Tagging Up With Protein Microarrays

Molecular Systems Biology, an open access journal, has an impressive new functional protein microarray paper. The authors identified a large number of targets for a yeast ubiquitin transferase (enzymes which transfer a protein tag, ubiquitin, onto other proteins), and the data has a good ring to it.

Some background: protein microarrays are a much more complicated subject than nucleic acid microarrays. One way to split them is by intent. Capture arrays have some sort of affinity capture reagent, most likely antibodies, on the chip surface. If properly designed, built & calibrated they represent a very highly multiplexed set of protein assays. Reverse-phase protein arrays spot fractionated, but unpure, proteins from biological samples on an array.

In contrast, functional protein microarrays attempt to represent a proteome on a chip as individually addressable spots in order to study aspects of that proteome. A number of groups have worked on functional protein microarrays, but there are a limited number of commercial sources, with perhaps the most successful being Invitrogen, which offers human and yeast arrays. If you'd like a great beach book on the subject, a new volume covers a wide array of topics, with Chapter 22 ("Evaluating Precision and Recall in Functional Protein Arrays") definitely my favorite.

Functional protein arrays present a huge challenge. In the ideal case the proteins would be produced, folded correctly and deposited on the slide in such a way that an assay can be run on every protein in parallel. This is a tall order, with lots of complications. Proteins may not fold correctly during expression or may unfold in the neighborhood of the slide surface, the post-translational state of the protein may be variable and is unlikely to capture all possible states of the protein, and the protein may not have key partners which are important for its function.

Despite these, and many other concerns, protein microarray experiments have been published describing various feats. Protein-protein interaction experiments to discover novel interactions (such as this one) or create comprehensive binding profiles (such as this one) are probably the most prevalent use, but the arrays can also be used to discover DNA binding proteins, identify novel enzymes, assay phenotypic differences of mutants, develop novel infectious disease diagnostic strategies, and identify the targets of protein kinases. [links are a mix of open access & paid access; apologies)

A wide variety of ingenious methods have been used to produce functional protein microarrays. The Invitrogen arrays are spotted from purified expressed protein and expected to bind randomly, but some other approaches ensure that the majority of protein molecules bind in a defined way. Some approaches actually synthesize the proteins in situ, and one group even deposited proteins on spots using a mass spectrometer!

Protein microarrays have had their growing pains. The amount of active protein found in a spot can vary widely. One study of protein-protein interactions failed to recover most of the known interactors of the bait protein. Since the bait is primarily a phosphoprotein binding protein, one possible explanation is that the insect-expressed human proteins were not in their correct phosphorylation state. However, poor recall of known substrates was also observed in protein kinase substrate searches run in both human and yeast (see Chapter 22 of the Predki book). Even without worrying about post-translational modification, coverage is an issue. While essentially the complete Saccharomyces proteome is available, the most extensive commercial human chip has less than 1/5th of the proteome and there are not (last I checked) commercial arrays for any other species.

The new publication wins on a bunch of scores. First, it is one of the handful of publications using such arrays which is not from one of the labs pioneering them, suggesting that they might work routinely. This publication uses the Invitrogen yeast arrays. Second, they did recover a lot of known substrates for their ubiquitinating enzyme. Third, the signals look very strong by eye, which has been the case for protein-protein interaction assays but much less so for protein kinase substrate discovery. Fourth, they batted 1.000 with novel positives from array in an independent in vitro ubiquitination assay and were able to verify that at least some of these are ubiquitinated by Rsp5 in vivo (by comparing ubiquitination in wt and Rsp5 mutant strains). Fifth, they performed a protein-protein interaction microarray assay with Rsp5 and the interaction results and ubiquitination results strongly overlapped.

Of course, I used to work at Ubiquitin Proteasome Pathway Inc (which is now touting a new drug with a new target in the pathway), and there I would have been digesting this paper until arrays danced in my dreams. Such assays offer an interesting possibility for greatly expanding our understanding of UPP players and functions -- many Ub transferases or Ub-removing proteases have no known substrates. While they have a lot of issues, functional protein microarrays are starting to make a difference in proteomics.