Risorse bibliografiche
Risorsa bibliografica obbligatoria
Risorsa bibliografica facoltativa
Scheda Riassuntiva
Anno Accademico 2015/2016
Scuola Scuola di Ingegneria Industriale e dell'Informazione
Insegnamento 097594 - PROTEOMICS LABORATORY
Docente Fasoli Elisa
Cfu 5.00 Tipo insegnamento Monodisciplinare

Corso di Studi Codice Piano di Studio preventivamente approvato Da (compreso) A (escluso) Insegnamento

Programma dettagliato e risultati di apprendimento attesi



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.
Gel filtration
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.
Electrophoretic Techniques
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.
Disc 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;
4. Resolution;
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
1. Carbamylation;
2. Deamidation;
3. Beta elimination of cysteine.
Sample pre-fractionation

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.


Laboratory Program


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.

Note Sulla Modalità di valutazione

Evaluation by: written examination.

Risorsa bibliografica obbligatoriaHamdan, M, Righetti, P.G, Proteomics Today: Protein Assessment and Biomarkers Using Mass Spectrometry, 2-D Electrophoresis and Microarray Technology, Editore: Wiley-VCH, Hoboken, Anno edizione: 2005
Risorsa bibliografica obbligatoriaJan-Christer Janson, Protein Purification, Editore: Wiley, Anno edizione: 2011

Mix Forme Didattiche
Tipo Forma Didattica Ore didattiche
laboratorio informatico
laboratorio sperimentale
laboratorio di progetto

Informazioni in lingua inglese a supporto dell'internazionalizzazione
Insegnamento erogato in lingua Inglese
Disponibilità di materiale didattico/slides in lingua inglese
Disponibilità di libri di testo/bibliografia in lingua inglese
Possibilità di sostenere l'esame in lingua inglese
Disponibilità di supporto didattico in lingua inglese
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