Collaborating Investigator/s

David Pagliarini (The Morgridge Institute for Research)

Summary

This project necessitated discovery lipidomics analyses for profiling of global lipid changes.

The biosynthesis of coenzyme Q presents a paradigm for how cells surmount hydrophobic barriers in lipid biology. Here, we reveal that this process relies on custom lipid-binding properties of COQ9. Overall, our results provide a mechanism for how a lipid-binding protein might access, select, and deliver specific cargo from a membrane to promote biosynthesis.

The collected discovery lipidomics data of this project served to advance LipiDex, the Center’s discovery lipidomics software tool, and enhanced its utility for analysis of a different organismal lipidome that could be repeated in the future projects.

Collaborating Investigator/s

Johan Auwerx (Ecole Polytechnique Fédérale de Lausanne)

Summary

To enable QTL mapping, an outstandingly large mouse cohort had to be characterized via quantitative lipidomics. The project’s focus on mitochondrial function required through characterization of low-abundance mitochondria-specific lipid species, such as cardiolipins, which was carried out in plasma to generate the most holistic view of the organism.

This project was a driver in the development of a high throughput quantitative lipidomics platform that was later built upon for the use in other studies.

Collaborating Investigator/s

Marv Wickens (University of Wisconsin-Madison)

Summary

This discovery-driven DBP required through and unbiased exploration of the relatively large knockout collection via multi-omic methodology.

The challenges of exploring this large multi-omic dataset propelled development of our web-based multi-omic data exploration and visualization approach that later culminated in the release of MS-Portal software, now customarily used for all multi-omic projects.

Collaborating Investigator/s

Alan Attie (University of Wisconsin-Madison)

Summary

The project extended the use of the Center’s high throughput label-free proteomics technology to novel and challenging tissue type, pancreatic islets, which required dramatically modifying sample preparation and handling procedures.

The developed sample preparation and handling procedures became a part of our streamlined and versatile methodology for high throughput label-free proteomics, now compatible with most sample types.

Collaborating Investigator/s

Johan Auwerx (Ecole Polytechnique Fédérale de Lausanne)

Summary

To enable QTL mapping, an outstandingly large mouse cohort had to be characterized via quantitative lipidomics. The project’s focus on mitochondrial function required through characterization of low-abundance mitochondria-specific lipid species, such as cardiolipins, in the most metabolically active tissue – liver.

This project drove development of the high throughput quantitative lipidomics platform that was later built upon for the use in other studies.

Collaborating Investigator/s

Dave Pagliarini (The Morgridge Institute for Research)

Summary

In-depth characterization of the large yeast knockout collection required holistic assessment of the knockout phenotypes via muti-omic methodology.

This project extended the use of our multi-omic approach to a new organism, Saccharomyces cerevisiae, and challenged it by increasing the demands for the number of samples analyzed, all of which required adopting our sample preparation and quantitation methodologies to rapidly and reproducibly profile many unique lipidomes, metabolomes, and proteomes.

Collaborating Investigator/s

Thomas Raife (University of Wisconsin-Madison)

Summary

The study necessitated examination of red blood cells whose contents are notoriously intractable for proteomic analysis.

Each year over 90 million units of blood are transfused worldwide, calling for optimized blood management and storage. During storage, red blood cells undergo degenerative processes resulting in altered metabolic characteristics which may make blood less viable for transfusion. However, not all stored blood spoils at the same rate.

We conclude that individuals can inherit a phenotype composed of higher or lower concentrations of proteins that can result in vastly different red blood cells storage profiles which may need to be considered to develop precise and individualized storage options. Beyond guiding proper blood storage, this intimate link in heritability between energy and redox metabolism pathways may someday prove useful in determining the predisposition of an individual toward metabolic diseases.

This work extended the use of our label-free quantitative methodology to this particular challenging sample type, permitting similar studies in the future.

Collaborating Investigator/s

Henrik Zetterberg (University of Gothenburg)

Summary

This project provided a real-world test bed for our emergent 5-plex isotopic N,N-dimethyl leucine (iDiLeu) technology that enables construction of a four-point internal calibration curve to determine the absolute amounts of target analytes.

The project successfully demonstrated the great utility of DiLeu tags for absolute quantitation of protein amounts in the most difficult sample types – clinical samples, paving the way for future uses of the reagent in other less complex situations.

Collaborating Investigator/s

William Ricke (University of Wisconsin-Madison)

Summary

The project required detection of protein changes in urine – a protein-poor sample type that is precious and difficult to obtain in in sufficient quantities.

DiLeu tagging allowed to pool samples from multiple animals, thus decreasing the amount of protein and urine needed to be obtained from any single animal and simplifying the sample collection. This strategy for reduction of required sample amounts could be used in the future studies using even more challenging and precious samples.

Collaborating Investigator/s

Luigi Puglielli (University of Wisconsin-Madison)

Summary

The project propelled the use of DiLeu quantitative technology, analyzed acetylated proteins, and measured metabolite flux.

AT-1/SLC33A1 is a key member of the endoplasmic reticulum (ER) acetylation machinery, transporting acetyl-CoA from the cytosol into the ER lumen where acetyl-CoA serves as the acetyl-group donor for Nε-lysine acetylation. Dysfunctional ER acetylation has been linked to both developmental and degenerative diseases. Collectively, our results suggest that AT-1 acts as an important metabolic regulator that maintains acetyl-CoA homeostasis by promoting functional crosstalk between different intracellular organelles.