Isolation, identification, and quantitation of biomolecules using high pressure liquid chromatography and mass spectrometry
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Greene, Landon Ray.
Isolation, identification, and quantitation of biomolecules using high pressure liquid chromatography and mass spectrometry. Retrieved from
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TitleIsolation, identification, and quantitation of biomolecules using high pressure liquid chromatography and mass spectrometry
Date Created2014
Other Date2014-10 (degree)
Extent1 online resource (xii, 109 p. : ill.)
DescriptionThe research performed in this dissertation involved isolation, identification, and quantitation of biomolecules using high pressure liquid chromatography and mass spectrometry. NanoRNAs, 2 – 4 nucleotide RNA oligomers, have been shown to prime transcription initiation in vivo. However, the particular oligonucleotides and absolute abundance could not be determined without the use of specific and sensitive analytical techniques. The goal of this research was to develop an analytical procedure to isolate, identify, and quantify nanoRNAs, in vivo, in Escherichia coli cells. A sequence of experiments was performed. A high performance pressure liquid chromatography (a.k.a. high pressure liquid chromatography) method was developed to isolate nanoRNA oligomers. This method was coupled with tandem mass spectrometry for detection. The combined HPLC-MS/MS method identified nanoRNAs in E. coli cell cultures. Experiments were performed to optimize the extraction of RNA oligomers from cell cultures to improve quantitation. An optimized extraction procedure and solid phase extraction method were developed which provided reproducible and quantitative analysis of nanoRNAs. The analysis of 5’, 3’-hydroxyl uridine-adenosine and 5’, 3’-hydroxyl uridine-adenosine-uridine was performed in E. coli cells. The results supported the qualitative hypothesis concerning the presence of these nanoRNAs. However, the absolute quantitation of nanoRNAs had significant error. Other research in this dissertation involved proteins. A novel monoclonal antibody, Das-1, was generated during ulcerative colitis research. This antibody has been used as a biomarker of various pre-cancerous and cancerous conditions of the gastrointestinal tract. However, the properties and structure of the specific antigen of mAb Das-1, a colonic epithelial protein (CEP), are unknown. The goal was to develop an isolation procedure to purify CEP. A three step procedure was developed. First, a size exclusion method was used to provide purification of CEP based on size. Second, a strong anion exchange method separated CEP based on overall surface charge. Finally, a hydrophobic interaction chromatography method provided purification based on hydrophobicity. The sequential purification of CEP using the three step procedure developed was tracked using several immunoassays. The results demonstrated significant purification of CEP.
NotePh.D.
NoteIncludes bibliographical references
Noteby Landon Ray Greene
Genretheses, ETD doctoral
Languageeng
CollectionGraduate School - New Brunswick Electronic Theses and Dissertations
Organization NameRutgers, The State University of New Jersey
RightsThe author owns the copyright to this work.