NEW YORK (GenomeWeb) – Researchers from VIB and Ghent University have devised a new peptide tagging method that allows for easier development of selected-reaction monitoring mass spec assays for targeted protein quantitation.
Detailed in a paper published this week in Nature Scientific Reports, the method uses specific peptides as tags for proteins of interest. These tags can be added to endogenous proteins using a CRISPR/Cas9-based approach. The target proteins can then be quantified using SRM assays to these peptide tags, eliminating the need to develop SRM assays to each specific protein target.
MRM mass spec is an increasingly popular tool for protein quantitation as it offers advantages of specificity and multiplexing compared to conventional immunoassays. Developing SRM assays remains complicated and time consuming, however, as researchers must carefully choose the optimal peptides and transitions to monitor in order to ensure the best results.
By tagging all target proteins with the same peptide, the VIB/UGent researchers get around this assay development issue as only assays to these peptide tags are needed, not assays to each individual protein of interest.
"Since CRISPR/Cas9 became available you can introduce tags in the genome and tag endogenous proteins," Sven Eyckerman, a VIB/UGent researcher and senior author on the paper, told GenomeWeb. "And in that case, if you use SRM [for protein quantitation] you need to only build one or a few SRM assays to measure these proteotypic peptide [tags] that we have developed. So it is kind of a universal assay."
To develop the tags, Eyckerman and his colleagues combed through the proteome of the hypertherophile Pyrococcus furiosus, looking for peptides that were unique to this organism and not present in any common model organisms. They also evaluated candidate peptides according to a variety of other criteria, including how amenable they were to detection by LC-MS/MS and how efficiently they undergo trypsin digestion.
Ultimately, the researchers settled upon two peptide tags, which they then used to tag proteins of interest using CRISPR/Cas9. Eyckerman noted that one of his group's primary interests is protein-protein interaction, and that the new technique provides advantages for such research in that it enables study of these interactions in endogenous proteins while also providing good quantitative data.
"People have been doing protein tagging for years," he said. "But in most cases they have been doing this by expressing a protein using a vector in a cell line or a system they are interested in and overexpressing [the protein]."
Such approaches, he said, did not allow for good quantitative measurements and were also complicated by artifacts arising from overexpression of the proteins being studied.
Use of the peptide-tagging technique, which the researchers named PQS (for Proteotypic peptides for Quantification by SRM), potentially provides a better look at "how [interacting proteins] behave if you start doing these [experiments] on the endogenous level and you start quantifying them," Eyckerman said.
With that in mind, he said he and his colleagues intended first to use the technique to re-examine some well-studied protein-protein interactions. For instance, in the Nature Scientific Reports study they used the method to look at the interaction between the proteins p53 and MDM2, which plays a prominent role in cancer biology. Looking at the effect of the small molecule Nutlin3, which disrupts this interaction, and the protease inhibitor MG132, which affects the proteins' stability, the researchers were able to identify significant changes in the amount of p53-bound MDM2 in response to treatment with these agents.
Being able to collect good quantitative data on the endogenous level, "you start seeing things that people haven't seen before, and you open up new biology," Eyckerman said.
For instance, "You can start to define the physicochemical characteristics of this interaction," he said. "You can start to define the affinity of the interactions … and if you at some point want to interfere with these interactions this kind of knowledge is very important. So it allows you to really characterize in detail the protein interactions in a detail that has not been available before, and that is really a combination of the quantitative measurements and working on the endogenous level."
The VIB/UGent researchers are also working to develop antibodies to their peptide tags that could be used for upfront enrichment in SISCAPA-style mass spec assays to increase sensitivity. Here, too, the tags' universality are a potential advantage, Eyckerman said, noting that "you don't need to develop [an antibody] for every protein you want to measure, you only need to develop it for the tags."
With only two peptide tags, the technique is limited in the number of proteins it can measure in an assay. Another limitation is the CRISPR/Cas9 technology, which Eyckerman noted is relatively effective at knocking out genes of interest but somewhat less so when it comes to making insertions.
However, he said, "The technology is moving so fast that it will become possible in a few years to tag a large number of genes, and then of course it will be great to have a panel of these peptide tags available, so we are definitely going to push that forward."
The group might also seek to develop the tags as a commercial product if there is sufficient researcher interest, he said.
As with all tagging methods, one potential downside is the possibility that addition of the tags will affect the behavior of the target proteins. "There is always this question," Eyckerman said.
Another limitation, he suggested, is the SRM mass spec technique itself. While effective, and increasingly commonplace, the method "is still kind of a high-end analysis technique that is pretty difficult," Eyckerman said. "It is not trivial."
"But it is a new approach, and the method opens the way to thinking of other ways of doing this," he said. "There is clearly an opportunity there, quantifying proteins using [tagging enabled by] CRISPR/Cas9. Maybe targeted proteomics is in the end not the way to go forward because it is kind of cumbersome, but it gives you things to think about."