Development of LC-MS Based Metabolomics and Lipidomics Techniques

• Author / Creator

  Tatlay, Jaspaul

• The analysis of thousands of metabolites and lipids for discovery of metabolite biomarkers for disease monitoring and diagnosis requires the highest sensitivity and robust methods. Metabolomics is the study of metabolites found in the body, which can be used to discover biomarkers of disease or monitor interactions in our body. In my thesis, I developed liquid chromatography mass spectrometry (LC-MS) methods that analyze small volumes of samples for metabolomics and profile the changes in lipids in serum samples collected from Parkinson’s disease (PD) patients and healthy controls to find PD biomarkers. Separation techniques have improved the field of metabolomics and lipidomics by increasing sensitivity, throughput, and resolution. In particular, the use of nanoliter flow liquid chromatography (nLC) and ultra-high performance liquid chromatography (UHPLC) have shown great promise. nLC takes the regular flow of the LC and reduces it to micro- or nano liter flow. With the decreased flow, the column size and connections need to be reduced as well. The advancements of these technologies have increased the coverage of the metabolome and lipidome. I used nLC-MS to improve the overall analytical performance of metabolomic profiling for handling of samples with small volumes. I first optimized nLC-MS for high performance chemical isotope labeling (CIL) metabolomics. CIL is a method used to chemically label metabolites to increase their retention on reversed phase (RP) separation, to improve ionization efficiency, and to allow for relative quantification. CIL is a sensitive technique, although when sample volumes are limited or the concentration of samples is low, the use of nLC is needed. nLC is used to increase coverage of the CIL metabolome and reduce sample consumption through the use of analyte trapping. iii Recent advancements in mass spectrometry, specifically in quadrupole time-of-flight (QTOF) instruments, have allowed for higher resolution, faster collection rates and improved sensitivity. This new generation of instrumentation allows us to examine the metabolome and lipidome further. In lipidomics, LC-MS is used to analyze thousands of lipids to get a better understanding of the lipidome and their roles in diseases. We used an ultra-high resolution quadrupole time of flight (UHR-QTOF) instrument connected to UHPLC to profile PD samples to identify potential biomarkers. Ultra-high resolution allows for higher mass precision and increased confidence in identification. Using statistical analyses we determined five lipids to distinguish the diseased samples from the controls. In receiver operating characteristic (ROC) analysis, these potential biomarkers had an area under the curve (AUC) of 0.976 with sensitivity of 92% and specificity of 90%. The same panel of five lipids plus another compound were used to distinguish PDD from PD. ROC analysis of the 6 compounds gave an AUC of 0.958 with sensitivity of 87% and specificity of 94%. We also observed an increasing trend of the 5 common lipids in concentration, suggesting the potential of using these biomarkers for not only diagnosis of PD, but also tracking PD progression into PDD.

• Subjects / Keywords

  • Metabolomics
  • Lipidomics
  • Mass Spectrometry
  • UHPLC
  • Liquid Chromatography
  • Dansylation

• Graduation date

  Spring 2016

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/R3GX45980

• 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.