Proteomics analysis

ROSANO, GERMÁN L

Rosario

Congreso; 49th meeting of the Society for Cryobiology; 2012

The proteome is the full set of proteins expressed in a cell or organism at a given time and condition. Proteomics analysis provides a snapshot of the proteome and allows the identification and quantification of virtually any protein. Also, proteome variation under different conditions can be detected (comparative proteomics). Proteomics techniques can be divided between gel-based methods (2D electrophoresis, 2DE) and mass spectrometry (MS)-based methods. 2D electrophoresis is the workhorse of proteomics, with an unparalleled resolution and ease of use. The workflow of a 2DE protocol is simple regardless of the origin of the sample. Although technically straightforward and with superior resolution, 2DE is not without pitfalls and limitations. A 2DE analysis may not detect low-abundant proteins and hydrophobic membrane proteins. Moreover, reproducibility among laboratories is still an issue. On the other hand, there have been major advances in MS-based techniques in the last decade. Combined with the resolving power of liquid chromatography, it is possible to detect scarce and abundant proteins in complex mixtures simultaneously. If cells are subjected to different treatments, then the proteomes can be compared by the newly developed techniques of ICAT, SILAC and iTRAQ. When absolute quantification is needed, an AQUA protocol should be considered. However, the expensive equipment required is a high-entry barrier for most laboratories. Also, MS-based technologies produce great loads of raw data. Its processing and analysis is still a major bottleneck. Correct sample preparation is essential to both groups of techniques. Protein extraction, avoidance of proteolysis, sample buffer formulation and elimination of impurities are key steps that need to be perfected before running a 2DE or an MS protocol. Only by standardizing and refining these procedures, then the results can be considered reliable. Despite some issues that need to be overcome, proteomics has reached a level of maturity comparable to other “omics”, such as genomics and transcriptomics. Advances in instrumentation have allowed high throughput analysis with high resolution and sensitivity in shorter times. Thanks to this, proteomics analysis is living in a golden era, making a deep impact in biological research, especially in systems biology.