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Proteomics: Building protein microarrays on the spot

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NAPPA is an alternative approach to microarray proteomics, which may soon be used for the study of small molecule-protein interactions, identifying binding targets for individual compounds.

The cDNA microarray has become commonplace in the modern lab, providing a potent tool for large-scale genomic studies. More recently, several groups have worked toward the development of similar arrays for the study of proteomics, particularly with an eye toward functional and interactional studies, or the characterizing changes in protein levels in a given biological sample. Many of these early approaches have mirrored the process of cDNA microarray production, starting with individual protein synthesis, followed by purification and spotting onto the array.

Although these strategies have met with a measure of success, they have yet to be broadly embraced by the research community. Joshua LaBaer, Director of the Harvard Institute of Proteomics (Boston, MA), suggests that there are specific complications to this approach that make it somewhat unpalatable. “I think the biggest problem is, it’s going to be hard, if you want an array of a thousand or ten thousand proteins, to purify that many proteins and then spot them on the array,” he says. “We’ve been interested in these arrays for years now...[but] we felt that this was just not going to be a viable approach in the long run.”

LaBaer’s team recently developed an alternative approach to microarray proteomics, known as the nucleic acid programmable protein array (NAPPA). NAPPA relies on seeding the array with cDNAs of interest, cloned as fusion proteins with a glutathione S-transferase (GST) tag. The entire batch of clones can then be subjected to in vitro transcription and translation; polyclonal anti-GST antibodies, which have been fixed onto the array directly adjacent to the plasmid spots, immediately trap the proteins generated by these reactions. The resulting protein array is rapidly generated, with no apparent cross-contamination. Importantly, the prepared DNA chip can be safely stored at room temperature for months, with no apparent negative impact on the quality of protein synthesis and binding.

After some initial trials, in which LaBaer’s group confirmed the selectivity of the system by detecting binding of the Cdk inhibitor p16 to Cdk4 and Cdk6, but not to Cdk2, they proceeded with a larger scale analysis, characterizing the binding relationships between twenty-nine different proteins involved in the formation of the human DNA replication complex. Query proteins were HA-tagged, to allow for detection with fluorescently labeled antibodies. 110 different interactions were detected, including 47 previously identified interactions. Of the interactions that had previously been characterized through careful biochemical analysis, 85% were confirmed via NAPPA. The final data allowed for the generation of a complex interaction map, detailing the binary associations among these various replication factors. Some known interactions were missed, in part because of the binary nature of the assay, which misses associations dependent on a ‘bridging’ factor; however, in a separate assay, LaBaer’s team demonstrated that these tertiary interactions could be confirmed by presenting both binding partners simultaneously to the appropriate bait.

Although the initial work with NAPPA has revolved around the characterization of protein-protein interactions, LaBaer has made clear that this is not the primary vision he has for his technology. “We chose [this] as a first step in part because we really wanted to validate the tool,” he says, “and I think that since there was a lot already known about the interactions within the human DNA replication complex, we had a nice setting where we could identify interactions between proteins that were already known to interact, and make sure that our system was doing what it was supposed to do.” Nonetheless, he adds, “there are already existing methods for [studying this], so the need for that in the protein microarray format is not as high.”

Instead, LaBaer’s team is currently looking to apply NAPPA to the study of small molecule-protein interactions, identifying binding targets for individual compounds and assisting in the drug discovery process. “You can imagine you’ve got a kinase inhibitor, and you want to get a sense of which kinases it binds to – and which ones it doesn’t – in a rapid assay. You could even imagine it as a tool for medicinal chemistry, and modify the molecule to make it more selective, if you want.”

Additionally, LaBaer is examining NAPPA’s potential role in clinical research, as a means for biomarker detection and for the characterization of immunodominant antigens from various pathogens. By generating chips consisting of genes expressed by a given pathogen or tumor sample, then treating these chips with serum from an affected patient, it becomes possible to isolate individual proteins that are triggering an early immune response, revealing potentially valuable information for diagnosis and the development of new vaccines.

Michael Eisenstein

References

Ramachandran, N. , Hainsworth, E. , Bhullar, B. , Eisenstein, S. , Rosen, B. , Lau, A.Y. et al. Self-Assembling Protein Microarrays. Science 305, 86–90 (2004). | PubMed |

Links

Harvard Institute of Proteomics