Development of Chemical Isotope Labeling (CIL) LC-MS Techniques for Untargeted Analysis Milk Metabolomics and Targeted Analysis of Propionic Acid

• Author / Creator

  Mung, Dorothea, YZ

• Metabolomics is the study of all the small molecules in a given sample or organism. It is used to understand individual variations caused by the relationship between the individual’s genetics, exposome and metabolic activity. There is interest in using metabolomics for personalized care leading to the merging of metabolomics with other fields such as nutritional sciences. These nutrimetabonomic studies focus on the individual’s metabolic response to a food or diet. As a note, metabolomics aims to study the effects of a stimuli on a metabolome while metabolomics involves metabolome fingerprinting of various samples. They are considered interchangeable with the more common use of the word metabolomics to describe most studies. Metabolomics can be used in a targeted or non-targeted manner. For non-targeted metabolomics, the entire set of metabolites is of interest. However, the metabolome is complex containing metabolites with various chemical properties. Profiling the metabolome with one analytical platform is impossible. However, the metabolome can be grouped by similar chemical properties (submetabolomes). Chemical isotope labeling (CIL) can be used to target submetabolomes by addition of an isotope tag to improve separation, ionization efficiency and provide the ability for relative or absolute quantification. In this way, the entire metabolome can be comprehensively studied using one or few analytical platforms such as positive mode reverse phase liquid chromatography mass spectrometry (RPLC-MS). In this work, I used CIL LC-MS to profile the amine and phenol submetabolome of cow milk. Milk is one of the main food groups in North America and is consumed multiple times a day. I first determined a sample cleanup protocol involving protein precipitation with methanol and lipid removal with dichloromethane. Dansylation labeling was utilized to target the amine and phenol submetabolome of cow milk. The differences between brands, batches and fat percentages of cow milk was investigated. Once the metabolome for cow milk was determined, the short-term effects of cow milk consumption on the human urine metabolome was investigated. Each individual was analyzed independently due to large inter-individual differences that masks metabolome changes when the volunteers were analyzed together. By comparing the urine dataset with the previously determined cow metabolome, subtle changes to the urine metabolome after milk consumption were able to be detected with confidence. The ability of metabolomics to generate metabolome fingerprints can also be useful for quality control or product authentication purposes. Human milk is known as the best source of nutrition for an infant. However, not all mothers are able to produce milk. Human milk sharing has become popular due to easy access and low cost of online purchasing. With the high demand, there is potential for human milk adulteration via dilution with foreign milk in order to increase profits. I used CIL RPLC-MS to profile the metabolome of human milk along with 5 potential human milk adulterants (soy, goat, cow, almond, infant formula). Once the metabolome fingerprints were obtained, the human milk was investigated with varying levels of adulteration (5%, 10%, 25%, 50% and 75%). Overall, I was able to detect as low as 5% adulteration of human milk with foreign milk When utilizing metabolomics in a targeted manner, a particular metabolite (i.e. propionic acid) or class of metabolites (i.e. short chain fatty acids) are of interest. Absolute quantification can be done for targeted metabolomics of one or a few metabolites if their respective standards are available. In this work, I used p-dimethyaminoacyl (DmPA) bromide to target and absolutely quantify propionic acid. The labeling method was optimized and a RPLC-MS method was tailored for the analysis of propionic acid.

• Subjects / Keywords

  • Metabolomics
  • Chemical Isotope Labeling
  • Dansylation

• Graduation date

  Fall 2017

• Type of Item

  Thesis

• Degree

  Master of Science

• DOI

  https://doi.org/10.7939/R39S1M23R

• 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.