蛋白质组学技术的详细讲座(非常详细).ppt

Quantitative Proteomics: Applications and Strategies October 2013 A little history… 1985 – First use: up to a 3 kDa peptide could be ionized 1987 – Method to ionize intact proteins (up to 34 kDa) described Instruments have no sequence capability 1989 – ESI is used for biomolecules (peptides) Sequence capability, but low sensitivity 1994 – Term «Proteome» is coined 1995 – LC-MS/MS is implemented «Gold standard» of proteomic analysis 2DE-based approach 2DE-based approach “I see 1000 spots, but identify 50 only.” Fenn et al., Science 246:64-71, 1989. LC-MS MS-based quantitation Inlet Ion Source Mass Analyzer Detector MALDI ES Time-of-Flight Quadrupole Ion Trap Quadrupole-TOF LC Peak intensities can vary up to 100x between duplicate runs. Quatitative analysis MUST be carried on a single run. Ion Intensity = Ion abundance MS measure m/z m/z Intensity Sample 2 Sample 1 Isotopic Labeling Unlabeled peptide: Labeled peptide: a) Enzymatic Labeling Metabolic Labeling SILAC X 3 X 3 Cells in normal culture media Ong SE et al., 2002 Chemical Labeling Gygi SP et al., 1999 ICAT (Isotope-Coded Affinity Tag) ICAT Thiol-specific group = binds to Cysteins ICAT Thiol-specific group = binds to Cysteins Quantitation at MS1 level m/z Intensity Double sample complexity, i.e. instrument have more “features”to identify, i.e. decrease in identification rate iTRAQ (isobaric Tag for Relative and Absolute Quantitation) Sample prep Total mass of label= 145 Da ALWAYS RecognizesArg or Lys iTRAQ iTRAQ Multiplexing Metabolic VS Chemical Labeling Summary Kolkman A et al., 2005 Label-free Mobile phase A A = 5% organic solvent in water B = 95% organic solvent in water B C18 column, 25cm long 20 s Label-free Strassberger V et al., 2010 Summary Summary Take home message Quantitation can be done gel-free Labeling can be performed at protein or peptide level,during normal cell growth or in vitro Quantitation can be achieved at MS1 or MS2 level Method choice depends on experimental design,