Development of Chemical Isotope Labeling Liquid Chromatography Mass Spectrometry on Quadrupole-Orbitrap Mass Spectrometers for Metabolome Analysis

• Author / Creator

  Wang, Chu-Fan

• Chemical isotope labeling liquid chromatography-mass spectrometry (CIL LC-MS) is a powerful technique in metabolomics, offering extensive coverage of the metabolome. This method involves dividing metabolites into sub-groups based on their functional groups and subjecting them to specific labeling reactions. By employing well-designed labeling reagents, CIL LC-MS enables improved LC separation and MS detection, the use of differential isotope labeling further allows for accurate relative quantification. To date, a four-channel labeling protocol has been developed in our lab, i.e., profiling amine-/phenol-, carboxyl-, hydroxyl-, and carbonyl-sub-metabolomes, achieving a remarkable 86%-95% coverage of non-lipid metabolites in the databases of MCID, HMDB, KEGG, YMDB and ECMDB. While quadrupole time-of-flight (QTOF) MS has been commonly utilized in CIL LC-MS, this thesis research focuses on implementing CIL LC-MS analysis on Q-Orbitrap MS, with an emphasis on maximizing instrumental performance for metabolomics.
  Chapter 2 compared the effects of different instrument types (TOF MS vs. Orbitrap MS) on metabolite detectability, considering detection sensitivity, dynamic range, ionization efficiency, and ion transportation efficiency. Instrument type was found to significantly impact metabolite detection in CIL LC-MS, many common peak pairs were detected from the two instruments; however, there were a significant number of unique peak pairs detected in each type of instrument, necessitating careful comparison of data generated using different instruments.
  Chapter 3 introduced a segmented spectrum scan method using Orbitrap MS in CIL LC-MS to enhance metabolite detection efficiency. During the LC data acquisition, the full m/z range was divided into multiple segments with the scanning of each segment to produce multiple narrow-range spectra. This approach realized a substantial increase in detectable metabolites while maintaining accurate relative quantification and precise peak ratio measurements.
  Chapter 4 evaluated the performance of the segment-scan-assisted CIL LC-MS across different sample types such as feces, urine, blood, cell extracts, saliva, etc. A 120-m/z segment scan was established as a routine approach to further improve the overall MS detection of dansyl-labeled metabolites on Q-Orbitrap MS.
  Chapter 5 explored the fragmentation patterns of dansyl-labeled amine-/phenol-containing metabolites using high-energy collisional dissociation (HCD) on quadrupole-Orbitrap MS, with a focus on MS/MS-based spectral elucidation to gain insights into the structure of dansyl-labeled metabolites.
  Overall, this contributes to the optimization and understanding of CIL LC-MS analysis on Q-Orbitrap MS for metabolomics, advancing the capabilities of metabolite detection and identification in complex biological samples.

• Subjects / Keywords

  • Chemical isotope labeling
  • LC-MS
  • Orbitrap
  • Metabolomics

• Graduation date

  Fall 2023

• Type of Item

  Thesis

• Degree

  Doctor of Philosophy

• DOI

  https://doi.org/10.7939/r3-ddy4-km97

• License

  This thesis is made available by the University of Alberta Libraries with permission of the copyright owner solely for non-commercial purposes. This thesis, or any portion thereof, may not otherwise be copied or reproduced without the written consent of the copyright owner, except to the extent permitted by Canadian copyright law.