Codice identificativo insegnamento: 052374
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.