Comprehensive Workplan for Expression and Analysis of Missing Proteins in Chromosome 16
This workplan outlines the strategies to express and analyze 60 missing proteins from Chromosome 16. It includes acquiring clones from the ASU collection, necessary subcloning into the pANT-cGST vector, and plasmid isolation through bacterial growth and miniprep. The plan emphasizes validation through DNA sequencing, protein expression using IVTT and GST enrichment, and MRM method development for targeted assays. Additionally, bioinformatics integration and updates from ENCODE, NeXtProt, ProteomicsDB, and other databases will refine the understanding of these proteins.
Comprehensive Workplan for Expression and Analysis of Missing Proteins in Chromosome 16
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Presentation Transcript
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
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
