Proteomics Methods

By Dr. Tomislav Meštrović, MD, Ph.D.

Proteomics describes the global analysis of proteome, which can be defined has as the protein content of a cell, a tissue or an entire organism in a defined state. Since the inception of this field 30 years ago, the complete characterization of all proteins has been its fundamental goal.

Due to the complex nature of the proteome, the constant development of new methods and techniques for the chromatographic separation and detection (but also sample cleanup, fractionation and preconcentration) becomes a crucial prerequisite for the correct identification of peptides and proteins. Simultaneous analysis of several thousands of different proteins from complex biological samples is often required in modern proteomics.

Methods of protein study

Three methods for separation of complex protein or peptide samples are preferred in proteomics: denaturing polyacrylamide gel electrophoresis (PAGE) or sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE), two-dimensional gel electrophoresis, and high-performance liquid chromatography (HPLC). Other useful tools include capillary electrophoresis and affinity chromatography.

No protein separation technique is more widely used than SDS-PAGE, mostly due to its simplicity, reproducibility, as well as acceptable instrument and consumable expenses. This method (initially described by Laemmli in 1970) separates proteins based on their primary structure or size, but not amino acid sequence. SDS-PAGE can be used to check the purity of samples and to estimate molecular weights for unknown proteins.

Around the same time at which SDS-PAGE was introduced, O’Farrell and his team applied isoelectric focusing to protein samples prior to SDS-PAGE; the result was the concept of two-dimensional (2-D) gel electrophoresis. This method has impressive separation capabilities and can easily interface with immunoblotting techniques.

High-performance liquid chromatography (HPLC) represents a separation technique developed for the analysis of organic molecules and ions. Instrumentation for HPLC research in proteomics is undistinguishable from conventional HPLC instrumentation, i.e. pumping systems, separation columns and detectors are used both for conventional analysis and for proteomics research.

Modern technologies in proteomics

Mass spectrometry has feasibly become the core technology in proteomics. The application of techniques based on mass spectrometry for the qualitative and quantitative analysis of global proteome samples derived from complex mixtures had a pivotal role in our understanding of cellular function.

In short, mass spectrometry represents an analytical tool useful for measuring the mass-to-charge ratio of one or more molecules present in a sample. These measurements are then often used to calculate the exact molecular weight of the components in question. Mass spectrometers can be employed to identify unknown proteins by determining their molecular weight determination, as well as to determine structure and chemical properties of selected molecules.

The techniques that are most often used are electrospray ionization (ESI) and matrix-assisted laser desorption/ionization (MALDI). Both of them are representatives of so-called soft ionization techniques in which ions are created with low internal energies and thus undergo little fragmentation.

Protein microarray technology has progressed rapidly for the identification, quantification and functional analysis of proteins in applied proteome research. Multiplex assays enable the precise characterization of proteins and the study of complex protein-protein interactions, but also peptides, low molecular weight compounds, oligosaccharides or DNA.

The next generation of microarrays with a capability for high-throughput, ultrasensitive, low-cost biomarker analysis will most probably involve a combination of nanotechnology, surface enzyme reactions, microfluidic networks and advanced data analysis tools. This will undoubtedly accelerate protein biomarker discovery and characterization of disease-specific pathways.

Further Reading

• All Proteomics Content
• What is Proteomics?
• Proteomics Uses
• Protein-Protein Interactions
• Interpreting Proteomics Data