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DTU Team Testing Orbitrap Fusion's Phosphoproteomic Capabilities as Part of Thermo Collaboration

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Thermo Fisher Scientific and the Department of Systems Biology at the Technical University of Denmark last week announced a collaboration on technology and application development related to research into cellular protein signaling.

As part of the collaboration, Thermo Fisher has placed instrumentation including an Orbitrap Fusion Tribrid mass spectrometer into a new network biology lab headed by DTU researcher Rune Linding.

Launched at the American Society of Mass Spectrometry annual meeting in June, the Fusion combines in one device a quadrupole for precursor selection with both an Orbitrap and ion trap mass analyzer (PM 6/14/2013).

Offering significant improvements over past Orbitrap models in terms of resolution and scan speed, the instrument has received plaudits from some advanced-access users for enabling dramatic increases in multiplexing capabilities and peptide IDs. Linding told ProteoMonitor this week that his lab in its early use of the machine has seen similar benefits, citing in particular its suitability for phosphoproteomics work.

Specifically, Linding said, the improved ease and performance of the Fusion's Easy-ETD ion source has allowed his team to considerably advance their localization of protein phosphorylation sites identified via mass spec analyses. Such localization data, he noted, is key to modeling protein signaling networks, and, he said, the inability to obtain good localization data has in the past significantly hampered their work in this area.

"Probably around 20 percent to 40 percent of the [phosphoproteomic] data we generate typically gets lost because we can't localize the phosphorylation sites," he said. "We're basically just losing data due to the lack of localization [data]."

His lab's early work with the Fusion indicates that the instrument may help improve this situation, Linding said.

The ETD [function] seems to work very nicely, which was not really the case with the older generation [Orbitrap] instruments," he said. "We have preliminary data that suggests that [the Fusion's ETD capability] is solving a lot of those [phosphosite localization] issues, and we now have much better data for calling which [amino acid] residues are modified."

Beyond the matter of phosphosite localization, Linding said his lab is also interested in using the machine for intact protein analysis and analysis of protein complexes. In particular, he said, "we have a big project on kinases ongoing where we want to look at the signaling complexes, and this instrument will allow us to do that much better than the previous generations [of Orbitrap]."

The instrument's improved sensitivity will likely also let the researchers use smaller samples for their proteomic analyses – a key consideration for work with clinical samples in diseases like cancer where samples availability is often limited.

Using the Fusion "we are able to look at very tiny tumors – tumors that are only a few weeks old in a mouse model, for instance," he said. "So really a very small amount of tissue. We will need to see how it goes over the next few months once we have been through our pilot experiments to see how far we can actually go with the instrument ... but so far we are very impressed by the results."

Linding said that compared to the Orbitrap Velos, which his lab previously used, the Fusion thus far appears much more stable and robust, although, he added, it remained to be seen whether this apparent improvement would hold up over time. He cited his experience with Thermo Fisher's Q Exactive instrument, noting that, while his team has been happy with the instrument, its quadrupole requires more frequent cleaning than they had expected.

"These are things that you only learn once you start using a new instrument intensely," he said. "So we will see."

The new DTU network biology lab has four Q Exactives in addition to the new Fusion. While his group's research has in recent years focused primarily on deep proteome-wide profiling, Linding said that they are now beginning to move into more targeted methods, using the Q Exactives for selected-reaction monitoring-style assays as well as data independent acquisition workflows.

His lab recently hired a student specializing in DIA mass spec from the group of Swiss Federal Institute of Technology Zurich researcher Ruedi Aebersold. While Aebersold's lab has done most of its DIA work on AB Sciex's TripleTOF 5600 platform, Linder said that he plans for the time being to continue using the Q Exactive for such experiments.

"If there is a new instrument from [AB] Sciex then of course we will look at it, but so far we have been pretty happy with the Q Exactive," he said. The addition of the new researcher's DIA expertise will allow the DTU team "to get a better feel for where we are [with their Q Exactive work] in comparison to the [TripleTOF 5600]," he added.

Linding and his colleagues will also likely consider obtaining a new triple quadrupole platform as they continue to move into more targeted proteomics work, he said.

The core area of Linding's research is what he calls "biological forecasting" – essentially an effort to predict cell behavior as a function of molecular networks. On the clinical side, his team is focused primarily on cancer and metastatic progression, attempting to understand the signaling networks that drive metastatic processes. They have also begun to investigate diabetes and plan to start research into conditions like depression for which "almost nothing is known about the networks involved," he said.

The researchers are undertaking these efforts in collaboration with a number of other labs within DTU's Center for Biological Sequence Analysis and Department of Systems Biology, as well as several outside research groups throughout the US and UK, Linding said. In addition to mass spec-based proteomics, they are applying approaches like single cell mass spectrometry -- using DVS Sciences' CyTOF mass cytometer – as well as metabolomics, robotic imaging, and phenotypic screening.

The lab will also benefit from CBSA's extensive supercomputing resources, Linding said, noting that DTU's strength in this area was perhaps one incentive driving the collaboration from Thermo Fisher's end. "We have very strong software and algorithm development, and there are not that many [academic] labs that can give them feedback on that side of things," he said.

The network biology lab will be housed in a new building being constructed by DTU to house its proteomics and high-throughput biotechnology resources, Linding said. Funding for the mass spec instrumentation has come from the university and outside private foundations as well as research grants.

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