BIORGANIC CHEMISTRY

The aim of the course is to provide students with strategies for the characterization of complex protein mixtures by proteomic methods. Particularly, proteomic investigations are based on the use of highly efficient separation techniques for proteins such as reversed phase high performance liquid chromatography (RP-HPLC) or two dimensional polyacrylamide gel electrophoresis (2D-PAGE) combined with biochemical methods, enzymatic digestion and MALDI end ESI mass spectrometry in order to obtain data suitable for searching, by specific software (Mascot, PEAKS, etc.), genomic, protein or ESTs (expressed sequence tags) databases..

Amino acids and proteins

Amino acids: what is an amino acid; acid-base properties of amino acids; isoelectric point; reactivity of amino acids; common and rare amino acids; essential and non-essential amino acids; from amino acids to peptides and proteins: properties of the peptide bond.

Proteins: primary, secondary and tertiary protein structure. Proteins with quaternary structure; conformation and protein functions; denaturation of proteins.

Methods for separation of protein mixtures

Two-dimensional electrophoresis (2D electrophoresis): sample preparation; first dimension: isoelectricfocusing (IEF); second dimension: electrophoresis on polyacrylamide gel (SDS-PAGE); visualization of protein spots (silver staining and Coomassie blue)

Liquid Chromatographic techniques for the separation of protein mixtures (RP-HPLC)

Mass spectrometry characterization of peptides and proteins

The "soft" ionization techniques: Matrix Assisted Laser Desorption / Ionization (MALDI) and Electrospray (ESI). Tandem mass spectrometry (MS / MS) for the characterization of a peptide sequence.

How to study proteins by mass spectrometry

Characterization of the amino acid sequence of a protein by mass spectrometry; from a single protein to complex protein systems: the proteomics “era”;

Characterization of complex protein systems

Protein identification by top-down and bottom-up approaches; gel-based and gel-free approaches. Interpretation of MS/MS data: de novo Sequencing; protein identification by homology by BLAST (Basic Local Aligment Software Tool). Protein database search by mass spectrometry data: Mascot and PEAKS software. Quantitative Proteomics: SILAC, ICAT and iTRAQ approaches and label-free approaches. Examples and practical applications.

About Bioinformatics

Biological databases; database structure; methods for aligning biological sequences; Dot Matrix and Replacement Matrices; search for similarities in databases using heuristic algorithms: FASTA and BLAST; Multiple Sequence Alignment: The ClustalW software.

1. Chimica e Propedeutica Biochimica – Bettelheim; Brown etc. – EDISES

2. Chimica Organica – W.H. Brown; C.S. Foote; EDISES

3. Computational Methods for Mass Spectrometry Proteomics – I. Eidhammer; K. Flikka; L. Martens; S.O. Mikalsen; WILEY

4. Proteomics in Practice: A Laboratory Manual of Proteome Analysis – R. Westermeier; T. Naven; WILEY

5. Slides of the lessons