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- DATA DEPENDENT ANALYSIS -

sHPP. - DATA DEPENDENT ANALYSIS -. Miguel Marcilla Goldaracena. Proteomics Unit. Centro Nacional de Biotecnología. CELL LINES AND FRACTIONATION TECHNIQUES. CCD18 (Fibroblast). RAMOS (Lymphoid). MCF7 (Epithelial). • SDS-PAGE • Basic rpHPLC. • SDS-PAGE.

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- DATA DEPENDENT ANALYSIS -

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  1. sHPP - DATA DEPENDENT ANALYSIS - Miguel Marcilla Goldaracena Proteomics Unit. Centro Nacional de Biotecnología

  2. CELL LINES AND FRACTIONATION TECHNIQUES CCD18 (Fibroblast) RAMOS (Lymphoid) MCF7 (Epithelial) • SDS-PAGE • Basic rpHPLC • SDS-PAGE Systematic Comparison of Fractionation Methods for In-depth Analysis of Plasma Proteomes Cao et al. Journal of Proteome Research. 2012. “High pH reverse-phase HPLC exhibited the highest peptide resolution and yielded the best depth of analysis with detection of the largest number of known lowabundance proteins (…)” Jurkat (Lymphoid) • SDS-PAGE • Basic rpHPLC

  3. SDS-PAGE Trypsin Digestion 15+1 slices MCF7 Protein Extract Basic rpHPLC LC-MS Analysis 5600 triple TOF (ABSciex) ProteinPrecipitation Trypsin Digestion 15 fractions

  4. SDS-PAGE vs BASIC rpHPLC (MCF7) 634 3260 2632 6526 unique proteins Basic rpHPLC (3894) SDS-PAGE (5892)

  5. SDS-PAGE vs BASIC rpHPLC (FDR <1%, peptidelevel) SDS-PAGE (15+1 slices) Basic rpHPLC (15 fractions)

  6. Proteins Identified inMCF7 cells Protein Coding Genes

  7. TARGET PROTEINS IDENTIFIED IN MCF7 CELLS “Known” proteins “Unknown” proteins

  8. SDS-PAGE vs BASIC rpHPLC (MCF7) SDS-PAGE RIPA Basic rpHPLC Jurkat SDS-PAGE SDS 4% Basic rpHPLC 5600 triple TOF (ABSciex) SDS-PAGE Urea CHAPS Basic rpHPLC CellLysis Fractionation LC-MS Analysis

  9. SDS-PAGE vs BASIC rpHPLC (Jurkat) 2733 6160 1166 Basic rpHPLC (8893) SDS-PAGE (7326) 10059 unique proteins

  10. Proteins Identified from Jurkat cells Protein Coding Genes

  11. TARGET PROTEINS IDENTIFIED IN MCF7 CELLS “Known” proteins “Unknown” proteins

  12. CONCLUSIONS • Data dependent LC-MS analysis allows the characterization of a significant number of proteins coded in chromosome 16 (More than 50% of “known” proteins are readily detected) • Peptide fractionation by reversed phase chromatography at basic pH is a valuable alternative to SDS-PAGE and allows the identification of a higher number of proteins

  13. Carmen GonzálezAdánAlpizar SeverineGharbiRosanaNavajas Alberto Paradela Salvador Martínez Alberto Medina

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