Scientific Overview

Rationale and Objectives

Rationale

Proteomics is the scientific discipline of characterizing and analyzing the proteome. The term proteome describes an entire protein complement expressed by the genome in the lifetime of a given cell, tissue or organism, including isoforms, polymorphisms and modifications, protein-protein interactions and the structural description of proteins. Proteomics encompasses the identification, characterisation and quantification of the proteome.

The dominant proteome analysis workflow utilizes a few important steps. They are: protein extraction from the cells, protein/peptide separation, site-specific protein enzymatic digestion and mass spectrometry (MS)/bioinformatics analysis.

The extraction of proteins from cells is a critical step in proteomics studies. The extraction of soluble proteins is simply performed by lysing the cells and collecting the supernatant. Different cell lysis methods are easily accessible in literature data.

Protein separation by two-dimensional polyacrylamide gel electrophoresis (2-D PAGE) provides high-resolution separation of complex protein mixtures. 2-D PAGE can potentially separate several thousand proteins in a single experiment (on average, between 5000 and 15000 per cell type). During 2-D PAGE the proteins are firstly separated according to their pI (first dimension), followed by their separation according to their molecular weight (second dimension). Alternatively, a mixture of proteins or peptides can be separated by high performance liquid chromatography (HPLC). HPLC separation is based on the differences in interactions of compounds (analytes) between two phases, mobile (liquid) and stationary, as the compounds travel through a column under high pressure. In reverse-phase HPLC mode, separation is performed according to the hydrophobicity of analytes (proteins/peptides). The HPLC systems are capable of detecting proteins of very low abundance and allow the analysis of complex protein/peptide mixtures in a single run. Where HPLC separation is followed by mass spectrometry/bioinformatics analysis, the method is known as Shot-gun proteomics.

On the other hand, protein spots separated by 2-D PAGE can be excised from the gels, properly rinsed, and in-gel digested, while in HPLC separation the proteins are usually digested in-solution, and then separated. Both methods (2-D PAGE and HPLC) in conjunction with MS analysis and followed by peptide mass database search, represent very powerful tools for protein identification and quantification.

MS is one of the most sensitive methods for the structural characterisation and protein identification. MS technique measures the mass to charge ratios (m/z) of gas-phase ions with extremely high sensitivity and resolution. The most common ionisation techniques are electrospray ionisation (ESI) and matrix-assisted laser desorption ionisation (MALDI), which allow the transfer of large, polar, thermally labile biomolecules into the gaseous phase for mass analysis (e.g. peptides).
Further, peptides can be fragmented in the mass spectrometer by tandem mass spectrometry (MS/MS), and the resulting MS/MS spectra are usually used to retrieve the corresponding peptide sequence from the database. Protein identification is accomplished by matching the list of observed m/z in MS and MS/MS spectra with a calculated list of all the expected peptide masses for each entry in the protein database.

Proteomics can be viewed as an array of biological or clinical assays and has a great impact on modern biology and medicine. Proteomics technology is applied to various standard biochemical and molecular biology studies: biomarker discovery, protein/protein interactions, biotypization, cell functioning, proteins or “small molecules” mechanism of action and so on.

Objectives

About the Course

For this Innomol FP7-REGPOT-2012-2013 - Proteomics Workshop Course the objective includes: understanding the fundamentals of mass spectrometry, with a focus on different separation techniques for peptides and proteins (multidimensional nano- and capillary-liquid chromatography/1-D and 2-D SDS-PAGE), cell lysate handling (sample homogenization) and protein/peptide enrichment strategies, peptide and protein analysis by MALDI-TOF/TOF and ESI-Q-TOF, bioinformatics data-analysis, followed by database searches, de novo sequencing and high-throughput data processing.

Further reading

• K. F. Geoghegan, M. A. Kelly, Mass Spectrom. Rev. 24 (2005) 347.
• B. Domon, R. Aebersold, Science 312 (2006) 212.
• S. E. Ong, B. Blagoev, I. Kratchmarova, D. B. Kristensen, H. Steen, A. Pandey, M. Mann, Mol. Cell. Proteomics 1 (2002) 376.
• Figeys, D, Proteomics: The basic overview, Industrial Proteomics: Applications for Biotechnology and Pharmaceuticals (2005), WILEY (ISBN: 978-0-471-45714-5)