Codice identificativo insegnamento: 052374
Programma
sintetico:
Introduction of proteomics and of protein
structure
Description of experimental workflow of protein extraction and
purification.
Determination of protein content via protein assays.
Chromatography:
definition and explanation of different phases and general experimental
parameters (resolution, retention time)
Description of different types
of chromatographic techniques and explanation of their application:
-
size exclusion chromatography (SEC)
-hydrophobic interaction
chromatography (HIC)
- reverse phase chromatography (RPC)
- ion
exchange chromatography (IEX)
- affinity chromatography (AC) and
immunoaffinity chromatography (IAC)
Electrophoretic techniques:
definition and description of polyacrylamide gel electrophoresis (PAGE),
differences between native PAGE and SDS-PAGE, western blotting analysis.
Description of bidimensional electrophoresis (2DE), understanding of
isoelectrofocusing (IEF) concept. Example of specific 2DE, like 2D
fluorescence difference gel electrophoresis (DIGE).
Mass spectrometry
(MS) analysis: explanation of MS parameters, description of different mass
spectrometers (e.g. ion trap, quadrupole, orbitrap). Understanding of mass
spectrum and of MS software, useful for data elaboration and for final
protein identification.
Quantitative proteomics: isotope coded affinity
tag (ICAT), isobaric tag for relative and absolute protein quantitation
(iTRAQ), stable isotope labeling amino acids in cell culture (SILAC).
Example
of proteomic research from literature.
The part above represents all
theoretical topics, exclusively explained by professor. The course will
comprise also an experimental part in laboratory, during which students
will apply specific protocols for proteomic analysis.
In detail, the
Laboratory lessons will include:
- extraction method and precipitation
protocol for protein purification
- application of specific assay for
protein quantification
- SDS-PAGE analysis
- 2DE analysis
- cut of
protein bands for MS analysis and application of MS protocol
- injection
of peptide solution in nLC-XL ion trap and mass spectrometry analysis
-
acquisition of RAW file and protein identification using specific software
-
final elaboration of MS results.