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A trio of scientists at the University of Wisconsin-Madison is launching a new research center to advance mass spectrometry-based proteomics in biomedical research, with the help of a grant from the National Institutes of Health and the National Institute of General Medical Sciences.

Under the five-year, $6 million P41 grant, Josh Coon, David Pagliarini, and Lingjun Li have started the National Center for Quantitative Biology of Complex Systems (NCQBCS), based at UW.

“The center aims to increase the pace and throughput of quantitative proteomics to rival that of genomic sciences,” Pagliarini told GenomeWeb. “Measuring proteins and their modifications brings you one step closer to the actual biochemistry. There are plenty of cases where understanding the genomics and transciptomics don’t get you there, and having protein-level data gets you closer to the biological problems actually being studied.”

Using mass spectrometry technology developed by Coon and Li and insight on post-translational modifications and their role in metabolism and cellular signaling from Pagliarini, the researchers will pursue projects that will push high-throughput proteomics technology development and address novel biological questions in a bidirectional manner. The center has already established a slate of starter projects with scientists at UW, other universities, and even Genentech.

“The idea is we develop these technologies in the context of these biomedical drivers,” Coon said. “The technology development is accelerated, the biology is accelerated. There’s a push and pull between the problems and the technologies.”

It’s an idea central to the P41 type of grant funding the center, Coon said. There are about 40 such grants funding development centers for a range of technologies, including mass spec, nuclear magnetic resonance, and crystallography. The NCQBCS will join a handful of other mass spec-focused centers. “Our tech is focused on building technology for faster, thorough proteome analysis,” Coon said. “We want to do comparative proteomics on a fast timescale and very deeply.”

With Coon, Li, and Pagliarini all working in Madison, Wisconsin, the germ for the idea to build a center like NCQBCS came out of their mutual collaboration and a realization that they could be doing more.

Coon and Pagliarini had previously collaborated on a number of quantitative proteomics projects involving mitochondria, Pagliarini’s area of expertise.

An early project that informs a lot of the research NCQBCS wants to do is a study, published in Cell Metabolism in 2012, of quantitative proteome mapping to look at mitochondrial proteins and their post- translational modifications. “Having spatial resolution and having info about modification is something you can’t get from [protein] sequence data alone,” Pagliarini, who added that the study relied on the kind of chemical tagging technology for quantitative proteomics Li is developing.

“[Li’s research] was synergistic to quantitative labeling strategies I had been pursuing,” Coon said. “At some point we realized what we were doing is scalable and already comprised a large piece of what a grant like this would look like,” he said. “We were already on this pathway and we believe that one of the best ways to accelerate this was to put it together in this center proposal.”

Li’s tags will be at the core of the technology development side at NCQBCS. Similar to isobaric tags for relative and absolute quantification (iTraq) or tandem mass tags sold by several companies such as Sciex, Thermo Fisher Scientific, Applied Biosystems, and Sigma Aldrich, Li’s technology involves chemical tags that can serve as barcodes for samples, enabling multiplexing far beyond what iTraq or TMT can offer. Thermo Fisher offers TMT kits with up to 10-plex capability and Sigma Aldrich offers eight-plex iTraq kits.

“We’re trying to bring this multiplexing tech to next level, where we combine it with a new chemical tags design and advancement of high-resolution instrumentation like [Thermo Fisher’s] Orbitrap,” Li said. “We’re look at 21- or 39-plex, and that can actually be increased further using some isotopic variants.”

With 39-plex, the researchers could eliminate the overlapping problem, where each sample’s proteins don’t overlap with successive detections and thus yield few proteins to actually compare. “There’s a stochasticity to what proteins are detected in any given experiment,” Coon said. “We could potentially compare proteomes from 40 mice in a single experiment. If you do 40-in-one, you have all those proteins from all 40 samples.”

It’s this kind of technology advance that will, ideally, yield new biological insights. One of NCQBCS’ collaborators is Ralph DeBerardinis of the University of Texas – Southwestern Medical Center at Dallas. Like many of the proposed projects, his involves metabolism and post-translational modifications. Specifically, he’s interested in whether those kinds of modifications are responsible for the metabolic changes seen in many types of cancer cells.

“We have a tremendous amount of information about all the metabolic variability of different cells or tumors, but to a large extent, we don’t understand why they behave the way they do,” DeBerardinis said. “We hypothesize that post-translational modifications will explain a lot of this.”

Histones are not the only proteins that are acetylized, phosphorylated, or methylated. Many other proteins receive these post-translational modifications, which might make an enzyme in a tumor more or less active.

“Our first challenge is to characterize the changes and figure out how those pathways are reprogrammed,” DeBerardinis said. “There are lots of proteomics labs that can characterize proteomes once you know what you’re looking for. What’s unique about Josh [Coon] is he can cast a wide net and help figure out which modifications affect metabolic pathways that might be beneficial to the tumor cell. “Their ability to cast a wide net over the changes in protein function and protein abundance is incredibly powerful,” he said. “If we know two cell lines are different, they can quickly return to you abundances of metabolic enzymes and post-translational modifications. Their high-throughput, unbiased technology really enables this type of work.”

That coverage not only provides loads of data on a metabolic pathway of interest, but also “accessory” data, which can lead to new questions and new projects.

“You just don’t know what you’re going to get, DeBerardinis said. “In 2016, now the best experiment you can design will help you test your hypothesis but also gives you accessory information.”

DeBerardinis’ project is just one of the10 so-called “driving biological projects” that will push Coon and his colleagues to improve their technology.

And even if they’re successful, their work won’t be done yet. The last part of the grant provides them money to bring in scientists from other institutions to learn the new methods and disseminate them.

“If you’re going to build this center, NIH wants to know who is going to benefit,” Pagliarini. “It has to have a big footprint and a larger reach. We have the skill sets to expand the scale of the technology and we know how to reach people,” he said.

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