV) is an alphavirus, which is propagated in nature via a complicated life cycle involving insect vectors and mammalian hosts. The outer protein shell is composed of the E1 and E2 glycoproteins organized in trimers of heterodimers. However, it is not well known how these structural proteins interact with each other to form outer shell. In this study, we use cross linking agents to covalently stabilize the interacting parts of the proteins and attempt to identify the cross linking sites. This study allows us to locate areas where these proteins interact with each other or with another copy of themselves and allow us to gain both structural and possibly functional information about the virus.	Version: 0 Downloads86 File Size: 0 bytes Platform: None Home Page: Not Specified Rating: (0 Votes)

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Toward Diagnostic Proteomics using Mudpit Technology:

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Submitter: brettsp	Released: Thu, 12-Jun-2008

Download Now Overview • A preliminary effort has been devoted to develop a diagnostic proteomics tool using Mudpit (or Multi-dimensional LC-MS/MS) technique. • Novel SIM LC-MS/MS method has been employed to monitor PSA level in serum, which lowered the detection limit from ~30ng in 30μg serum (48μg/ml) to <3ng in 30ug serum (5μg/ml) in the whole serum sample. • Removal	Version: 0 Downloads79 File Size: 0 bytes Platform: None Home Page: Not Specified Rating: (0 Votes)

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Mass Spectrometric Characterization of Phosphorylation Sites

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Submitter: brettsp	Released: Thu, 12-Jun-2008

Download Now Overview RsbV1 and RsbV2 in Chlamydia trachomatis (CT), the presumed substrates of Ct RsbW kinase, were overexpressed, purified, and specifically phosphorylated in vitro by Ct RsbW, a putative serine protein kinase. Preliminary identification of phosphorylated peptides were carried out by MALDI-TOF mass spectrometry analysis of tryptic peptides of RsbV1 and RsbV2 before and after alkaline phosphatase treatment. Almost full sequence coverage and exact phosphorylation sites were obtained by cLC-MS/MS approach. Phosphorylation sites of RsbV1 and RsbV2 were identified as Ser 56 and 55 respectively, which are aligned perfectly with phosphorylation site of RsbV and SpoIIAA from Bacillus subtilis suggesting a partner switching mechanism as a signaling pathway for intracellular development of the parasite.	Version: 0 Downloads94 File Size: 0 bytes Platform: None Home Page: Not Specified Rating: (0 Votes)

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Label-Free Relative Quantitation of Developmentally Expressed Soluble and Membrane Proteins in the R

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Submitter: brettsp	Released: Thu, 12-Jun-2008

Download Now The pathways involved in the processes of fruit development and ripening are unique to plants and vary between species. Developmental, physiological, anatomical, biochemical and structural differences contribute to the operation of unique pathways, genes and proteins. Many of these have been characterized in tomato, a climacteric-ripening fruit; however non-climacteric ripening such as that in citrus is poorly understood. Improved understanding of non-climacteric fruit maturation may yield benefits both for public health and agricultural economics.This study aims to identify and quantify developmentally regulated proteins in two stages of "Washington naval" oranges (Citrus sinensis) fruit development; at early stage II, a still-green, smaller, acidic orange; and at stage III, a larger, sweeter, juicier, less acidic orange. Accurate LC-MS/MS data were acquired with a Finnigan LTQ-FT or LTQ for the differentially expressed proteins in the two developmental stages, then label-free techniques were employed to quantify differences between proteins of the two subsets: Both label-free spectral counting and comparison of peak intensities using the Sieve program. Statistical analyses were conducted with Spotfire software. A citrus genome-wide ESTs database and the NCBI-nr (green plants) database were used to identify proteins, which were subsequently classified according to their putative and assigned functions to known biosynthetic pathways. A number of proteins were found to differ between the two stages of orange, suggesting significant shifts in some pathways; these may suggest mechanisms for the metabolic changes affecting the quality of the fruit and resistance to disease. These studies may also reflect some “synergism” between different pathways during maturation of the fruit.	Version: 0 Downloads159 File Size: 0 bytes Platform: None Home Page: Not Specified Rating: (0 Votes)

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ABRF 2008 Facility Poster

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Submitter: brettsp	Released: Thu, 12-Jun-2008

Download Now acid analysis, Edman sequencing and tandem mass spectrometry techniques including protein identification, post-translational modification discovery, label free quantitation and protein cross-linking analysis. The facility utilizes state of the art instrumentation, including a Thermo LTQ-FT Ultra. Our group utilizes open source software and uses techniques and protocols that are made publicly available. Using open source software allows users to analyze their own data without significant monetary investment. We also offer data analysis and sample preparation classes to UC Davis and the community at large, including a week long hands on summer short course. Our group operates as an open core facility withithe Genome Center on the UC Davis campus	Version: 0 Downloads87 File Size: 0 bytes Platform: None Home Page: Not Specified Rating: (0 Votes)

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