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

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Presentation on theme: "- DATA DEPENDENT ANALYSIS - Proteomics Unit. Centro Nacional de Biotecnología sHPP Miguel Marcilla Goldaracena."— Presentation transcript:

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

2 MCF7 (Epithelial) MCF7 (Epithelial) RAMOS (Lymphoid) RAMOS (Lymphoid) CCD18 (Fibroblast) CCD18 (Fibroblast) CELL LINES AND FRACTIONATION TECHNIQUES SDS-PAGE Basic rpHPLC SDS-PAGE Jurkat (Lymphoid) Jurkat (Lymphoid) SDS-PAGE Basic rpHPLC 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 (…) 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 (…)

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

4 rpHPLCSDS-PAGE FDR (Peptide level) 1 % # PSM1358235186 # Peptides1237925772 # Proteins38945892 Mean Coverage (peptides/protein) 3.24.4 63426323260 Basic rpHPLC (3894) SDS-PAGE (5892) SDS-PAGE vs BASIC rpHPLC (MCF7) 6526 unique proteins

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

6 Proteins Identified in MCF7 cells Protein Coding Genes

7 TARGET PROTEINS IDENTIFIED IN MCF7 CELLS Known proteinsUnknown proteins

8 SDS-PAGE vs BASIC rpHPLC (MCF7) Jurkat RIPA SDS 4% Urea CHAPS SDS-PAGE Basic rpHPLC SDS-PAGE Basic rpHPLC SDS-PAGE Basic rpHPLC Cell LysisFractionationLC-MS Analysis 5600 triple TOF (ABSciex)

9 Basic rpHPLC (8893) rpHPLCSDS-PAGE FDR (Peptide level) 1 % # PSM6224858100 # Peptides5451341382 # Proteins88937326 Mean Coverage (peptides/protein) 6.05.6 116627336160 SDS-PAGE (7326) SDS-PAGE vs BASIC rpHPLC (Jurkat) 10059 unique proteins

10 Proteins Identified from Jurkat cells Protein Coding Genes

11 TARGET PROTEINS IDENTIFIED IN MCF7 CELLS Known proteinsUnknown 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 GonzálezAdán Alpizar Severine GharbiRosana Navajas Alberto Paradela Carmen GonzálezAdán Alpizar Severine GharbiRosana Navajas Alberto Paradela Salvador Martínez Alberto Medina Salvador Martínez Alberto Medina

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