Master of Science, The Ohio State University, 2024, Chemistry

Multiplexed biomarker detection has proven to be important in offering a more effective and accurate disease diagnosis compared with single biomarker detection. Mass spectrometry (MS), an analytical technique that provides sensitive and specific analyte detection, has long been employed for signal transduction of immunoassays toward disease detection. Unlike with direct detection of biomarkers, the use of small molecule mass reporters allows the use of simpler and less expensive portable mass spectrometers. Our research group has previously used quaternary ammonium species as mass reporters for MS-based immunoassays performed on inexpensive paper substrates. However, the number of unique masses that could be generated with the quaternary ammonium species was limited by the commercial availability of the starting materials, which in turn limited the multiplexing ability of our MS-based immunoassay platform. The aim of this thesis was to develop isobaric peptide probes that employ small peptides as mass reporters. The isobaric peptide probes were developed by designing and characterizing peptide mass reporters to investigate their fragmentation patterns in tandem MS (MS/MS) and their sensitivity via nano-electrospray ionization (nESI). Results showed that peptides containing two arginines (Arg) that sequester mobile protons and a cleavage site of aspartic acid (Asp) and proline (Pro) provide two dominant b- and y-type ions in MS/MS. The position of the Asp-Pro bond determines the mass of the corresponding b- and y-type ions. By changing the position of the Asp-Pro amino acids with a particular peptide, we can generate different isobaric peptide probes that fragment to give distinct diagnostic ions. The limit of detection (LOD) and quantification (LOQ) of the optimized peptide design (AcIRNPTIDPINR MW 1350.5 Da) in nESI MS/MS were 0.35 ng/mL (0.26 nM) and 1.03 ng/mL (0.77 nM), respectively. In preparation for the use of the isobaric peptides in immunoassay, we synthesi (open full item for complete abstract)

Committee: Abraham Badu-Tawiah (Advisor); Heather Allen (Committee Member); Vicki Wysokci (Committee Member) Subjects: Analytical Chemistry; Chemistry

Master of Science, The Ohio State University, 2022, Chemistry

We hypothesized that programmable recognition of non-canonical pair (NCP) sites could be achieved by triplex hybridization with bifacial peptide nucleic acids (bPNA) that side-chain display a combination of native RNA bases and synthetic bases melamine and ammeline. To test this hypothesis, we designed and synthesized bPNAs with different base combinations on the side chains. We then conducted biophysical gel shift assays and functional fluorescence activation assays to screen the NCP binding capability of these bPNAs. For gel shift assays, we constructed DNA and RNA libraries with 16 different NCP internal bulge sites built in the duplex. The Cbf-labeled probes were screened against constructed DNA and RNA libraries and visualized on native polyacrylamide gel. Cbf signals were normalized against the positive control sample which forms the known T-M-T or U-M-U triples. Band intensity was used to reflect the binding of bPNA to individual NCP sites. For the functional assays, spinach RNA aptamer and cognate DFHBI fluorescent module were used to construct a light-up aptamer system. Our results indicated melamine recognition U x C site besides U x U site, and we identified ammeline as a general base which could recognize U x G and C x C sites. Guided by the biophysical screens, spinach aptamer with NCP bulges were designed and used to functionally screen for bPNAs binding to NCP sites within RNA scaffolds. This assay effectively and sensitively translates NCP binding affinity into fluorescence readouts. The results from fluorescence activation assays were consistent with biophysical data. Further testing with previously reported bPNAs was done on spinach-derived platforms and resulted in similar conclusions, establishing the fluorescent aptamer based activation assay as a way to screen for NCP interactions.

Committee: Jane Jackman (Committee Member); Dennis Bong (Advisor) Subjects: Biochemistry; Chemistry

Doctor of Philosophy (PhD), Ohio University, 2016, Chemistry and Biochemistry (Arts and Sciences)

Ambient ionization methods allow the ionization of untreated samples in the open environment. In this dissertation, two different ambient ionization techniques, desorption electrospray ionization (DESI) and probe electrospray ionization (PESI), has been developed and coupled with liquid chromatograph (LC) and electrochemistry (EC) and their analytical applications have been explored and discussed. Liquid sample DESI generally employs a DESI probe to spray solvent with high voltage to ionize sample as the sample solution is delivered to the ion source by a piece of fused silica transfer capillary. A new splitting interface, a PEEK capillary tube with a micro-orifice drilled in the capillary wall, was used to connect with LC column for applying DESI ionization. A small portion of LC eluent emerging from the orifice can be directly ionized by DESI with negligible time delay while the remaining analytes can be online collected. Furthermore, online derivatization using reactive DESI is possible for additional application such as supercharging proteins. Since splitting via an orifice introduces negligible dead volume and back pressure, the performance of the LC/DESI-MS with the focus of using ultra-fast LC for analyzing sample was further evaluated. Using a monolithic C18 column, metabolites in urine can be separated within 1.6 min, online monitored by DESI and collected as purified samples. Negative ions can be directly generated for acidic analytes in acidic LC eluent by DESI during the LC/MS analysis process using a spray solvent with alkaline pH. In addition, DESI-MS is found to be compatible with ultra-performance liquid chromatography (UPLC) for the first time. The 45 s separation of drugs can be achieved via UPLC/DESI-MS under high temperature. The combination with EC further broadens LC/MS applications. UPLC-MS combined with EC via DESI was first developed for the structural analysis of proteins/peptides that contain disulfide bonds. Using this combine (open full item for complete abstract)

Committee: Hao Chen (Advisor); Peter Harrington (Committee Member); Shiyong Wu (Committee Member); Shigeru Okada (Committee Member) Subjects: Analytical Chemistry; Chemistry