Skip to main content
Premium Trial:

Request an Annual Quote

Lunenfeld Researchers Using SRM-MS to Quantify Kinetics of Protein-Protein Interactions

Premium

By Adam Bonislawski

A research team led by scientists at the Samuel Lunenfeld Research Institute of Toronto's Mount Sinai Hospital has devised a technique combining affinity purification and selective-reaction monitoring mass spectrometry to quantify the dynamics of protein-protein interactions.

With it they studied the signaling dynamics of the GRB2 protein — a key participant in a number of tyrosine kinase receptor signaling pathways — identifying 90 GRB2-associated proteins and tracking their context-specific and time-dependent interactions.

The work, detailed in a paper published last week in the online edition of Nature Biotechnology, demonstrates GRB2's importance in a wide range of protein-interaction networks and provides what could be a broadly applicable method for quantifying the signaling dynamics of similar adaptor proteins, said study author and Lunenfeld researcher Nicolas Bisson.

Past studies quantifying the dynamics of protein-protein interactions have typically relied on peptide labeling methods like SILAC. These techniques, though, are difficult to apply to human tissue and have limited multiplexing capabilities.

The Lunenfeld researchers instead chose SRM mass spec, allowing them to perform label-free quantitation of the GRB2-binding proteins they identified.

In the case of both methods the primary aim, Bisson said, is improving reproducibility, a key challenge in such quantitative interaction studies and in protein quantitation work more generally.

"From one experiment to another or even within the same experiment, the problem is reproducibility," he told ProteoMonitor. "There are proteins that will show up in one experiment and then not another one. And it's not because they're not there, it's just a matter of the [mass spec's] sensitivity."

Adding to the difficulty is the stochastic nature of mass-spec instruments, — the fact that the peptides to be sequenced in a given run are selected more or less randomly, making it difficult to rely on these measurements for quantitative information, Bisson noted.

SRM mass spec addresses these problems by allowing researchers to select specific precursor and fragment ion pairs for monitoring, providing more sensitive, reproducible, and quantitative measurements of the corresponding peptide and protein across different samples and experiments.

Because SRM is a targeted approach, the researchers first needed to identify the GRB2 binding proteins they wanted to monitor, which they did using HEK293T cells expressing a 3xFLAG-tagged GRB2 fusion protein, isolating 108 different GRB2 interactors from the cell lysate and identifying them via LC-MS/MS on an AB Sciex QSTAR Elite.

They then developed SRM assays for these interactor proteins on an AB Sciex QTRAP 5500 machine, successfully devising assays for 90 of the 108. Using these assays, the researchers were able to pull out GRB2 interaction complexes via affinity purification and quantify the levels of interacting proteins over a range of time points and in response to a variety of different stimuli.

In particular, they investigated the effect of epidermal growth factor on the GRB2 network, stimulating the cells with EGF for durations of 0, 1, 3, 10, 30, and 100 minutes and analyzing the composition of the GRB2 complexes at each time point.

This analysis identified 13 proteins showing increased association with GRB2 in response to EGF, which the scientists were able to sort into three groups — an EGFR-like group, a SHIP2-like group, and an IRS4-like group — based upon the distinct kinetics of their binding to GRB2.

The study also characterized the various GRB2 complexes formed in response to six different endogenous growth factors — EGF, fibroblast GF, hepatocyte GF, insulin-like GF-1, insulin, and platelet-derived GF-B-chain dimer — identifying growth factor-specific GRB2 complexes as well as several likely core GRB2 signaling proteins shared across the majority of complexes.

In the study, the researchers touched only lightly on the biological implications of their findings, Bisson noted, adding "there are a lot of interesting findings regarding GRB2 that we're following up on."

In particular, he said, the work identified some GRB2-associated proteins capable of binding to both its SH2 and SH3 domains, a finding that could help explain the robustness of GRB2 networks.

Beyond the specific GRB2-related findings, the study demonstrates the broader utility of AP-SRM as a tool for studying the networks surrounding such hub proteins, Bisson said. He added that his lab is currently investigating several other proteins using the technique but declined to specify what they were.

The primary drawback to the technique is the time required for method development. Building SRM assays is a time-consuming process in which researchers must identify the peptides on which to base assays for a given protein; determine the best transition to explore using mass spec; and develop and optimize methods for performing the separation and the assay.

Generating assays for the 90 proteins measured in the GRB2 study took several months, although he noted that he could now likely do it in around a month, Bisson said.

One development that could shorten the SRM process is the emergence of resources like the SRMAtlas, a collection of single reaction-monitoring assays developed by researchers at the Institute of Systems Biology and the Swiss Federal Institute of Technology.

Containing more than 170,000 single reaction monitoring assays Bisson said one each for at least five proteotypic peptides for each of the 20,300 human genes currently annotated as protein-encoding — the atlas aims to offer a set of standardized SRM assays that proteomics researchers can use to more easily investigate proteins of interest.

Bisson said, however, that given the relatively small scale of the GRB2 work, he preferred to keep the assay development in-house.

"We know the elution times [on their specific platforms] and we work hard to have reproducible chromatography, so for working on this scale I'd rather work based on my own data," he said.

"If I was working on a larger scale, though — and soon I probably will be working on quite a larger scale — then an atlas like [the SRMAtlas] would be very useful for sure," Bisson added. "Because you can't develop [SRM] assays against 3,000 proteins."

Another tool that could potentially ease SRM assay development is the new SWATH data acquisition method introduced by AB Sciex at last month's American Society of Mass Spectrometry annual meeting for use on its TripleTOF 5600 instrument (PM 06/10/2011).

The technique, which the company developed in coordination with ETH Zurich's Ruedi Aebersold, co-leader of the SRMAtlas project with the ISB's Rob Moritz, allows researchers to do targeted analyses of specific proteins by searching reference spectra captured by the machine against the reference spectra in the SRMAtlas.

Such a technique "could potentially work" as an alternative to conventional SRM methods, Bisson said, noting that several of his Lunenfeld colleagues are currently working to assess how reproducible the SWATH method is compared to standard SRM.

"It's not the same, but it's quite good, so you could apply that as well — not do the SRM [upfront] but do it retroactively," he said. "I don't think that as we speak it will replace SRM, but maybe in the future it will be better."

"But how much better will SRM be then? How much will triple quads have improved? It's hard to predict," he said.


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

The Scan

US Booster Eligibility Decision

The US CDC director recommends that people at high risk of developing COVID-19 due to their jobs also be eligible for COVID-19 boosters, in addition to those 65 years old and older or with underlying medical conditions.

Arizona Bill Before Judge

The Arizona Daily Star reports that a judge weighing whether a new Arizona law restricting abortion due to genetic conditions is a ban or a restriction.

Additional Genes

Wales is rolling out new genetic testing service for cancer patients, according to BBC News.

Science Papers Examine State of Human Genomic Research, Single-Cell Protein Quantification

In Science this week: a number of editorials and policy reports discuss advances in human genomic research, and more.