Development of nanoparticle-based biochip system for detection of post-translational modifications of proteins

Abstract

Analysis of protein modifcations is essential for understanding the fidelity of most biological functions in living systems as well as for developing therapeutics and diagnosis. Highly sensitive detection of protein-modifying enzymes or post-translational modifications (PTMs) of proteins was described here by using the nanoparticle-based biochip systems. In the first approach, the powerful use of nanoparticles (AuNPs) in secondary ion mass spectrometry (SIMS) was demonstrated. As a result of the secondary ion mass singal enhancement of peptides by AuNPs, the sensitive detection of phosphorylation and proteolytic acitivity was attained, and the control of the orientation and length of peptides on non-biofuling surfaces improved the detection efficiency. Unlike traditional matrix-assisted laser desorption/ionization MS (MALDI MS), this nanoparticle-enhanced SIMS (NE-SIMS) enalbed matrix-free analysis and label-free chemical imaging with high sensitivity and selectivity. In the second approach, energy transfer between quantum dots (QDs) and AuNPs was employed for analyses of proteolytic acitvity and protein glycosylation. While the photoluminescence of donor QDs immobilized on glass was quenched by the AuNPs, the protolytic activity or protein glycosylation modulated the energy transfer between QD and AuNP with high specificity. Since the AuNPs can be used as common energy acceptors even in a distance of more than 10 nm, the conjugation of AuNPs and QDs with different colors enabled multiplexed assays of proteases and glycoproteins with higher sensitivity, which the conventional fluorescence resonance energy transfer (FRET) system does not provide. In addition, the chip-based energy tranfer system enabled reliable analysis with no aggregation of nanoparticles as well as with very small reaction volume, which led to a reliable detection in a high-throuput manner. In these regards, nanoparticle-based biochip system will be promising for diagnosis of PTM-associated d...