A Wider Net for Catching Proteins

microwestern

Illustration by Clint May

Most people who have spent any length of time in a laboratory know the pain and frustration of Western blots. There’s probably a little bit of PTSD in every cell biologist related to gels falling apart, leaky electrophoresis chambers, or bands that should be there but aren’t, causing you to wonder which of the preceding 40 steps went wrong.

But don’t hate the method, hate the human error – Western blots, for all the agony they’ve caused, have been one of the most widely-used and productive lab methods of the past 30-some years. Used to detect the amount of protein in a given cell or tissue sample, Western blots have furthered our understanding of the intricate machinery of the cell, from the assembly line that builds it to the defects that can lead to cancer and other diseases. More specific than another protein assay, mass spectrometry, Western blots are the weapon of choice for laboratories that want to characterize the amount and status of a specific protein.

Nevertheless, Western blots have their limits, and the key word is “protein,” singular. Due to the limited size of a Western blot gel and the expense of the antibodies needed to “visualize” the proteins within, blots can only assay, at most, a handful of proteins in each run. Given that the protein networks of cells can contain hundreds or even thousands of proteins, that’s like trying to figure out the image on a puzzle by looking at only one piece at a time. The search was on for a better method, one that could take a snapshot of hundreds of proteins from a cell sample simultaneously.

Such a breakthrough was announced over the weekend in the journal Nature Methods, where a team of scientists led by Richard Jones, assistant professor at the University of Chicago’s Ben May Department for Cancer Research and the Institute for Genomics and Systems Biology described a promising new technique: micro-western arrays.

“When you walk into a dark room and don’t have much information, it’s difficult to predict where everything is going to be,” Jones said. “If someone can simply turn on the light, you don’t have to progress one step at a time by bumping into things. With this new technology, you can potentially see everything at the same time.”

Jones and his colleagues took advantage of the fact that the “hundreds-at-a-time” problem has already been solved for another target: genes. Since the 1990’s, scientists have used devices called microarrays to measure the expression of hundreds or thousands of genes at once, an innovation that has allowed genomics to take the lead in biology. As a result, scientists began to learn a lot more about the instructions for proteins that they knew about the proteins themselves, as “proteomics” lagged behind due to the restrictions of the Western blot.

The forward leap of Jones’ group was to apply the large-scale capabilities of DNA microarrays to the specificity of Western blots, producing the micro-western array. Essentially, the micro-western array is made up of over one hundred miniaturized Western blots, pre-printed on a specially-designed gel. Scaling down the size makes simultaneous comparison of a bunch of proteins possible, and dramatically reduces the cost – mere nanoliters of cellular sample and antibodies are needed for each individual blot.

Such a method could dramatically affect both the laboratory and the clinic, Jones said. Scientists can now look at a large chunk of a protein network at once, seeing how the activation or inhibition of one part of the network cascades down through the other proteins. One can also use a micro-western array to run dozens of different treatments at once on a small group of proteins, comparing the effects of many different concentrations of a potential toxin, for example. In the clinic, a single tissue sample collected from a biopsy can be stretched much further and tested for hundreds of proteins instead of just one or two, enabling more specific diagnosis and more individualized treatment.

“In the clinic, you’re limited by the fact that typically most cancers are diagnosed by one or two markers; you’re looking for one or two markers that are high or low then trying to diagnose and treat an illness,” Jones said. “Here, we can potentially measure a collection of proteins at the same time and not just focus on one guess. We’ve never been able do that before.”

Jones’ paper does more than just describe the method; they also took it for a test drive. One cancer cell line frequently studied in the laboratory contains an elevated amount of a protein called epidermal growth factor receptor, or EGFR. EGFR is normally activated to trigger the proliferation of skin cells, but when it is overactive, it can lead to the formation of tumors. Jones, in partnership with scientists from the Massachusetts Institute of Technology, sought to look at the dozens of proteins inside the cell who are affected by activation of EGFR, a task that would take months or years with a standard Western blot, but which could be done quickly and efficiently with the micro-western array.

“We started asking questions about what we could do that no one else could previously do,” Jones said. “We could actually reproducibly see 120 things at a time rather than looking at 1 or 2 or 5.”

Their experiments led to a more complete picture of the EGFR network, the Rube Goldberg-esque tree of activity triggered by turning on just the one protein. Notably, Jones and his team observed that activating EGFR also activated ten other receptors, a finding that could explain why some cancers are so slippery in response to treatment.

“If a cell were able to distribute its signal like this to other oncogenic receptors, if you were to target a cancer with an inhibitor to one of those it would essentially have ten ways out of avoiding your therapy,” Jones said.

In a broader sense, the micro-western array could take proteomics out of the shadow of its more popular brother, genomics, making the kind of large-scale studies that have been applied to DNA possible for proteins. That forward leap will create new headaches for researchers, but of a different kind – rather than dealing with crumbling gels, scientists studying proteins will have to figure out new, computer-based ways of analyzing mountains of complex data about the interactions of hundreds of proteins. But if understanding the machinery of the cell is the ultimate goal, that’s a good problem to have.

About Rob Mitchum (526 Articles)
Rob Mitchum is communications manager at the Computation Institute, a joint initiative between The University of Chicago and Argonne National Laboratory.
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