Course Program a.a 2015-2016
A review of basic concepts
1) Acid-base properties of amino acids;
2) The peptide bond; the alpha helix;
3) Primary to quaternary structures on proteins.
1) Some definitions;
2) The proteome complexity;
3) The tools of the trade.
Strategies in chromatographic protein purification;
1. Types of chromatographic beads;
2. Band broadening and peak shape;
3. Resolution and how to modulate it;
4. The Van Deemter equation.
1. Ionizing groups in proteins;
2. Electrophoretic titration curves;
3. Buffers for cation & anion exchangers;
4. Volatile buffers.
1. Various types of resins;
2. Distribution coefficients (Kd & Kav);
3. Measurements of Mr of proteins;
4. Desalting and buffer exchange;
5. Final polishing step.
Reversed-Phase, High Performance Liquid Chromatography (RP-HPLC)
1. Mechanism of protein/peptide adsorption;
2. Types of stationary phases;
3. Effects of pH, TFA & temperature on elution patterns;
4. Two-dimensional techniques.
Hydrophobic interaction chromatography (HIC)
1. Types of stationary phases;
2. Mechanism of protein adsorption;
3. Effects of pH, types of salts and their gradients on elution patterns;
Affinity chromatography (I)
1. Lectin Affinity chromatography;
2. Immuno Affinity Chromatography;
3. Thiol Disulphide Covalent Chromatography (TDCC);
4. Dye-Ligand Affinity Chromatography (DLAC);
5. Immobilized metal-ion affinity chromatograph (IMAC).
Affinity chromatography (II)
1. Cell Affinity chromatography;
2. Protein A Affinity Chromatography;
3. Hydroxy apatite chromatography.
1. Mechanism of Chromatofocusing;
2. Types of resins;
3. Types of buffers;
4. Examples of protein separations.
1. An historical overview;
2. From moving boundary to zone electrophoresis;
3. Discontinuous techniques;
4. Focusing techniques;
5. Two-dimensional techniques
6. Capillary zone electrophoresis.
1. Ferguson plots;
2. Mr measurements;
3. Polyacrylamide gel structure;
4. Pore size and geometries.
SDS PAGE (Sodium dodecyl sulphate polyacrylamide gel electrophoresis)
1. Mode of SDS binding and exceptions;
2. Mr measurements;
3. Porosity gradient gels;
4. Blotting techniques;
5. Immuno-detection after blotting.
Isoelectric focusing (IEF) – Immobilized pH gradients (IPG)
1. The carrier ampholyte buffers;
2. The transient state;
3. The steady state;
5. The Immobiline chemicals;
6. Modelling of ultra-narrow and wide IPG gradients.
1. Kohlrausch regulating function;
2. Zone sharpening;
3. Concentration effects;
4. Analytical instrument: the Tachophor;
5. Preparative instrument: the Tacofrac.
Capillary zone electrophoresis
1. The electroendoosmotic flow;
2. Micellar Electrokinetic Chromatography;
3. Peptide separations;
4. Protein Separations;
5. SDS in sieving liquid polymers;
6. Capillary isoelectric focusing.
Sample preparation (I)
1. Protein solubilization;
2. Sample clean-up;
3. Cell disruption/lysis;
4. Sample fractionation.
Sample preparation (II)
1. Reduction and alkylation of proteins;
2. Alkylation with iodoacetamide derivatives;
3. Alkylation with unsaturated compounds;
4. Artefacts due to improper sample handling.
Sample preparation (III). Expected and Unexpected Artefacts
3. Beta elimination of cysteine.
Prefractionation via SEC, Cation and Anion exchangers, Reversed phase, HIC, HILIC resins.
Evaluation of the results.
Mass spectrometry in proteome analysis
MALDI-TOF; Ion traps; Orbitrap, Velos, triple-quadrupole
Analysis of post-translational modifications (PTM) of proteins.
Detection of low-abundance proteins in proteomes via combinatorial peptide ligand libraries (CPLL)
1. Chemistry of CPLLs;
2. examples in food and beverage analysis;
3. Biomarker discovery in biological fluids;
4. Analysis of the secretome in tissue culture of cancer cells.
The lab course will consist mainly on the following experiments:
1) SDS-PAGE (sodium dodecyl sulphate, polyacrylamide gel electrophoresis single dimension) followed by Coomassie Blue staining;
2) Isoelectric focusing in conventional carrier ampholyte buffers;
3) Two-dimensional maps of proteins in mini-gels, followed by staining with Coomassie Blue;
4) Data acquisition and map analysis via the PDQuest software.
5) Digestive protocol for mass spectrometry analysis
6) Protein identification via nanoLC-MS/MS analysis and proteomic data bases search.