Date of Award
Matthew A. Tarr
Cell surface proteins play an important role in understanding cell-cell communication, cell signaling pathways, cell division and molecular pathogenesis in various diseases. Commonly used biotinylation regents for cell surface mapping have shown some potential drawbacks such as crossing the cell membrane, difficult recovery of biotinylated proteins from streptavidin/avidin beads, interference from endogenous biotin and nonspecific nature of streptavidin. With aim to solve these problems, we introduced sulfo-N-hydroxysuccinimidyl (NHS) ester functionalized magnetic nanoparticles containing cleavable groups to label solvent exposed primary amine groups of proteins. Silica coated iron oxide magnetic nanoparticles (Fe3O4@SiO2 MNPs) were linked to NHS ester groups via a cleavable disulfide bond. Additionally, the superparamagnetic properties of Fe3O4@SiO2 MNPs facilitate efficient separation of the labeled peptides and removal of the detergent without any extra step of purification. In the last step, the disulfide bond between the labeled peptides and MNPs was cleaved to release the labeled peptides. The disulfide linked NHS ester modified Fe3O4@SiO2 MNPs were tested using a small peptide, and a model protein (bovine serum albumin) followed by liquid chromatography-tandem mass spectrometry analysis (LC-MS/MS) of labeled peptides. In the next step, disulfide linked, NHS ester modified Fe3O4@SiO2 MNPs (150 nm) successfully labeled the solvent exposed cell surface peptides of Saccharomyces cerevisae. Electron microscopic analysis confirmed the cell surface binding of NHS ester modified Fe3O4@SiO2 MNPs. Mass spectrometric analysis revealed the presence of 30 unique proteins containing 56 peptides.
Another MNPs based labeling reagent was developed to target solvent exposed carboxyl acid residues of peptides and proteins. The surface of Fe3O4@SiO2 MNPs was modified with free amine groups via a disulfide bond. Solvent exposed carboxyl groups of ACTH 4-11 and BSA were labeled by using1-Ethyl-3-(3-dimethylaminopropyl)carbodiimide (EDC) chemistry. Upon cleaving the disulfide bond, labeled peptides were analyzed by LC-MS/MS.
The MNPs containing labeling reagents offers specific labeling under physiological conditions and rapid magnetic separation of labeled peptides prior to mass spectrometric analysis. The ability of large Fe3O4@SiO2 MNPs to specifically attach to cell surface makes them a potential candidate to study the surface of variety of different cell types and complex proteins surrounded by lipid bilayer.
Patil, Ujwal S., "Magnetic nanoparticles containing labeling reagents for cell surface mapping" (2015). University of New Orleans Theses and Dissertations. 2049.