Proteomics Laboratory

It should be noted that this is a limited number access course. The mandatory procedure for access request is here http://www.ccsbio.polimi.it/?page_id=27

Prerequisites

Proteomics requires general knowledge on inorganic and organic chemistry, biochemistry and biology.

Course objectives

Description of analytical technologies, applied in laboratory, for the extraction/isolation, the identification and the characterization of proteins. Students will learn the analytical features of experimental methods and they will be able to choose the most appropriate for different applications. The main effort will be devoted in the laboratory experiments, where students will apply some of technologies described during lessons.  By a brief explanation of commonly used and innovative analytical protocols, students will be able to directly apply to protein samples, understanding all experimental steps and solving any related difficulties. Finally students will have the possibility to use specific instrumentations, like for example system for protein electrophoresis, and to perform entire mass spectrometry analysis for the protein identification.

Program course

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.

It should be noted that this is a limited number access course. The mandatory procedure for access request is here http://www.ccsbio.polimi.it/?page_id=27 

Learning methodology

The Proteomics Laboratory course is formed by direct teaching lessons, during which the professor will use powerpoint files, and by experimental training in Proteomics Laboratory. During these final lessons, all students, previously divided into small groups, will directly perform proteomic analysis after receiving all information about experimental protocols and security rules.

Proteomics materials

All slides, discussed during lessons, will be organized in powerpoint files and uploaded on Beep web site, before lessons.

Final exam and evaluation:

Knowledge verification will carried out with a final written test, formed by both multiple-choice questions and questions required a more detailed answer. It will be possible to evaluate the student’s ability to remember and to connect the different topics of course, but also to understand the degree of comprehension, gained through personal study.

In detail, it will be evaluated:

• the completeness of knowledge,
• the elaboration of relations between different topics, developing a deep critical capacity,
• the appropriate use of terminology.

The final exam will be composed by questions with different and specific scores, related to their difficulty, and the final evaluation will be performed considering all of them and the performance during the experimental training in laboratory.