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Proteomic Study Suggests Greatly Expanded Role for Phosphorylation in Yeast Central Metabolism

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Researchers at the Swiss Federal Institute of Technology have completed a phosphoproteomic study of yeast central metabolism that provides evidence of a significantly larger role for protein phosphorylation than previously known.

The study, detailed in a paper published this week in Molecular Systems Biology, examined phosphorylation levels in the 204 enzymes involved in yeast central carbon and amino acid metabolism, newly identifying five of these proteins as phosphoregulated and bringing to 14 the number of known phosphoenzymes in these networks.

The results greatly expand the role that phosphorylation is thought to play in regulating yeast metabolism, Uwe Sauer, an ETH Zurich research and author on the paper, told ProteoMonitor. He suggested as well that the study was a good example of proteomics' ongoing evolution from a discipline focused on basic cataloging of proteins to a tool for exploring functional and biological questions.

The project, Sauer said, was largely inspired by the observation that many observed metabolic responses are poorly explained by transcriptional regulation, suggesting that post-transcriptional processes like post-translational modifications likely played a key role.

"We come from a metabolism and functional angle," he said, noting that the project was a collaboration between him and co-author and ETH Zurich colleague Ruedi Aebersold, who performed the proteomic and phosphoproteomic analyses. "And in my lab we have done plenty of transcript analyses, but they don't seem to indicate that transcriptional regulation is very important in modulating the activity of these networks. So that was the reason we thought phosphorylation might be one of the mechanisms."

To investigate the role of protein phosphorylation, the researchers quantified total protein levels and phosphorylation levels for each of the 204 enzymes involved across five different nutritional conditions, correlating this proteomic data with metabolic flux data.

The nutritional conditions investigated, Sauer said, were canonical states widely used in metabolic research: minimal media cultures grown either aerobically in glucose, galactose, or ethanol; a minimal media culture grown anaerobically in glucose; and a complex media culture grown in yeast extract peptone.

These states, the authors wrote, "cover different modes of metabolic operation – from glycolysis to gluconeogenesis, from fermentative and respire-fermentative to fully respiratory."

The researchers quantified the proteins by mass spec, using selected-reaction monitoring on a Thermo Fisher Scientific TSQ Vantage to measure total protein expression and shotgun proteomics on a Thermo Scientific hybrid LTQ-FT and LTQ Orbitrap to quantify phosphorylation levels.

Having good data for both measurements was crucial, Sauer noted, as it allowed the researchers to determine changes in phosphorylation levels in the context of protein expression changes. "If you don't know how much protein is there, just knowing the phosphorylation changes isn't enough information," he said. "Under many conditions the protein abundance changes dramatically… and if the protein is going up [a corresponding rise in phosphorylation] doesn't necessarily mean anything. So we needed [both pieces of data] … otherwise we would have been able to get a quantitative assessment of the degree of phosphorylation."

Sauer added that in the future the study might be improved upon by using the more quantitative SRM-MS technique not only for measuring total protein levels but for the phosphoproteomic measurements, as well.

"It would be beautiful if we could do the phosphoproteomics with [SRM] mass spectrometry," he said. "That would make a huge difference. It would make the [results] even better."

Even with the combination of SRM and shotgun data, however, the researchers were able to identify as phosphoregulated five enzymes not previously known to be so. This ups the total number of enzymes in this pathway known to be phosphoregulated from nine to 14.

Of these five, the researchers validated their findings on three enzymes – the proteins Pda1, Pfk2, and Gpd1 – via targeted mass spec, metabolomics, and physiological flux analysis using mutants lacking the relevant phosphosites.

Although most of the phosphosites involved had been previously identified, their functional importance had not been established, Sauer said.

In the case of Gpd1, for instance, "people believed for quite some time that it was transcriptionally regulated, and a lot of modeling had been done assuming it was transcriptionally regulated," he said.

In the case of Pfk2, he added, "no one had ever even seen that it was phosphorylated" before.

Sauer said that the project demonstrated proteomics' growing role in hypothesis-driven research, noting that "what both [Aebersold] and I were hoping for was that we would go beyond [the protein] cataloguing" that much proteomics work has traditionally focused on.

"These catalogues are very important, and they help us navigate," he said. "But it is very hard to generate hypotheses [from them]."

The "critical element" for such research is an experimental structure that relates quantitative proteomics data to some measure of biological function, Sauer said. "And in this particular case we have a way to quantify function in a metabolic network by what we call flux. By relating this flux function to the abundance of phosphor-enzyme species, we can start generating hypotheses."

He observed that as more and more of the technical challenges that once faced proteomics are resolved, questions of "functional relevance are more and more important."

One recent and high-profile sign of the shift in this direction was an editorial published this month in the Journal of Proteomics announcing that the journal was changing its standards to require that the studies it publishes include the "formulation of an adequate testable biological hypothesis."

"Although high-quality research on non-model organisms, as well as papers describing significant technical advances in biological mass spectrometry and proteomics, will continue to be welcome in the pages of Journal of Proteomics, this journal will implement in the Instructions for Authors the requirement for original papers to include a paragraph describing how the reported methodology or proteomics findings significantly advance, respectively, the field and the understanding of the biological process investigated," wrote Juan Calvete, the journal's editor-in-chief.

"Not everything is perfect, but [proteomics] is really strong these days in what it can do," Sauer said. "So this [push toward functional relevance] is clearly moving forward."

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