Methodology Development For Mass Spectrometric Analysis Of Protein Posttranslational S-Fatty Acylation

Protein posttranslational S-fatty acylation essentially refers to the covalent attachment of 16-carbon palmitic acid or other long chain fatty acids to the free thiols of cysteine residues present in proteins. Compared with other lipid modifications, S-fatty acylation is reversible and thus regulable. It plays important roles in protein trafficking, membrane targeting and anzymatic activities. Because S-fatty acylation usually occurs on low abundance membrane proteins and membrane associated cytosolic proteins, this work is focused on the development of new methods for selective enrichment of S-fatty acylated peptides.(1) Synthesis and characterization of magnetic Fe3O4/Au nanoparticles. In this work, Fe3O4 nanoparticles were first prepared by a chemical co-precipitation method and then Au was coated on the surface of Fe3O4 nanoparticles through the reduction of of HAuCl4 by sodium citrate. It has been experimentally demonstrated that the resultant Fe3O4/Au nanoparticles have a uniform particle size. They can not only be separated by an external magnetic field but also can be well suspended in aquesous solutions.(2) Enrichment and mass spectrometric analysis of S-fatty acylated peptides based on magnetic Fe3O4/Au nanoparticles. Taken advantage of the specific bonding of Au with free thiol groups, an affinitive method has been developed in this work for rapid and selective isolation of targeted S-fatty acylated peptides from complex biological context under an external magnetic field. In combination with mass spectrometric techniques of highly sensitive MALDI-Q-TOF, isolated S-fatty acylated peptides can then be further identified.(3) Controlled fabrication and characterization of nanoporous alumina membrane. In this work, nanoporous alumina membranes were fabricated through an electrochemical anodization method. Mechanisms of the nanopore formation and the experimental conditions have been investigated. It has been revealed that the resultant anodized nanoporous alumina membranes are highly ordered and can be used for future analysis of biological samples.