Proteomic Biology Using LC/MS - Large Scale Analysis of Cellular Dynamics and Function

Introduction. Chapter 1. Overview of Proteomics. What is the proteomics? 1-2 Proteomic analysis using two-dimensional gel electrophoresis (2DE) and mass spectrometry . 1-2-1 Protein separations by 2DE. 1-2-2 Development of the technologies for protein identification . 1-2-3 The protein identification based on gel separation and mass spectrometry. 1-3 Strategies for characterizing an entire proteome and understanding the proteome function. 1-3-1 Modification-specific proteomics. 1-3-2 Activity-based profiling. 1-3-3 Sub-cellular (Organelle) proteomics. 1-3-4 Machinery [complex (interaction)] proteomics. 1-3-5 Dynamic proteomics. Chapter 2. Proteomic Tools for Analysis of Cellular Dynamics. 2-1 LC-BASED PROTEOMICS TECHNOLOGIES. 2-1-1 LC system for peptide separation . LC-MS interface. ESI apparatus . Micro- and nano-capillary columns. Packing materials. 1D-LC system. Application of 1D-LC-MS/MS to shotgun analysis of moderately complex protein mixtures. Experimental example 2-1. 2D LC-MS system. Experimental example 2-2. 2-1-2 Application of LC-MS methods to functional proteomics. Sub-cellular (organelle) using a cell-surface modification reagent and cell fractionation. Experimental example 2-3 . Modification proteomics. Phosphorylation site mapping. Experimental example 2-4. Glycosylation site mapping. Experimental example 2-5. Ubiquitinated protein identification by using ubiquitin-specific antibody . Experimental example 2-6. 2-2 Development of quantitative proteomics. 2-2-1 Isotope labeling for quantitative analysis using MS. in vivo labeling. in vitro labeling. 2-2-2 Quantitation strategies for LC-MS analysis of isotope labeled peptide mixture and software for computer analysis. 2-2-3 Label free quantitation software. 2-2-4 Absolute quantitation. Method using stable isotope labeled reference peptides. Method without using internal standards. Chapter 3. Dynamics of Functional Cellular Machinery: From Statics to Dynamics in Proteomic Biology. From statics to dynamics in proteomic biology. 3-1 DYNAMIC ANALYSIS OF CELLULAR FUNCTION. 3-1-1 Strategy for dynamic analysis of cellular machineries (multi-protein complexes). Approach collecting time dependent data. Approach utilizing stage specific protein association. 3-1-2 Methods for the isolation of a cellular machinery/multi-protein complex. Affinity-tag purification. Affinity chromatography with an antibody- or a protein-immobilized column. Immuno-affinity purification using antibody-fixed beads. Experimental example 3-1. Pull-down purification using immobilized protein beads. Experimental example 3-2. 3-1-3 Cellular machinery (multi-protein complex). 3-2 Dynamics of Ribosome Biogenesis . 3-2-1 Snapshot Analyses of Preribosomal Particles in Yeast . 3-2-2 Snapshot Analyses of Preribosomal Particles in Mammals. Experimental example 3-3. 3-2-3 Quantitative (dynamic) analysis using isotope labeled reagents. Experimental example 3-4. 3-2-4 Orthogonal Comparison of the Process of Ribosome Biogenesis. 3-3 Dynamic analyses of Sub-cellular structures. 3-3-1 Proteome dynamics of the nucleolus.