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Meet the duo diving ultra-deep to reveal the inner life of the T cell 

This article featured in Illuminate Newsletter Spring ‘23
Key Researchers
Toby Dite
Senior Research Officer
Research Officer
Sections in this article
L-R: Collaborators Dr Julia Marchingo and Dr Toby Dite, in WEHI’s Proteomics Facility.

Postdoctoral researchers Dr Julia Marchingo and Dr Toby Dite are among the first recipients of a Co-Lab: Platform Collaboration Grant, established to support ambitious collaborations between WEHI’s platform technology and laboratory scientists, and funded by a generous bequest from the Estate of John Thompson and Mary H Thompson.

Julia

The focus of my research as an immunologist has been immune cells called ‘killer’ T cells, which act as a key defence against viruses and cancer by killing affected cells.

I’ve been working to understand how the response of T cells differs when they are responding to a viral infection they can clear, compared to when the response goes wrong and they fail to eliminate the virus.

The best way to learn what’s truly going on in a T cell is to study the proteins that are made and broken down over the course of a real immune response.

Proteins are the main functional units of the cell. Every cell contains thousands of types of proteins present in different numbers, from one copy to millions of copies per cell. Each protein has a specific role, from catalysing chemical reactions to regulating gene expression, so knowing the composition and interactions of the proteins is the key to determining the cell’s capabilities and functions.

Proteomics is the study of all the proteins produced by a cell. Existing proteomics technology can give us information on up to about 7000 of the most abundant proteins, but there can be over 12,000 protein types in any given cell, so we’re missing a lot of interesting biology we can’t quite detect.

Knowing more about what’s happening in the world of proteins will allow us to identify potential targets for controlling T cell immunity, to help find new ways of treating disease.

We’re not messing around here – we want to know everything! And as if that wasn’t challenging enough, we want to look at T cells in a real immune response, which doesn’t give us much starting material to work with.

That’s where Toby’s technical expertise comes in. Our Co:Lab project came out of a fortuitous conversation about a new piece of equipment in the Proteomics Facility. I wondered if it could help with diving deeper into the T cell proteome, but Toby came up with an idea for a different way to supercharge the proteomics technology, to reveal far more than has previously been possible.

In the long term, as well as enabling our own research, we’re also hoping our Co:Lab project can build a resource others can tap into.

Many researchers at WEHI are studying cell types that aren’t common; if we can build a pipeline to do whole-cell proteomics efficiently on a far smaller number of cells than usually needed, that’s something a lot of people would be able to use.

Toby

Even the latest methodologies at our disposal in the Proteomics Facility weren’t up to the task for what Julia’s trying to do – basically, she’s trying to see not just a lot of things in the cell but everything!

The core technology we use in proteomics is mass spectrometry. Proteins in a tissue sample are first broken down into smaller units called peptides. The peptides are then electrically charged and their masses, and the masses of their fragments, are detected and analysed.

From this, we can identify which proteins are present in the tissue sample and in what quantities.

To see more proteins, you usually have to start with more material. But conventional mass spectrometry requires massive numbers of cells – literally millions – and far more than Julia has to work with when she’s studying T cells.

Our goal for this project is to be able to work with 100,000 cells or fewer, so we had to come up with a technique that would allow us to answer Julia’s questions in detail, while working within the biological restrictions.

Julia and I have been friends for several years and did overlapping stints as postdocs in Scotland but we hadn’t worked closely together before we made our Co:Lab pitch. I admit that after talking to her I went into ‘massive nerd mode’ thinking about the technical challenges!

Our solution was to ‘hot-wire’ existing proteomics methods. Essentially, we’re combining two techniques – gas-phase fractionation (GPF) and data-independent acquisition (DIA) – in a way that hasn’t been done before.

Using this new GPF-DIA strategy lets us dramatically increase the number of peptides and proteins we can profile, to delve deeper into the proteome than ever before.

We’re using powerful instruments, a powerful technique and powerful software, but combining them in a unique way that will let us see things that have never been seen up to now.

Collaboration is really where science is at today – it’s no longer about a single researcher sitting at the bench and teaching themselves the techniques they need to use.

When the question is big and broad and important, collaboration is really the only way to answer it, and the Co:Lab approach at WEHI is a great way to respond to that, by bringing platforms and big ideas together.

This funding makes it possible to “go big” on projects that require technological innovation to address a fundamental biology question… or in our case, to go ultra-deep!

First published on 12 September 2023
This article featured in Illuminate Newsletter Spring ‘23
WEHI Authors
Toby Dite
Senior Research Officer
Research Officer
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