Superparamagnetic nanoparticle based (10nm) isolation of plasma membrane for high resolution mass spec. analysis of the proteome, lipidome and glycome

The more than 20.000 human genes give rise to many more proteins due to alternative splicing, post-translational modification, etc. Within the c ell/neuron, all these proteins are temporally and spatially organized. S everal genome-wide approaches like micro-arrays attempt to reveal patter ns of interactions, signaling cascades, etc. However, they ignore in gen eral the location of the proteins and where they are active, which is ge nerally considered as a central feature of cellular organization. In rec ent years, proteomics is becoming increasingly important in revealing th e organelle s composition. Currently, catalogs of proteins are availab le for different types of subcellular organelles. However, because of th e difficulties inherent in purifying organelles, most reliable proteomic s data are available only for specialized organelles including centrosom es, mitochondria and synaptic vesicles and the real challenge is yet to obtain a proteome catalog of endocytic organelles. Recently it has been recognized that the best protein assessment was reached using highly enr iched fractions of ER and Golgi, suggesting that the purification approa ch is still the most preferable. Here we present, the uptake of nanomete r sized particles with superparamagnetic properties as an innovative app roach to isolate endosomal subcompartments using magnetic chromatography . Moreover, magnetic nanoparticles (MNPs) covalently coupled to differen t biomolecules (ligands, growth factors, antibodies, toxins, etc.), in c ombination with a pulse chase uptake paradigm, can provide a unique way to temporally resolve major endosomal compartments along selective degra dative or recycling routes. Ferrite MNPs were prepared by a thermal decomposition method, widely use d for the synthesis of monodisperse nanoparticles with controllable size . This method makes use of a hydrophobic surfactant, to passivate the su rface, which results in nanoparticles that are solely dispersible in non -polar solvents. For use in biological applications, these nanoparticles need to be made water-dispersible. Here, we present a new procedure to decorate ferrite MNPs with diverse functionalities, based on the exchang e of the hydrophobic surface ligands with silanes bearing different endg roups. Amino-, carboxylate- and poly(ethylene glycol)-terminated silanes , being respectively positively, negatively and neutrally charged, were found to render the MNPs stable with respect to aggregation and keep the m highly dispersed in biological media. Due to their unique surface func tions, these MNPs show great potential for use in endosomal proteomics. In the first preliminary experiments, the MNPs were interacted with HELA cells to monitor their cellular uptake. It was shown that their uptake was strongly dependent on the type of coating and the presence of fetal calf serum in the medium. Future experiment will focus on the pulsed cha se uptake of MNPs and the covalent grafting of biomolecules to the nanop articles surface to allow for specifically directed cellular uptake.status: publishe