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.

Collaborating Investigator/s

Wei Xu (University of Wisconsin-Madison)

Summary

The project required detection and quantification of a rare and low-abundance post-translational modification – arginine methylation. Here we established a methodology for indirect detection and quantification of arginine methylation using DiLeu tags, extending the utility of this quantitative technology to analyses of this rare PTM that could be repeated in the future.

Collaborating Investigator/s

David Pagliarini (University of Wisconsin-Madison)

Summary

Mitochondrial phosphorylation is abundant and dynamic, yet its regulation and contribution to organellar function is poorly understood. To address this, we coupled quantitative phosphoproteomics with classic biochemistry to study the mitochondrial phosphatase Ptc7p. We found that Ptc7p deletion perturbs mitochondrial phosphorylation, partially inactivates citrate synthase, and decreases organellar function.

Collaborating Investigator/s

Subhojit Roy (University of Wisconsin-Madison)

Summary

To reveal proteomic differences between various brain regions, especially deep characterization of tissues was required that could not be achieved using standard single-shot label-free proteomics method. The methodology used in the project for the first-time combined label-free quantification and extensive peptide fractionation demonstrating that highly reproducible quantification of over 10K proteins in human tissues could be achieved this way. This combination of techniques could be used in the future when particularly in-depth characterization of samples is desired.

Collaborating Investigator/s

Jim Wells (University of California, San Diego)

 Summary

The effectiveness of the engineered kinase could only be accessed via quantitative phosphoproteomics that required highly reproducible measurements over a large dynamic range. This application of the NeuCode technology expanded its use beyond the whole proteome characterization to characterization of post-translational modifications.

Collaborating Investigator/s

Donald Kirkpatrick and Anwesha Dey (Genentech, South San Francisco, CA)

Summary

In this project we introduced neutron-encoded (NeuCode) amino acid labeling of mice as a strategy for multiplexed proteomic analysis in vivo. Using NeuCode, we characterized an inducible knockout mouse model of Bap1, a tumor suppressor and deubiquitinase whose in vivo roles outside of cancer are not well established. NeuCode proteomics revealed altered metabolic pathways following Bap1 deletion, including profound elevation of cholesterol biosynthetic machinery coincident with reduced expression of gluconeogenic and lipid homeostasis proteins in liver. Bap1 loss increased pancreatitis biomarkers and reduced expression of mitochondrial proteins. These alterations accompany a metabolic remodeling with hypoglycemia, hypercholesterolemia, hepatic lipid loss, and acinar cell degeneration. Liver-specific Bap1 null mice present with fully penetrant perinatal lethality, severe hypoglycemia, and hepatic lipid deficiency. This work revealed Bap1 as a metabolic regulator in liver and pancreas, and it established NeuCode as a reliable proteomic method for decip hering in vivo biology.