Multi-omic Mass Spectrometric profiling of a large CRISPR knockout collection elucidates functions of uncharacterized mitochondrial proteins and their role in human disease
Monday, November 4, 2019 - 4:15pm
Icahn 101
Quantitative & Computational Biology

Abstract: Proteomics and other omic approaches constitute a powerful and versatile toolkit for probing biological systems. Encompassing many layers of biological information, multi-omic data holistically describe a living system and its response to perturbations. Mitochondria – central hubs of cellular metabolism – are particularly amiable to multi-omic profiling, as myriad metabolites, lipids, and proteins co-function in these organelles and jointly orchestrate responses to various changes. Despite the recent surge in mitochondria- and metabolism-focused research, numerous mitochondrial processes and their key players remain poorly understood, as are their contributions to human health and disease. To shed new light on this essential organelle, we acquired a custom collection of >200 human cell lines that contained 116 unique CRISPR-Cas9 single-gene knockouts of functionally characterized (“sentinel”) and uncharacterized mitochondrial genes. “Sentinel” proteins were selected to include members of primary mitochondrial pathways (e.g., citric acid cycle, fatty and amino acid metabolism, oxidative phosphorylation, etc.) and to prominently feature proteins with known human disease associations. Using shotgun proteomics, metabolomics, and lipidomics, we performed multi-omic profiling of the cell lines in biological triplicate, collecting over 1,500 LC or GC-MS/MS experiments, 10,000,000 tandem mass spectra. Altogether we consistently monitor the abundances of >11,000 diverse biomolecules in each of the 166 cell lines. These data To facilitate visualization and exploration of the wealth of collected data, we created a custom web-based portal that performed bioinformatic analyses and on demand generated common graph types, e.g., volcano plots, bar charts, and correlation profiles. The remarkable size, depth, and high quality of the dataset afforded a comprehensive view of cellular responses to mitochondrial gene disruptions, permitting correlation of molecular perturbations across numerous cell lines in search of commonalities between uncharacterized and “sentinel” proteins. Thus far, capitalizing on multiple data types, we have designated a protein as a new member of co-factor Q biosynthetic pathway, placed another at the mitochondria-peroxisome interface, and identified a new player in the formation of the MICOS complex. These early findings showcase the tremendous potential for discovery our data and multi-omic profiling in general hold. 

About Dr. Coon:

Josh Coon

"I grew up in rural Michigan and during these formative years greatly enjoyed flyfishing and woodworking. Putting the latter interest to practical use, I constructed several riverboats (for fishing) while in high school and college.  Chemistry interested me, especially Analytical Chemistry, as it offered an avenue to continue “building”.  Not boats, but chemical instrumentation. To escape the cold I joined the Chemistry graduate program at the University of Florida and worked with Willard Harrison. Professor Harrison didn’t just guide my research, he taught me how to write, present, and think like a scientist. He was a gentleman in every sense of the word. Upon graduation in 2002, I moved to Charlottesville, Virginia to join the laboratory of Professor Don Hunt. At Virginia I met John Syka.  Don and John both shared a passion for science that was as infectious as it was inspiring.  Together we worked to develop electron transfer dissociation (ETD).  ETD worked just as we had hoped and the dissociation technique is now commonly used for proteomics and has been commercially introduced by no fewer than four major instrument vendors.  In 2005 I moved to Wisconsin to start my own program. And though we have been productive and impactful with ~ 250 published manuscripts, I am most proud to have produced 25 Ph.D. scientists, and our academic family continues to grow. The overarching mission of my program is to (1) facilitate expedient, comprehensive analysis of proteins and metabolites by innovating new mass spectrometric technologies and (2) apply these techniques to advance biomedical research." - Josh Coon

Link to Coon Labs website: https://coonlabs.com/