PEG And Ethanol Based Two Step Non-denaturing Method For Human Serum Albuminome Analysis

BackgroundHSA is a negatively charged, highly soluble protein, comprisingapproximately50%of the total serum protein concentration. HAS bindsboth exogenous and endogenous compounds, which plays the role intransportation. The depletion of high-abundant proteins is often the initialstep in serum-based proteomics studies where the discovery of potentialprotein biomarkers, which are often in low abundance. However, depletionof high-abundant proteins results in the removal of low-abundant proteins,which can affect on the analysis of samples and has the potential itself to beused as a biomarker source. To this end, researchers have begun to focus onthe proteins and peptides, which are binding to the high-abundant proteins,particularly the albuminome.Enrichment of HSA is commonly achieved through the use ofantibody-or dye-based spin or LC columns, or chemical-based methods.But these methods are expensive, protein’s degeneration and non-specificbinding, are not suitable for the screening of a large number of clinical sample.ObjectiveThe purpose of this study was to separate and purify human serumalbumin using polyethylene glycol and ethanol. Establishing a separationand purification method, which has higher purification efficiency, operationprocess is simple, lower costs and suitable for large sample separation andpurification.Methods1. Enrich HSA using polyethylene glycol and ethanol.(1)Blood was drawn from median cubital vein and serum was separatedby centrifugation. Then serum was centrifuged at room temperature fordelipidation and stored until analysis.(2)Prepare PEG solution of different concentrations and analysis theoptimal concentration for removing immunoglobulin by means of Westernblot.(3)Choosing the optimal concentration of PEG, then continuingenriching HSA using ethanol solution of different concentrations andanalysis the impact of PEG for separating albuminome by Western blot.2. Comparison between the two methods.(1)Enrich human serum albuminome by the use of Protein GSepharose beads and ethanol.(2)Comparing the albuminome separated by the two methods by 2-DE.(3)Comparing the albuminome separated by the two methods byLC-MS/MS.Results1. Establishment of PEG-EtOH method.By detecting the serum protein precipitation separated by PEG, itindicates the best PEG concentration for removing IgG is12%. Then enrichthe albuminome by ethanol and find that the PEG contained in thesupernatant do not affect the efficiency of ethanol, the best ethanolconcentration for enriching albuminome is42%.2. Comparation of the two methods.Comparing the2-DE maps of albuminome separated by two methods,and identify the different proteins by MALDI-TOF/TOF-MS. We foundthat the different proteins are Alpha-1-antitrypsin and Vitamin D-bindingprotein, which are high-abundant proteins. Then identify the albuminomeenriched by the two methods, and a total of53kinds of proteins or peptidescan be detected from the three groups.ConclusionThe polyethylene glycol and ethanol co-precipitation method couldefficiently separate human serum albuminome.