Skip to main content
Premium Trial:

Request an Annual Quote

Max-Delbruck Team Builds Protein-Array Platform to Study Proteome-Wide PTM Interactions

Premium

By Adam Bonislawski

Researchers at Berlin's Max-Delbruck Center for Molecular Medicine have developed an array-based platform for the proteome-wide screening of post-translational-modification-dependent protein interactions.

The system uses biotinylated peptides featuring PTMs of interest to probe macroarrays containing up to 7,390 unique proteins, quantifying their interactions via laser-induced fluorescence emission.

Thus far, the scientists have primarily used the technique to study the role of protein methylation in chromatin and gene regulation, but it should be broadly applicable across a range of PTMs, according to MDC researcher Achim Leutz.

The platform, described in a paper published last month in the online version of Nature Protocols, is intended to complement conventional mass-spec techniques that study PTM-dependent protein interactions while offering a less expensive and equipment-intensive approach, Leutz told ProteoMonitor this week.

Additionally, he said, the method will enable certain investigations not possible using mass spec – in particular, concentration-independent studies of PTM interactions that could provide insight into the binding patterns of lower-abundance proteins.

Mass spec-based approaches, Leutz noted, typically use cell lysates containing proteins expressed at varying levels. A significant advantage of the array technique "is that it normalizes the protein expressions."

"When you take a lysate and you have proteins that are very highly expressed versus those that are very low abundance, you have an internal competition" that can obscure interactions among the low-abundance analytes, he said.

On the array platform, by contrast, "every spot contains just one kind of protein, so the protein [expression] is normalized across the membrane and the [modified] peptides have equal chance to interact with any of them."

The array method is also useful for detecting weak PTM-protein bonds, Leutz said. Such interactions might be difficult to observe in a cell-lysate sample, but because the arrays contain "millions of copies of [an individual] protein on a single spot, it's difficult for [the peptide probe] to get loose because the neighboring proteins are all of the same type."

In recent work, the MDC team used the system to study the role of methylation in C/EBP transcription factors' regulation of gene expression, synthesizing dimethylated and unmethylated C/EBP peptides and screening them on the array.

The researchers detected a number of previously identified C/EBP interaction partners, including the proteins Brg1, BAF47/Ini-1, G9a, GATA1, CBP, and TIF1β, successfully distinguishing between those that preferentially interacted with the methylated peptide compared to the unmodified variant.

Screening with chemically synthesized peptides as opposed to full-length proteins allows the researchers to select specific modification sites for study. Particularly in the case of the methylation research it was important to use small peptide motifs, Leutz noted.

"If you consider a protein domain, it most likely has multiple arginines and lysines. And if you modify that [domain] then you would potentially modify multiple of those," he said. "So by screening with peptides you have really a defined interaction sequence, and the interaction is dependent on the modification."

A potential downside to this approach, Leutz noted, is the loss of protein folding that might affect interaction levels. However, he added, for other modifications like phosphorylation that can be added more selectively, larger peptides can be used.

"Basically you can use whatever length [peptide] your synthesis will work at," he said. "In the [methylation] screen we used 40-amino-acid peptides, which might already have some folding. We did 60-amino-acid [peptides] and those worked, as well. So you can probably also expand it to protein domains that carry certain modifications at one or two certain residues."

Leutz first tried to develop a screening method for PTM-dependent interactions 15 years ago using phage-expressed, radioactively labeled proteins. Finding little success, he dropped the project, returning to it two years ago after realizing that using arrayed proteomic libraries could streamline the process.

The array format also simplifies data analysis on the backend, Leutz said, noting that the PTM-protein-binding levels can be quickly be quantified and analyzed "using the same methods used for microarray gene-expression analysis."

The platform uses UniPEx macroarrays from biotech firm ImaGenes, which last December was acquired by Source BioScience (GWDN 12/1/10). One of its main limitations is the fact that currently these arrays contain only 7,390 proteins – about one-third of the human proteome.

Leutz predicted, however, that "in the near future all proteins will be available in an expressed form," which will enable researchers to screen against the entire proteome. He added that having "the protein arrays in a much smaller size" would also be a welcome improvement, as current arrays come in a 22-cm-by-22 cm format.

"One would like to deal with it like microarray screening, but there are no companies that have developed such arrays that we can use for what we are doing," he said.

According to Leutz, his group considered patenting the platform, "but it has so many procedures that have already been published that we didn't really think it made sense."

MDC is a research institute, "so we have no plans to offer it commercially [ourselves]," he said. "But we might consider working together with companies that are interested."


Have topics you'd like to see covered in ProteoMonitor? Contact the editor at abonislawski [at] genomeweb [.] com.

The Scan

Highly Similar

Researchers have uncovered bat viruses that are highly similar to SARS-CoV-2, according to Nature News.

Gain of Oversight

According to the Wall Street Journal, the Biden Administration is considering greater oversight of gain-of-function research.

Lasker for mRNA Vaccine Work

The Scientist reports that researchers whose work enabled the development of mRNA-based vaccines are among this year's Lasker Award winners

PLOS Papers on Causal Variant Mapping, Ancient Salmonella, ALK Fusion Test for NSCLC

In PLOS this week: MsCAVIAR approach to map causal variants, analysis of ancient Salmonella, and more.