Pushing The Limits Of Orbitrap Mass Analyzer

Researchers have pushed the limits of the Orbitrap mass analyzer, an instrument with relatively high resolution, to collect mass spectra of intact protein complexes with molecular weights approaching 1 million daltons (Nat. Methods, DOI: 10.1038/nmeth.2208). Extending the high resolution of the Orbitrap to large complexes that up to now could be mass-analyzed only at significantly lower resolution makes it possible to identify finer molecular details, which could open up opportunities in drug discovery.

Albert J. R. Heck of Utrecht University, in the Netherlands; Alexander Makarov of Thermo Fisher Scientific, in Bremen, Germany; and their coworkers carried out the study by modifying Thermo’s benchtop Orbitrap, the Exactive Plus. They changed the software to allow analysis at higher mass-to-charge ratios (m/z), adjusted the voltage settings, and increased the pressure in the collision cell.

They probed the instrument’s performance characteristics at high m/z using clusters of the salt CsI. They detected CsI clusters with a resolving power—a measure of the ability to separate two peaks—of 25,000 at m/z 5,000 and of 16,000 at m/z 10,000. In comparison, similar measurements on a quadrupole time-of-flight instrument had resolving power of only 5,000 across the m/z range. “The resolution that we get is not so much limited by the mass analyzer,” Heck says. “It’s limited by how well we can desolvate the ions.”

The group used the modified Orbitrap to measure a variety of biological macromolecular complexes. For example, they analyzed a 146-kDa monoclonal immunoglobulin G antibody at sufficient resolution to distinguish various forms with different numbers of sugar units. Analyses of the 801-kDa protein complex GroEL had sufficient resolution to count the number of adenosine diphosphate (ADP) and adenosine triphosphate (ATP) molecules bound to the complex.

“We see this mass difference of 80 Da between ATP and ADP on an 801-kDa complex,” Heck says, a difference of 0.01%. “We really can see individual small molecules binding to these large protein complexes.”

“The ability to count individual ATP nucleosides bound to a GroEL complex in its 70+ charge state is a remarkable result,” says R. Graham Cooks, a chemistry professor at Purdue University.

The new capability can lead to potential breakthroughs in drug discovery, says Brandon T. Ruotolo, a chemistry professor at the University of Michigan. “Multiprotein systems are amongst the most promising therapeutic targets. Detailed characterization of such massive multiprotein complexes is quite challenging, especially when searching for evidence of small-molecule interactions within such an assembly.”

The timeline for commercialization remains uncertain. “This is still a research-only implementation,” says Iain Mylchreest, vice president of R&D in the chromatography and mass spectrometry unit of Thermo. “We are investigating how we can potentially commercialize this in the future such that it would be turnkey and not a series of manual and nonstandard modifications.”