Arabidopsis thaliana seedlings grown in liquid culture were used to recover proteins secreted from the whole plant. The aim was to identify apoplastic proteins that may be lost during classical extraction procedures such as preparation of cell walls. The inclusion of polyvinyl-polypyrrolidone (PVPP) in the protocol of purification of secreted proteins allowed a more efficient identification of proteins after their separation by two-dimensional gel electrophoresis (2-DE) and mass spectrometry analyses. Improvement of identification was 4-fold. It is related to an increased number of detectable peaks on mass spectra increasing the percentage of sequence coverage, and the identification confidence. The role of PVPP was to trap phenolic compounds and to prevent their unspecific interactions with proteins. These experiments resulted in the identification of 44 secreted proteins, of which 70% were not identified in previous cell wall proteomic studies. This may be due to specific gene regulation in seedlings and/or to a better access to apoplastic proteins not bound to cell walls.
Several zwitterionic detergents differing in their polar heads, linker parts and hydrophobia tail were synthesized and evaluated for their efficiency in protein solubilizers for two-dimensional electrophoresis. A model system consisting of human red blood cell ghosts was used for this purpose. This study leads to the description of several new efficient detergents and allowed us to derive structural constraints for the design and synthesis of efficient detergents for two-dimensional electrophoresis. These constraints apply to the hydrophilic head (sulfobetaine but not carboxybetaine), to the hydrophobic tail (12 to 16 alkyl carbons long, linear alkyl or alkylaryl) and to the presence and nature of the linker between the hydrophilic head and hydrophobic tail.
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