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Novel Nanomaterials and Chromatographic System for Enhanced Separation and Characterization of Biomacromolecules and Nanoparticles

Wang, Yanhui
http://rave.ohiolink.edu/etdc/view?acc_num=osu1534174697101168

Abstract Details

2018, Doctor of Philosophy, Ohio State University, Chemistry.
With recent advances in technologies and methodologies, proteomics, which is the large-scale analysis of proteins, has been continuously developed in the field of bioinformatics, biotherapeutics and biomarker discovery. Top-down proteomics, which focuses on the analysis of intact proteins, has emerged within the last decade with significant advantages over the traditional bottom-up approach, such as the characterization of labile protein structures and the universal detection of all existing modifications. The front-end separation technologies for intact proteins are of the primary importance for the successful implementation of top-down proteomics. The work reported herein focuses the development of miniaturized liquid chromatography (LC) system and an effective and eco-friendly solvent system to address the challenges faced in intact protein separation and characterization. Electrospun nanofibers featuring effective chromatographic performance as the stationary phase of the ultrathin layer chromatography (UTLC) was developed in this work for the separation of amino acids and intact proteins. Nafion, a synthetic perfluorinated cationic polymer, was incorporated into a carrier polymer, polyacrylonitrile (PAN), to fabricate the nanofibrous stationary phase via electrospinning method. The separation of charged amino acids and proteins on the Nafion-PAN UTLC was based on the ion exchange mechanism (IEX). Design of experiments (DOEs) methods were applied to optimize the Nafion-PAN stationary phase and separation conditions. The nanofibers exhibited excellent mechanical stability and solvent compatibility. The separation of amino acids confirmed the feasibility of Nafion-PAN nanofibers as the ion exchange UTLC stationary phase. The separation of intact proteins has illustrated that Nafion-PAN stationary phase is also suitable for separation of large biomolecules. The retention of proteins on the Nafion-PAN UTLC largely depends on the properties of proteins including the net charge, hydropathicity, molecular size and structure. The Nafion-PAN UTLC demonstrated high separation efficiency for both amino acids and intact proteins. In addition to intact protein separation, a micellar liquid chromatography (MLC) system was developed using the polyacrylonitrile UTLC device for the size characterization of polyethylene glycol (PEG)ylated gold nanoparticle (AuNP), an emerging agent in cancer therapeutics, of which the cellular uptakes and cytotoxicity are highly dependent on its size. PEGylated AuNPs with different sizes in the range of 10-80 nm were well separated from each other. The developed method also permitted the separation of AuNPs capped with different molecular weight of PEG in the range of 2-20 kDa. Micellar mobile phases were adopted to provide a highly biodegradable chromatographic system. This method exhibited excellent separation performance with smallest plate heights < 2 µm and resolution of each pair of AuNPs > 1.5. Decent separations for all PEGylated AuNPs could be achieved within 5 min. This method was applied to monitor the transformation of AuNPs in serum protein, serving as a rapid and convenient tool for characterization of size distribution and modification of PEGylated AuNPs. Efforts towards improving the mobile phase system of intact protein separation were also made by adopting enhanced fluidity liquid chromatography (EFLC), which involves the addition of liquefied gas, such as carbon dioxide, to the conventional liquid mobile phases. The addition of liquefied CO2 provides increased diffusivity and decreased viscosity of the mobile phase, which inherently leads to more efficient separation. Herein, EFLC was first time applied to hydrophobic interaction chromatography (HIC) to study the impact of liquefied CO2 on the chromatographic behaviors of proteins. Since conventional HIC is known to preserve the native structure of proteins, the effects of liquefied CO2 on protein structures, charge state distributions (CSD) and ionization efficiencies would be more pronounced by adapting EFLC to HIC online coupled to electrospray ionization mass spectrometry (ESI-MS). In this work, EFLC offered improved chromatographic behaviors including a shorter analysis time, better peak shapes and a higher plate number. Liquefied CO2 proved to be an ESI friendly and “supercharging” reagent without sacrificing chromatographic performance, which can be used to improve peptide and protein identification in the large-scale application. The EFLC system was also applied to reversed phase chromatography (RPC) for intact protein separation. The EFLC solvents utilizing liquefied CO2/methanol/water mixture could be considered as a “greener” alternative to traditional water/acetonitrile mobile phase in LC with better separation efficiency and peak symmetry. Various mobile phase additives were compared under RPC mode to provide the optimum condition for integrated EFLC-MS system for intact protein separation and characterization.
Susan Olesik (Advisor)
Philip Grandinetti (Committee Member)
Abraham Badu-Tawiah (Committee Member)
240 p.
Analytical Chemistry

Recommended Citations

Citations

  • Wang, Y. (2018). Novel Nanomaterials and Chromatographic System for Enhanced Separation and Characterization of Biomacromolecules and Nanoparticles [Doctoral dissertation, Ohio State University]. OhioLINK Electronic Theses and Dissertations Center. http://rave.ohiolink.edu/etdc/view?acc_num=osu1534174697101168

    APA Style (7th edition)

  • Wang, Yanhui. Novel Nanomaterials and Chromatographic System for Enhanced Separation and Characterization of Biomacromolecules and Nanoparticles. 2018. Ohio State University, Doctoral dissertation. OhioLINK Electronic Theses and Dissertations Center, http://rave.ohiolink.edu/etdc/view?acc_num=osu1534174697101168.

    MLA Style (8th edition)

  • Wang, Yanhui. "Novel Nanomaterials and Chromatographic System for Enhanced Separation and Characterization of Biomacromolecules and Nanoparticles." Doctoral dissertation, Ohio State University, 2018. http://rave.ohiolink.edu/etdc/view?acc_num=osu1534174697101168

    Chicago Manual of Style (17th edition)

osu1534174697101168
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This open access ETD is published by The Ohio State University and OhioLINK.