Biomineral forming organisms employ unique sets of proteins and other biomacromolecules to generate the specific structures and properties of their mineralized parts. To date, two major bottlenecks exist for obtaining a detailed understanding of the role of proteins in morphogenesis of the silica-based cell walls of diatoms. Firstly, a method is lacking for isolating the intracellular organelle for biosilica formation, called the silica deposition vesicle (SDV). Secondly, only a few silica biomineralization proteins have been identified so far. In this project we will drastically increase the number of characterized silica biomineralization proteins by detailed proteomics analyses of biosilica isolated form three closely related but morphologically distinct diatom species (Thalassiosira pseudonana, T. oceanica, Cyclotella cryptica).
To achieve this we will employ a combination of conventional and homology-driven proteomics methodologies to circumvent the paucity of available protein sequences from diatoms and overcome imperfections of genomes annotation. This requires extending the capabilities of Mass Spectrometry driven BLAST (MS BLAST) software to enable searches against non-annotated DNA databases. Identified proteins will be subjected to unbiased screening for post-translational modifications and sequence polymorphisms taking advantage of (i) the high mass resolution of the Orbitrap mass spectrometer, and (ii) previously developed in-house software tools. A central aim of this project is isolation of the SDV using a combination of subcellular fractionation techniques including free flow electrophoresis and fluorescence assisted microparticle sorting, two techniques that have not yet been utilized for this purpose. Successful isolation of the SDV would not only allow for the characterization of the complete set of proteins and other biomolecules, but would also open up the possibility to study SDV biogenesis in vitro.