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WG1. 2014 workplan

WG1. 2014 workplan. Expression of 60 missing proteins. 1.1. Adquisition of clones from the ASU collection 1.2. Subcloning in the pANT-cGST vector if necessary 1.3. Plasmid isolation: Bacterial growing and miniprep plasmid DNA isolation

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WG1. 2014 workplan

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  1. WG1. 2014 workplan • Expression of 60 missing proteins. • 1.1. Adquisition of clones from the ASU collection • 1.2. Subcloning in the pANT-cGST vector if necessary • 1.3. Plasmid isolation: Bacterial growing and miniprep plasmid DNA isolation • 1.4. Clone validation: DNA sequencing with modified T7 primers • 1.5. Protein expression: IVTT expression and GST enrichment • 1.6. MRM method development and assay: MRM peptide/transitions selection, sample testing and MRM method refine • Shotgun analysis of expressed proteins: Characterization of missing proteins expressed through shotgun proteomics • 3. MRM of expressed proteins in biological samples • 3.1. Bioinformatics selection of the appropriate biological matrix • 3.2. Biological sample preparation and assay

  2. WG1. 2014 workplan • 4. DB analysis: actualization and bioinformatic data integration to refine the available information of the Chr16 missing proteins • 4.1. ENCODE crossreference: actualization of the transcription data of missing proteins available in ENCODE experiments (Corrales) • 4.2. NeXtProt update: checking of the list of missing proteins. Actualization and bioinformatic data recovery (Pino) • 4.3. ProteomicsDB crossreference: check of the available proteomics information for missing proteins • 4.4. Non coding genes update: actualization of the missing protein lists with the non coding genes information (J. Vázquez) • 4.5. Peptide Atlas: checking for missing protein peptide spectra • Protein expression for top down proteomics: Selection of proteins for top down proteomics, scale up expression reaction and protein purification. • 5.1. Protein expression • 5.2 Top down proteomics • Synthetic peptide standarization: Verification of MRM data obtained in the sp-HPP WG2 trhough the comparison with the spectra and retention time of synthetic peptides • 6.1. Query to the MRM DB to select the peptides for synthesis • 6.2. Peptide synthesis (heavy or light?): select either light or heavy peptide for either verification or verification and quantitation respectively • 6.3. MRM data verification: comparision of the spectral data and retention time

  3. WG1. 2014 workplan

  4. WG1. 2014 workplan

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