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.