Exclusive Interview: Characterizing Protein-Protein Interactions in Cancers Using AP-SWATH

Dr Anne-Claude Gingras, a Senior Investigator and Lea Reichmann Chair in Cancer Proteomics at the Luenfeld-Tanenbaum Research Institute, is a recognized authority in the field of proteomics. Her current research focuses on the cellular mechanisms involved in diseases, including cancer and cerebral cavernous malformations. Dr Gingras spoke exclusively to SelectScience about the state-of-the-art technology being used by her research group and her recent paper, published in Nature Methods, which details a quantitative approach for interaction proteomics.

Research in the Gingras group focuses on experimental and computational methods to improve the field of interaction proteomics by gaining a better understanding of the ways in which protein complexes are built, localized and regulated.

Current projects investigate changes in protein-protein interactions resulting from disease-associated mutations, specifically looking at those mutations that have been linked to cancers and cerebral cavernous malformations (a disease characterized by brain ’caverns‘, in which blood accumulates due to leaky capillaries that can cause symptoms from headaches to seizures and cerebral hemorrhage).

In order to investigate proteomics associated with these diseases, the group uses a state-of-the-art mass spectrometer. Dr Gingras highlights the advantages of using mass spectrometry with SWATH analysis over traditional methods for quantifying protein interactions.

“I see a mass spectrometry as a super-fast and accurate, quantitative Western blot. Mass spectrometry is essentially an amazing digital quantitative reader of interactomes. So long as you design your experiment in a way in which you have nicely embedded negative control, you know that you’re actually not making calls for interactions that don’t really exist. The question then becomes really simple: How do you design an efficient pipeline that enables you to look systematically at the differences, in terms of what they interact with? SWATH is a very appropriate way to do that.”

Collaboration strategy
A recent collaboration with AB SCIEX to develop new mass spectrometry tools for proteomics research resulted in the publication of ‘Mapping differential interactomes by affinity purification coupled with data-independent mass spectrometry acquisition’. The strategy described in this Nature Methods paper adapts the quantitative approach of SWATH data-independent acquisition with interaction proteomics, and is now being used by the Gingras group to systematically investigate the consequences on interactions of the introduction of disease-associated mutations in the coding sequence of a protein. SWATH™ Acquisition 2.0 technology is incorporated in AB SCIEX’s high-resolution TripleTOF® 6600 mass spectrometer.

According to Dr Gingras, SWATH analysis will also help to improve future research in her laboratory.
“The benefit that it’s brought to us is targeted proteomic-style quantification with very, very tight accuracy, but without having to set up the targeted proteomic experiment and without having to worry about whether you lose some interactions with some partners, specifically because you just don’t look at them.
“So, it has allowed us to comprehensively look at our interactomes. The fact that we’re enriching it again is making our lives much easier than many of the other customers that AB SCIEX is dealing with. So, for us it’s a very nice pipeline. We’re working really hard, not only with AB SCIEX, but also with a lot of people in academia, to improve some of the steps. For example, how you create the list of what you’re going to extract in terms of quantitative information. There are a lot of improvements that can and will happen.”