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.

Collaborating Investigator/s

John Kao (University of Wisconsin-Madison)

Summary

The DBP provided a test bed for our DiLeu based metabolite tags.

Mass spectrometry-based stable isotope labeling has become a key technology for protein and small-molecule analyses. We developed a multiplexed quantification method for simultaneous proteomics and amine metabolomics analyses via nano reversed-phase liquid chromatography-tandem mass spectrometry (nanoRPLC-MS/MS), called mass defect-based N,N-dimethyl leucine (mdDiLeu) labeling. Paralleled proteomics and amine metabolomics analyses using mdDiLeu were systematically evaluated and then applied to pancreatic cancer cells.

The innovative outcome of this project was the isobaric labeling technologies for multiplexed metabolite quantification.

Collaborating Investigator/s

Wei Xu (University of Wisconsin-Madison)

Summary

Identification of CARM1 substrates in this project relied on the ability to confidently detect disappearance of methylation sites in CARM1 knockout cells, which could only be accomplished using highly reproducible multiplexed quantitative workflows. This project established novel methodology for direct global profiling of arginine methylation in vivo and refined enrichment and data generation practices necessary for effective analyses of this understudied PTM that could be easily reproduced in future studies.

Collaborating Investigator/s

Jon Odorico (University of Wisconsin-Madison)

Summary

The C-terminal domain of human CO1A1 provided a highly relevant platform to test ETD and its ability for hydroxyproline isomer detection. The project expanded the utility of ETD fragmentation to analyses of this important but understudied PTM.

Collaborating Investigator/s

Wei Xu (University of Wisconsin-Madison)

Summary

We leveraged our nascent ETD glycoproteome profiling technologies to study the glycosylation alterations in PKM2 KO cells vs. parental breast cancer cells. Being an early application of quantitative glycoproteomics using our ETD technologies, it has helped refine and develop our technologies.

Collaborating Investigator/s

Mark Burkard (University of Wisconsin-Madison)

Summary

The DBP necessitated very deep characterization of the phosphoproteome beyond the number of sites typically detected. The project prompted exploration of new phosphopeptide enrichment, fractionation, and data normalization approaches that are now in routine use.