Proteins are vital parts of living organism, as they are the main components of the physiological pathways of cells. Proteomics is considered the next step (with transcriptomics as the next step following proteomics) in the study of biological systems, and it is much more complex in comparison to genomics. The reason: genomics is more or less constant for an organism while the proteome differs not only from organism to organism but even from cell to cell of the same organ.
Current research strategies and methodologies applied for proteomics research are two dimensional gel electrophoresis combined with nano liquid chromatography and mass spectrometry.
For proteomics approach, where the sample amount is often limited, the interest in miniaturized LC systems was spurred by need to combine high efficiencies and low flow rates, the latter being of relevance to mass spectrometry (MS) detection of tryptic peptides. Additional advantages of nano HPLC systems are their economy of use, as smaller amounts of stationary-phase material are needed, while also smaller volumes of mobile phase are being consumed. Further, with the column miniaturization and the low flow rates, miniaturization also involves a reduction of the detector volume.
Due to the extremely high sample complexity of proteomics samples, the single dimension separation is often not sufficient since many analytes co-elute and influence the ionization (ionization suppression) of other molecules, a further sample “simplification” is needed. The “simplification” can be achieved by applying multidimensional separation methods such as using the ion exchange chromatography or the hydrophilic separation (HILIC) as the first separation and fractionate the run. Subsequently, the collected fractions are re-injected onto the reversed phase HPLC column, separated, and detected with MS. However, despite the combined separation power of the combined systems, the complexity of the proteome and of all posttranslational modifications requires for further developments and investigation into new stationary phases, new separation methods (mobile phases, buffer systems), and the development of new sample preparation techniques.
Methods & Techniques
Protein purification by precipitation, chromatographic separation and clean-up, online digests; protein sources can be tissue (blood, brain, lungs) of both human and animal origin, cell lysates etc.; chromatography for sample preparation and separation prior to mass spectrometric detection and subsequent bioinformatic data analysis
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