蛋白质组学技术的详细讲座(非常详细).pptx

Quantitative Proteomics: Applications and Strategies ;A little history…;;;;MS-based quantitation;Ion Intensity = Ion abundance;MS measure m/z;;Enzymatic Labeling;Metabolic Labeling;SILAC;Chemical Labeling;ICAT (Isotope-Coded Affinity Tag);ICAT;ICAT;Quantitation at MS1 level;iTRAQ (isobaric Tag for Relative and Absolute Quantitation);iTRAQ;iTRAQ;Metabolic VS Chemical Labeling;Summary;Label-free;Label-free;Summary;Summary;Take home message;Applications;Applications – Cell Biology;Applications – Cell Biology;Applications – Immunology;Clinical Proteomics;Interactomics;Signaling Pathways;Take home message;SILAC;SILAC;Importance of Dialyzed Serum;No alterations to cell phenotype;Why SILAC is convenient?;Why SILAC is convenient?;Catch 22;Proline Conversion!;Typical SILAC experiment workflow;Additional validation criteria;Triple SILAC;;Quantitative phosphoproteomics in EGFR signaling;MAP kinases activation;Spatial distribution of phosphorylation dynamics;Interactomics;Limitations;SILAC-labeled organism;Super-SILAC;Spike-In SILAC;Take home message;Label-free;Label-free;Label-free;Label-free;Label-free;Label-free;Label-free;Spectral Count;Spectral Count;;Area Under Curve measurement;Area Under Curve measurement;Importance of Resolution for label-free;Cox and Mann, Nature Biotechnol 26, 2008.;1. Retention time 2. Peak intensity 3. Monoisotopic mass accuracy;Regarding Label-free…;Data without Normalization;How this was demonstrated?;How this was demonstrated?;MaxQuant and Yeast;Label-free in primary cells;;Infection with Sendai virus(activate RIG-I PRR);Take home message;SRM / MRM;A little history…;So far, ID everything we can;Targeted analysis;Plasma dynamic range;Improving detection through tergeting;Biomarker;Discovery phase;Biomarker;Shorter gradient = More complex MS1;SRM (Selected Reaction Monitoring);Different transitions from same peptide;Performance with synthetic peptides;Shorter gradient = More complex MS1;Number of biomarkers discovered so far by MS;Spiking sinthet