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Annual Rhizomics Group Meeting

Annual Rhizomics Group Meeting. -current state on proteome analysis-. October-2010. Poales. Starting Point- Monocot Species. - Phylogenetically close. - Agronomic importance: invasive species. BCA assay. 12% SDS-PAGE 100ug proteins 20mA each gel. trypsin concentration: 06ng/uL.

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Annual Rhizomics Group Meeting

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  1. Annual Rhizomics Group Meeting -current state on proteome analysis- October-2010

  2. Poales Starting Point- Monocot Species - Phylogenetically close - Agronomic importance: invasive species

  3. BCA assay 12% SDS-PAGE 100ug proteins 20mA each gel trypsin concentration: 06ng/uL 30 linear ranges nrNCBI+translatedUniGene sequences for Oryza sativa Proposed Workflow for Label-Free Quantitative Proteomics protein quantitation pre-fractionation protein extraction gel slicing Phenol extraction followed by resuspension in 50mM Tris+2%SDS in gel digestion LC-MS/MS (LTQ) range:200-2000 m/z Top 7 database search (Sequest) spectra count (Scaffold) until database is available Filter: 1 peptide per protein 99-95% protein-peptide probability XCorr : 1.5-2-2-3.5-3.0

  4. “Temporary database” - Oryza sativa nrNCBI + translated UniGene random sequences (to estimate FDR) 175,586 entries (currently)

  5. BCA assay 12% SDS-PAGE 100ug proteins 20mA each gel trypsin concentration: 06ng/uL 30 linear ranges nrNCBI+translatedUniGene sequences for Oryza sativa Proposed Workflow for Label-Free Quantitative Proteomics protein quantitation pre-fractionation protein extraction gel slicing Phenol extraction buffer followed by resuspension in 50mM Tris+2%SDS in gel digestion LC-MS/MS (LTQ) range:200-2000 m/z Top 7 database search (Sequest) spectra count (Scaffold) until database is available Filters to remove false hits Combine results from all ranges Current version: massive uploading capacity

  6. Available: mass spectrometry data protein extract Protein Extracts and Mass Spectrometry Data

  7. Comparative proteome analysis of Acorus calamus underground tissues Number of detected proteins False discovery rate: 2.2%

  8. Comparative proteome analysis of Acorus calamus underground tissues Changes in expression: pair wise t-test 517 non-redundant proteins Elongation Zone vs Roots Apical Meristem vs Roots Apical Meristem vs Elongation Zone apical meristem 189 elongation zone 180 apical meristem 76 elongation zone 69 roots 40 roots 19 229 proteins significantly changed 180 proteins significantly changed 145 proteins significantly changed spectra count difference = #counts tissue A - # counts tissue B

  9. Medium-term goal 1) Mass spectrometry data proteins involved in invasive behavior 2) Comparative analysis with non-invasive species Calamus (mass spec data already available) Aloe Ginger Turmeric

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