ABSTRACT
Gold nanoparticles are utilized in a variety of sensing and detection technologies because of their unique physiochemical properties. Their tunable size, shape, and surface charge enable them to be used in an array of platforms. The purpose of this study is to conduct a thorough spectroscopic characterization of Au and functionalized hybrid Au@SiO2 nanoparticles under physiological conditions and in the presence of two proteins known to be abundant in serum, bovine serum albumin and human ubiquitin. The information obtained from this study will enable us to develop design principles to synthesize an array of surface-enhanced Raman spectroscopy-based nanoparticles as platforms for theranostic applications. We are particularly interested in tailoring the surface chemistry of the Au@SiO2 nanoparticles for applications in theranostic technologies. We employ common spectroscopic techniques, with particular emphasis on circular dichroism and heteronuclear single quantum correlation nuclear magnetic resonance (HSQC NMR) spectroscopy, as combinatorial tools to understand protein conformational dynamics, binding site interactions, and protein corona for the design of nanoparticles capable of reaching their intended target in vivo. Our results conclude that protein adsorption onto the nanoparticle surface prevents nanoparticle aggregation. We observed that varying the ionic strength and type of ion influences the aggregation and aggregation rate of each respective nanoparticle. The conformation of proteins and the absorption of proteins on the surface of Au nanoparticles are also influenced by ionic strength. Using two-dimensional [15N-1H]-HSQC NMR experiments to compare the interactions of Au and Au@SiO2 nanoparticles with 15N-ubiquitin, we observed small chemical shift perturbations in some amino acid peaks and differences in binding site interactions with ubiquitin and respective nanoparticles.
ABSTRACT
Plasma proteomic experiments performed rapidly and economically using several of the latest high-resolution mass spectrometers were compared. Four quantitative hyperfractionated plasma proteomics experiments were analyzed in replicates by two AB SCIEX TripleTOF 5600 and three Thermo Scientific Orbitrap (Elite/LTQ-Orbitrap Velos/Q Exactive) instruments. Each experiment compared two iTRAQ isobaric-labeled immunodepleted plasma proteomes, provided as 30 labeled peptide fractions, and 480 LC-MS/MS runs delivered >250 GB of data in 2 months. Several analysis algorithms were compared. At 1% false discovery rate, the relative comparative findings concluded that the Thermo Scientific Q Exactive Mass Spectrometer resulted in the highest number of identified proteins and unique sequences with iTRAQ quantitation. The confidence of iTRAQ fold-change for each protein is dependent on the overall ion statistics (Mascot Protein Score) attainable by each instrument. The benchmarking also suggested how to further improve the mass spectrometry parameters and HPLC conditions. Our findings highlight the special challenges presented by the low abundance peptide ions of iTRAQ plasma proteome because the dynamic range of plasma protein abundance is uniquely high compared with cell lysates, necessitating high instrument sensitivity.
