1 / 13

- 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.

shana-berry
Download Presentation

- DATA DEPENDENT ANALYSIS -

An Image/Link below is provided (as is) to download presentation Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author. Content is provided to you AS IS for your information and personal use only. Download presentation by click this link. While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server. During download, if you can't get a presentation, the file might be deleted by the publisher.

E N D

Presentation Transcript

  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

More Related