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Putting the power of proteomics in the hands of Flow Cytometry

The combination of simplicity and power is turning flow cytometry into the highest throughput protein analysis method yet developed.

Large-scale protein analysis, or proteomics, is still a relatively small discipline where the research front is driven by a few labs with unrestricted access to mass spectrometry (MS). MS is equivalent to a mainframe computer: very big, very sophisticated, and only a few people have the skills to use them.

What we are trying to create is something analogous to a personal computer, where everyone is able to conduct experiments with hundreds or even thousands of proteins a day. With MS this is possible, but the machines are very expensive and slow and very few people have access to them.

Finding an alternative to MS

There have been attempts to copy principles from so-called DNA microarray analysis to proteomics.  Thus, antibodies spotted onto slides have been used to capture fluorescently labelled proteins from samples such as cell lysates. This problem is that antibodies often cross-react since the assay format only reports on the total amount of protein bound by each capture antibody, the results will often be misleading.

Therefore, this chip technology never took off for proteomics.

The solution to this was implement a principle from western blotting, where the proteins are separated by size using gel electrophoresis. With this technique, different protein targets for the same antibody can be seen as discrete bands on the blot.  For antibody array analysis, the proteins must be in solution, so we used an instrument called GelFree, which yields liquid fractions with size separated proteins. Each fraction is measured with identical arrays.

However, the arrays are not chips but beads.

Putting the power of proteomics in the hands of flow cytometry

By using latex beads assigned with five-dimensional color codes, we can read 2,500 differently coloured antibodies with the flow cytometer. This technique is similar to the well-established and well-known Luminex. We have made our own version, using flow cytometry to measure molecules or proteins instead of cells (Microsphere Affinity Proteomics, MAP).

Because we have our own beads, we can afford to measure each sample with up to 100 copies of the array. When we do this we get two-dimensional analysis: one dimension is the amount of protein that is captured and the other is the size of the protein. These bead-based arrays with a multiplexing capacity of up to 5000 allow us the convenience, affordability and throughput needed to take proteomics to the next level.

The benefit of this is that it allows us to do the same thing we can do with a western blot with one antibody at a time. Now, we can do it with thousands of antibodies simultaneously, putting the power of proteomics in the hands of flow cytometry users.

Meeting the challenges of antibody research

We can use this technology to compare and test a huge number of antibodies. Not only does this make large-scale protein analysis much simpler, faster and more affordable, but also meets one of the most discussed challenges in proteomics: i.e. that antibodies do not necessarily bind in the way that is expected.

Often, researchers buy antibodies only to find that they do not do what they want them to do. It is estimated that in the US alone, universities waste $350 million each year purchasing antibodies that simply don’t work.

Another benefit is that when conduction a western blot, you typically take a picture of it and publish the results as an image. However, in our case, the results are numerical with the flow cytometry output being numerical. Being able to publish a spreadsheet with numerical data allows others to reuse the data.

In the antibody field, almost everything has been published in images. It is very difficult to work with 10,000 images, so the re-use of data is essentially impossible. But with spreadsheets you can download and analyse data, making it much easier to test reproducibility and compare cells from different bandwidths.

Going forward, there are two areas where we would like this technology to develop in collaboration with the antibody manufacturers.

Firstly, we want to use MAP as an antibody testing tool. We believe this will work extremely well and that companies will have the means to compare tens of thousands of antibodies. This will enable them to remove those which don’t work from catalogues, so that researcher will have superior tools to work with.

Secondly, we want to develop MAP into research kits. We hope that we can convince antibody manufacturers to work with us to develop multiplex technology into kits that researchers can use easily to measure thousands of proteins in each experiment.

We need to measure proteins to understand cell function and develop effective therapies. The kind of high throughput proteomics enabled by MAP will empower us to do this and to understand biology in a completely new way.

 

Fridtjof Lund-Johansen is Head of flow cytometry core facility at Oslo University Hospital, Norway.

 

 

Download the Flow Cytometry Congress: Europe agenda to discover the latest technological developments and applications of flow cytometry on cellular analysis and in cancer diagnosis and treatment. 

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