RESUMO
Magnetic supports are tested for use in batch affinity capture of proteins. Two types of magnetic polymer composites were used for solid phase synthesis and for the batch affinity chromatography of folate binding protein from a protein mixture. Gly-Gly-L-Methotrexate as well as other analogs were synthesized on magnetic supports consisting of either polyoxyalkyleneamine grafted onto polystyrene beads or a copolymer of polyethylene glycol dimethylacrylamide (PEGA). Both supports incorporated within their matrix sub-micron particles of paramagnetic magnetite. The peptide-methotrexate analogs were attached to the magnetic supports via a photocleavable linker. The bound methotrexate-peptide analogs were equilibrated with a protein mixture consisting of bovine albumin, chicken albumin, folate binding protein, lysozyme, lactoferrin and lactoperoxidase precursor in phosphate buffered saline (PBS) and then after magnetically separating and washing the supports of any unbound components the bound protein was removed either through the photocleavage of the tethered methotrexate-peptide ligand or via exchange with soluble methotrexate. In all cases, the photocleavage or exchange with soluble methotrexate released folate binding protein as the major affinity captured protein. Of the two magnetic supports tested, the PEGA based support was found to be superior to the polyoxyalkyleneamine grafted polystyrene support and comparable to beaded agarose in releasing bound folate binding protein. Of the two methods for removing bound protein, photocleavage of the covalently attached ligand was found to release exclusively folate binding protein as opposed to exchange with soluble methotrexate which released residual amounts of the non-specifically bound proteins bovine and chicken albumin, in addition to folate binding protein. Thus, use of the PEGA based magnetic support in conjunction with a photocleavable linker should help facilitate the automation of multiple parallel affinity chromatography for proteomics applications.
Assuntos
Proteínas de Transporte/isolamento & purificação , Cromatografia de Afinidade/métodos , Ferro/química , Metotrexato/análogos & derivados , Óxidos/química , Proteômica/métodos , Receptores de Superfície Celular/isolamento & purificação , Acrilamidas/química , Ligação Competitiva , Proteínas de Transporte/química , Óxido Ferroso-Férrico , Receptores de Folato com Âncoras de GPI , Magnetismo , Metotrexato/química , Estrutura Molecular , Fotólise , Polietilenoglicóis/química , Poliestirenos/química , Proteínas/química , Proteínas/isolamento & purificação , Receptores de Superfície Celular/químicaRESUMO
The recent abundance of genome sequence data has brought an urgent need for systematic proteomics to decipher the encoded protein networks that dictate cellular function. To date, generation of large-scale protein-protein interaction maps has relied on the yeast two-hybrid system, which detects binary interactions through activation of reporter gene expression. With the advent of ultrasensitive mass spectrometric protein identification methods, it is feasible to identify directly protein complexes on a proteome-wide scale. Here we report, using the budding yeast Saccharomyces cerevisiae as a test case, an example of this approach, which we term high-throughput mass spectrometric protein complex identification (HMS-PCI). Beginning with 10% of predicted yeast proteins as baits, we detected 3,617 associated proteins covering 25% of the yeast proteome. Numerous protein complexes were identified, including many new interactions in various signalling pathways and in the DNA damage response. Comparison of the HMS-PCI data set with interactions reported in the literature revealed an average threefold higher success rate in detection of known complexes compared with large-scale two-hybrid studies. Given the high degree of connectivity observed in this study, even partial HMS-PCI coverage of complex proteomes, including that of humans, should allow comprehensive identification of cellular networks.