Abstract
BackgroundThe proteomes of mammalian biological fluids, cells and tissues are complex and composed of proteins with a wide dynamic range. The effective way to overcome the complexity of these proteomes is to combine several fractionation steps. OFFGEL fractionation, recently developed by Agilent Technologies, provides the ability to pre-fractionate peptides into discrete liquid fractions and demonstrated high efficiency and repeatability necessary for the analysis of such complex proteomes.ResultsWe evaluated OFFGEL fractionator technology to separate peptides from two complex proteomes, human secretome and human plasma, using a 24-wells device encompassing the pH range 3–10. In combination with reverse phase liquid chromatography, peptides from these two samples were separated and identified by MALDI TOF-TOF. The repartition profiles of the peptides in the different fractions were analyzed and explained by their content in charged amino acids using an algorithmic model based on the possible combinations of amino acids. We also demonstrated for the first time the compatibility of OFFGEL separation technology with the quantitative proteomic labeling technique iTRAQ allowing inclusion of this technique in complex samples comparative proteomic workflow.ConclusionThe reported data showed that OFFGEL system provides a highly valuable tool to fractionate peptides from complex eukaryotic proteomes (plasma and secretome) and is compatible with iTRAQ labeling quantitative studies. We therefore consider peptides OFFGEL fractionation as an effective addition to our strategy and an important system for quantitative proteomics studies.
Highlights
The proteomes of mammalian biological fluids, cells and tissues are complex and composed of proteins with a wide dynamic range
The peptides are recovered in liquid phase, which is much more convenient for the others subsequent separation experiments like liquid chromatography
We focused on the 7 charged amino acids aspartate (D), glutamate (E), arginine (R), lysine (K), histidine (H), tyrosine (Y), cysteine (C) which mainly contribute to the overall charge of the peptides and used for the theoretical calculation of their pI [13]
Summary
The proteomes of mammalian biological fluids, cells and tissues are complex and composed of proteins with a wide dynamic range. The recent introduction of commercially available OFFGEL fractionator system by Agilent Technologies, provides an efficient and reproducible separation technique [1,2]. Human plasma is the most complex body fluid and contains a large number of proteins with a dynamic range of at least 9–10 orders of magnitude [6]. This complexity is a problem for proteomic analysis and it is necessary to develop efficient separation techniques to determine its precise protein composition
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