Development of Chemical Isotope Labeling Liquid Chromatography Mass Spectrometry for Cellular Metabolomics

• Author / Creator

  Luo, Xian

• Cellular metabolomics is the global study of a whole set of metabolites and its chemical processes in cells. Different analytical platforms, such as NMR and MS, have been employed for profiling the highly complex cellular metabolome and various data processing approaches have been developed. Compared with metabolomic profiling of biofluids, more challenges remain in cellular metabolomics. For instance, additional sample handling steps, including cell harvest, metabolism quenching and cell lysis, are always required. The sample handling step should be fully optimized and compatible for downstream analysis. In some situations, the amount of cells is extremely limited, but the sensitivity of most analytical platforms cannot meet the requirement for in-depth metabolomic profiling of such small amount of samples.
  Towards these challenges, the objective of my thesis is to establish efficient sample handling methods and develop high performance analytical platform for cellular metabolomics. First, a technique combining efficient cell lysis method and chemical isotope labeling (CIL) LC-MS for comprehensive metabolomic profiling of S. cerevisiae was developed (Chapter 2). This technique was used to investigate the metabolome change of S. cerevisiae under nitrogen starvation and ultrasonication stimulus (Chapter 2 and 3). Second, a rapid and efficient method was developed for harvesting and lysing adherent mammalian cells (Chapter 4). Third, high performance CIL nanoLC-MS was developed and used for high-coverage metabolomic profiling of small numbers of mammalian cells (Chapter 5). This technique was applied to profile the metabolome of circulating exosomes from pancreatic cancer patients (Chapter 6).
  Overall, this thesis describes the development of various approaches for handling different types of cells tailored to CIL LC-MS based metabolomics and the development of highly sensitive CIL nanoLC-MS and its application for analyzing small numbers cells and circulating exosomes.

• Subjects / Keywords

  • Chemical Isotope Labeling
  • Mass Spectrometry
  • Metabolomics

• Graduation date

  Fall 2018

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/R3ZP3WG51

• License

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