Cathy Mercado
Faculty Advisor: Aleksandra Skirycz
Year: 2021

“omniTICC: cell-wide untargeted protein-metabolite interaction mapping using ion exchange chromatography”

Project Summary:

Metabolites are small molecular intermediates or end products of metabolism, playing key roles in cells as energy sources, building blocks, but also signals and regulators. To exert their many functions, metabolites bind to proteins. Although there are many known protein-metabolite interactions (PMIs) it has become increasingly clear that these constitute just a tip of an iceberg.  Advances in mass spectrometry-based metabolomics and proteomics have allowed for the development of many different biochemical approaches to look for novel protein-metabolite interactions (PMIs). Co-fractionation mass spectrometry encompasses different strategies for untargeted, metabolome and proteome-wide characterization of the protein-metabolite interactome.  The aim of this work was to test suitability of the ion exchange chromatography to separate protein-metabolite complexes from free metabolites. The method was dubbed omniTICC from the Omni-target Identification by Chromatographic Co-elution. omniTICC method is based on a different charge of metabolites versus protein-metabolite complexes. Each experiment entails two samples. Protein-free metabolite sample (control) and cellular lysate sample (sample). All collected IEX fractions undergo metabolite and protein extraction, to be further analyzed with LC-MS. Obtained protein and metabolite intensities are used to plot elution profiles. Differential elution measured between control and sample points to a PMIs.  Moreover, Escherichia coli was used because it can be compared to the already known PMI dataset as a proof of concept. The results of the metabolite analysis indicate the validity of this method, with a difference in many metabolites e.g. tyrosine, deoxyinsosine, and leucylmethionine in the control to the sample. Progress on this project is on-going and collaborations to use other organisms, such as C. elegans, are in the works. A map network of possible PMIs provides valuable information for future studies.

My Experience:

This past summer I was able to participate in-person on a project applying biochemistry techniques to formulate an unbiased, system wide detection method to look for protein-metabolite interactions. In those ten weeks, I employed biochemistry techniques such as ion exchange chromatography with FPLC, Bradford assays, metabolite and protein extractions, and assisted with LC-MS, to name a few. Under the mentorship of the Skirycz lab, I was able to grow as a researcher by broadening my outlook to a research area I was not familiar with. Additionally, this program offered a look at the different research at the institute and allowed me to view the different career paths I can follow after pursing graduate school. My goal is to take the information I was given to apply for graduate schools that will give me the opportunity to learn new techniques as a grow as a scientist.