Depletion of abundant proteins from Bothrops jararaca snake plasma using chromatography for proteomics analysis

摘要

Plasma, the best known but perhaps the most complex biofluid, contains thewidest dynamic range of cellular protein species in the body. The 22 most abundant proteins constitute more than 99% of the mass of total plasma proteins. Therefore, depletion of the high abundant proteins becomes a critical step in the plasma proteome profiling, especiallywhen the limit peak capacity and low dynamic range of mass spectrometry analysis occur with protein identification. HiTrap Blue HP column is known for human albumin purification. So, the aim of this work was to deplete the most abundant protein, albumin, from the Bothrops jararaca snake plasma, with the intention of extending the dynamic range of proteome detection for greater coverage and diversity of protein identifications. In a one-step process, albumin was partially removed by affinity chromatography using a HiTrap Blue HP column and the collected fractionswere analyzed by 1D and 2D SDS-PAGE. The results showed that albuminwas depleted around 40% by 1D SDS-PAGE, while analysis by 2D resulted in a lower number of spots in the depleted plasma than non depleted plasma, 56 and 91, respectively.We concluded that HiTrap Blue HP column is effective to deplete plasmatic albumin from B. jararaca snake plasma; however, it depletes other proteins too. The perspective of this project is to identify spots’ proteins in order to analyze whether this purification step is as necessary as it is for human plasma. These results are important considering the limitation of materials, such as snake plasma and venom, and could also be useful to deplete highly abundant proteins from other animals’ plasmas.