Researchers from the University of Wisconsin-Madison have characterized the mitochondrial acetylome, finding significant changes in protein acetylation in response to calorie restriction and activity of the protein deacetylase SIRT3.
The study, which was detailed in a paper published last month in the online edition of Molecular Cell, offers new insights into acetylation's role in regulating the mitochondrial proteome and related metabolic processes, Josh Coon, a UW-Madison researcher and author on the paper, told ProteoMonitor.
The project also provides an example of the increasing ease and utility of large-scale quantitative proteomic experiments, and, in particular, the power of higher multiplexing, Coon added.
The researchers examined in biological triplicate the liver mitochondrial proteome in four different mouse types – wild type, wild type on a calorie-restricted diet, mice without SIRT3, and mice without SIRT3 on a calorie-restricted diet. Using isobaric tagging, immunoenrichment, two-dimensional chromatography, and mass spec analysis on a Thermo Scientific Orbitrap Elite, they quantified 3,285 acetylation sites – 2,193 of them from mitochondrial proteins – and calculated measurements of the relative acetyl occupancy across the four experimental conditions.
The data generated by the study suggests a number of questions for further investigation, Coon said.
"We noted large-scale dramatic changes to the acetylome when [mice] undergo calorie restriction or the knockout of SIRT3," he said. Calorie restriction has been observed to extend life spans in a number of species, and past research has indicated that SIRT3 could be a key player in this phenomenon.
"There are several key enzymes that were targets for acetylation that are specific to [calorie restriction or knockout of SIRT3] that are now being followed up on biologically," Coon said.
He added that beyond the potential mechanistic implications, the study was also notable for the amount of acetylation it observed.
"The mitochondria was known to be highly acetylated, but nobody has really reported that to the extent that we found," Coon said. "We found that there was at least one acetylation site on 60 percent of mitochondrial proteins."
Coon credited this expanded view of mitochondrial acetylation to his team's efforts to refine the immunoenrichment process and to the higher speed and sensitivity of the Orbitrap Elite compared to previous instruments.
"A lot of the depth you are seeing that people are getting nowadays compared to three or four years ago is in large part due to just having better instruments," he said.
Advanced instrumentation and reagents also improved the team's quantitative analysis, Coon noted. In particular, he cited the multiplexed isobaric tagging approach they used as key to providing confidence in the data they generated.
Isobaric labeling uses stable isotope tags attached to peptides of interest to enable relative or absolute quantitation of proteins via tandem mass spectrometry. Digested peptides are labeled with tags that fragment during MS2 to produce signals corresponding to the amount of peptide present in a sample.
These reagents are sold as tandem mass tags, or TMT, by Thermo Fisher Scientific through a licensing agreement with Proteome Sciences, and as isobaric tags for relative and absolute quantitation, or iTRAQ, by AB Sciex.
"We've been working a lot with isobaric tagging, so we're pretty good at getting that method to work well," Coon said. "And so we applied that approach to look at 12 animals — three experiments looking at four animals each."
"We got good overlap in the sites that we mapped, which meant that if we could map a site in all three experiments then we had it measured in 12 animals across four conditions," he said. "That meant we could calculate false discovery rates on whether the quantitative value we measured was significant or not. That was key in allowing us to identify the real important targets of either SIRT3 or calorie restriction."
The isobaric tags' multiplexing capabilities were key to achieving this high number of replicates, Coon said, noting that "if you had to do these experiments 12 times instead of just three, the labor, the instrument time, the cost, just gets really high and becomes unapproachable."
He added that he expects an "exponential growth" in the amount and quality of data that researchers will be able to generate from such experiments as reagents and instrumentation continue to improve.
"Five years ago we wouldn't have been able to touch this experiment," he said. "Three years ago it probably could have taken three months to do. And this year it took us a month to prepare and two weeks to run."
Coon added that he expected that in a few years researchers would be able to run the experiment on as many as 48 animals in the same amount of time.
"As these [isobaric] tags evolve to allow more multiplexing, you can perform very complex multiple variable quantitative [post-translation modification] specific experiments in a reasonably small time frame," he said.
The TMT method – which was used by Coon and his team in the Molecular Cell study – enables researchers to multiplex up to six samples, while AB Sciex's iTRAQ method can multiplex eight.
According to John Rogers, manager of MS reagents at Thermo Fisher, the company has recently developed TMT tags capable of multiplexing up to 10 samples. It plans to begin offering the 10-plex tags as a product through its custom reagents division by May or June of next year, he told ProteoMonitor, and then as a standard catalogue product sometime after that.
The new tags require the high resolution of more recent mass spec releases, Rogers said, noting that initially company researchers assumed they would be suitable for use only on the Orbitrap Elite, the current top-of-the-line model. However, he said, as they have shared them with other collaborators, they have found they also perform well with slightly older models like the Orbitrap Velos.