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FTICR Mass Spec Allows Detailed Sequence, Structural Analysis of Intact Protein Complexes


NEW YORK (GenomeWeb) – University of California, Los Angeles researchers have developed a workflow using Fourier-transform ion cyclotron resonance mass spec to analyze intact protein complexes.

Described in a paper published this week in Nature Chemistry, the method allows researchers to collect sequence and structural information on large protein complexes in the same experiment and could prove useful for a variety of applications including in drug development and structural biology, said Joseph Loo, professor of chemistry and biochemistry at UCLA and senior author on the study.

Among the study's most notable findings was establishing that FTICR instruments can be used for the analysis of large macromolecular complexes, Loo said.

"I think that previous to our work, FTICR had not been considered a platform that could address very large complexes," he said, noting that this was due to questions such as whether large ions with high mass-to-charge (m/z) ratios could be efficiently transmitted and trapped in these instruments as well as whether resolution would suffer when looking at large high m/z ions.

Typically, researchers have used QTOF mass specs for such work with Orbitraps moving into the field more recently. But, Loo said, FTICR instruments have certain advantages that, were it shown they could analyze large complexes, would make them an attractive option.

FTICR "still has superior mass resolution," compared to other mass spec technologies, Loo said. "And that is always beneficial for very large complexes."

Additionally, the architecture of FTICR machines makes it easy to implement a variety of different fragmentation techniques, and this versatility is important for effectively analyzing native protein complexes by mass spec, he said.

Proteomics has traditionally considered proteins as discrete entities within a sample, but proteins typically exist and function within cells as part of complexes, interacting with other proteins and molecules. Researchers have used a variety of approaches to better characterize protein interactions, ranging from yeast two-hybrid experiments and immunoprecipitation mass spec to approaches like cross-linking mass spec and cryo-electron microscopy.

Native mass spec likewise aims to analyze proteins as they exist within complexes and in their intact states.

Last year, a team led by Northwestern University researcher Neil Kelleher, published a paper describing an Orbitrap-based method for unbiased discovery and characterization of intact, endogenous protein complexes. That method used three stages of mass spec fragmentation: an MS1 stage to measure the mass spectrum of the intact protein complex they were analyzing, looking at one or more of its different charge states; an MS2 stage in which they used the mass spec's quadrupole to isolate one specific charge state and then ejected individual components of the complex into the HCD cell to be fragmented; and an MS3 in which the individual components were isolated via the quadrupole and identified and characterized.

They then took the data collected on the various subunits and pieced it back together to generate structural information about the complex as a whole.

The FTICR approach used by Loo and his colleagues, on the other hand, collects sequence information at the protein-complex level, which, the authors said, allows them to "directly link fragmentation mass spectra to the higher-order structure of the protein complexes."

Using FTICR allowed the researchers to explore a number of different fragmentation approaches, Loo said, noting that this was key to the work.

"This particular instrument is very versatile, and it gives you many options to put on these different kinds of methods for ion activation," he said. "We didn't know which method of activation would be best. For instance, certain types of complexes, because of the way that they're structured, because of the way that they form salt bonds that keep them very stable, you need a different method of activation in order to release those fragment ions. So, it was really a matter of having multiple methods of fragmentation that allowed us to get that kind of structural information that we were looking for."

In fact, Loo said, the success or lack thereof of particular fragmentation methods provides structural insights in and of itself.

"It points to a way, in the future, that when one has an unknown complex, stepping through the different forms of activation [and seeing which are effective] can give additional information about how that complex might be put together," he said. "One could almost set up a flowchart. Try this method first. If that doesn't fragment, then move on to this method. If it does fragment, that must be because [of a given structural feature]."

"Rather than going to crystallography or cryo-[electron microscopy], you could go through this flowchart to get some really unique spatial information before you go on to the other higher-resolution structural techniques that people use today," he said.

In terms of practical applications, Loo cited work his lab had done in Alzheimer's investigating a compound that appears to inhibit aggregation of the amyloid plaques characteristic of the disease.

"This ligand binds to the protein, and it prevents the protein from aggregating," he said. "We've been trying to understand how this ligand binds to the protein, and we've published several papers on using the [mass spec] technique to locate where that ligand is binding on the protein."

"These proteins don't crystallize very well, and so our information is very unique," he added. "It gives the people who are developing that compound a handle on what amino acids, what structures of amino acids, this ligand binds to. And perhaps we could use that for modeling efforts to understand how the drug actually prevents aggregation in those kinds of neurological diseases."

Such an approach is generally applicable, Loo suggested. "I can see pharmaceutical companies using this technique to pinpoint ligand-binding, to see where a small molecule drug binds on their [target] protein."

"We haven't considered therapeutic proteins like antibodies yet, but I could see that coming as well," he added.

Loo noted that while the study focused on FTICR, he expects Orbitraps, due to their much smaller footprint and greater simplicity of maintenance and operation, will probably become the instrument of choice for such work as Thermo Fisher Scientific and collaborating researchers continue to add to fragmentation methods to these machines.

"Orbitraps, once they become more flexible with respect to the users' needs [with regard to native mass spec], will probably be the way to go," he said. "But I don't want to shove FTICRs under the rug yet."