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ETH Zurich, University of Toronto Studies Apply SWATH Mass Spec to Protein Interaction Research

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A pair of studies published this week in Nature Methods demonstrate the potential utility of data independent acquisition mass spec for protein interaction research.

The studies, one led by researchers at the University of Toronto and the other by a team from the Swiss Federal Institute of Technology Zurich, combined affinity purification with the SWATH DIA technique on an AB Sciex TripleTOF 5600 to investigate changes in protein-protein interactions in response to various stimuli.

Their results suggest that DIA mass spec methods like SWATH offer the ability to track changes in protein complexes across a large number of proteins and with improved dynamic range and quantitative accuracy compared to traditional data dependent acquisition, or shotgun, mass spec approaches, ETH Zurich researcher Ruedi Aebersold, an author on both papers, told ProteoMonitor.

"The general idea that both papers are pursuing is that with our [protein] interaction measurements, we need to get beyond just stating that proteins interact and on to measuring how [interactions] dynamically change under certain conditions," Aebersold said.

To do this effectively, researchers need to be able to quantify the various interacting proteins across multiple time points and conditions, he noted. And, while shotgun proteomic methods can offer such quantitative data via measurements of precursor ion intensity and spectral counting, targeted proteomic techniques like selected-reaction monitoring mass spec or SWATH give the most accurate quantitative results when comparing a large number of samples, he said.

"The DDA dataset of a complex sample will typically identify a somewhat larger number of peptides than SWATH," Aebersold said. "But, when you do repeat analyses, which is what we are most interested in, there is a crossover point at which the somewhat smaller number of peptides identified per SWATH run [becomes] a higher number of consistently identified peptides."

He said that an analysis by targeted proteomics firm Biognosys – of which he is a co-founder – had determined this crossover point to be around three to four samples.

In addition to improved quantitative accuracy, because SWATH captures quantitative data on all the proteins detected in a sample, it also allows researchers the opportunity to re-interrogate datasets in light of new findings, Aebersold noted.

In the ETH Zurich-led study, the researchers analyzed the 14-3-3β scaffold protein interactome following stimulation of the insulin PI3K-AKT pathway. The 14-3-3 family consists of seven scaffold proteins that play regulatory roles in a variety of cellular functions. To investigate this interactome, the ETH Zurich researchers affinity purified 14-3-3β after PI3K inhibitor treatment, without treatment, and at four time points after IGF1 stimulation, analyzing the purified proteins via SWATH.

Via this analysis they identified 1,967 proteins across four orders of magnitude in three out of three biological replicates in at least one experimental condition. From this dataset they identified 567 high-confidence 14-3-3β interacting proteins whose dynamics they then characterized using their time-course data.

In particular, the researchers focused on the behavior of subunits shared by the protein complexes mTORC1 and mTORC2, using their data to corroborate previous findings that the majority of mTOR kinase interacting with 14-3-3β comes from mTORC2 as opposed to mTORC1.

The University of Toronto study used affinity purification followed by SWATH to profile the cyclin-dependent kinase CDK4 interactome and changes due to mutations in this protein linked to melanoma as well as to treatment with an HSP90 inhibitor.

In their study of the mutant CDK4 forms, the researchers found a variety of changes in these proteins' interactomes compared to that of the wildtype protein, including an increased association with HSP90 proteins and the CDC37 chaperone protein as well as a decrease in interactions with the INK family of CDK inhibitors.

Upon treatment of the CDK4 proteins with the HSP90 inhibitor, the researchers found that interactions between HSP90 and the protein CDC37 remained high in the mutant forms compared to wild-type.

As the authors noted, a rationale for the use of HSP90 inhibitors for treating tumors driven by mutant CDK4 is that such treatment could prevent recruitment of CDC37-HSP90. However, the preservation of these interactions in the face of treatment with an HSP90 inhibitor suggest, they said, that such tumors "may not benefit from treatment with HSP90 inhibitors or may require higher dosages to obtain the desired therapeutic results."

The University of Toronto team also presented in their study a new statistical approach for calculating protein fold-changes and determining the confidence of these calculations. The ETH Zurich researchers, meanwhile, used the OpenSWATH software developed in Aebersold's lab and which, Aebersold said, they plan to present in a forthcoming paper in Nature Biotechnology.

The two programs "do similar things," Aebersold said, noting that the Toronto team's software was designed to work with AB Sciex's proprietary PeakView product while OpenSWATH had been developed as an open-source tool. Several AB Sciex researchers were co-authors on the University of Toronto paper, including Ron Bonner and Stephen Tate, who helped develop the SWATH technique in concert with Aebersold's lab.

SWATH is designed specifically for use with AB Sciex's TripleTOF 5600 instrument. However, similar DIA methods are either offered or are being developed by a variety of vendors including Waters, Agilent, Thermo Fisher Scientific, and Bruker.

Last month, aforementioned proteomics firm Biognosys expanded the capabilities of its DIA data analysis program Spectronaut to include compatibility with Thermo Scientific's Q Exactive instrument.

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