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WVU Team Combines Ion Mobility with Trapping MS for Phosphoproteomics, Structural Analyses


NEW YORK (Genomeweb News) – Researchers at West Virginia University have combined ion mobility with a linear ion trap mass spectrometer to create a platform for proteomics and structural proteomics work.

Detailed in a study published last month in Analytical Chemistry, the instrument allows for pairing the additional separation power of ion mobility with the multistage tandem mass spectrometry capabilities of an ion trap device, Stephen Valentine, a WVU researcher and senior author on the paper, told ProteoMonitor.

As Valentine noted, ion mobility devices, which can separate and identify ions based on their movement through a buffer gas, are typically attached to time-of-flight instruments – as in the case of ion mobility-enabled mass specs from vendors like Waters and Agilent.

However, he said, attaching ion mobility to a trapping instrument "has advantages in that you can now store ions in the trap" and subject them to various forms of fragmentation and analysis.

"What this allows us to do is to select an ion of specific mobility, fill the trap, and if there is sufficient signal, do MSN on that particular ion," Valentine said.

The mass spec instrument itself is a Thermo Fisher Scientific LTQ Velos, which Valentine said his lab selected because at the time – two years ago – it was one of the leading ion trap models and had top of the line capabilities in terms of electron-transfer dissociation.

Combining ion mobility with ETD was one of the primary goals of the study, he said, noting that this allowed them to follow the ion-mobility based separation and fragmentation with ETD in the ion trap.

In the Analytical Chemistry paper the researchers put this combination to work for the analysis of phosphopeptides, using the upfront ion mobility to fragment and identify phosphorylated peptides and then using ETD in the ion trap to determine the location of the phosphorylation site.

"So we can fragment the peptide ions in the [ion mobility] drift tube, [then] use a gentle fragmentation to knock off [protein phosphorylations]," Valentine said.

The researchers were then able to identify ions that had lost phosphorylations by either their ion mobility characteristics or m/z and select them for further analysis with ETD, which provides good coverage along the peptide backbone, allowing for localization of phosphosites.

"So you can imagine setting up an algorithm that would go through the two-dimensional [ion mobility] dataset very rapidly, look for any [phosphorylation] loss, and then select out those particular ions for ETD analysis to identify the phosphorylation site," Valentine said.

The combination of ion mobility with the ion trap's ETD capabilities also allows for structural analysis of target proteins, something Valentine said he and his colleagues are currently pursuing.

In this work, the researchers use hydrogen-deuterium exchange in the drift tube followed by ETD on the ion trap. In hydrogen-deuterium exchange, added deuterium replaces hydrogen in the amides of a peptide backbone, and by analyzing the amides in which this exchange occurs, researchers can obtain structural information about a protein.

"We dope a little bit of [deuterium] into the buffer gas [in the drift tube] and allow the ions to exchange their exchangeable hydrogens for deuterium," Valentine said. "And as they exchange their hydrogens for deuteriums they will become heavier and we can measure their mass shift with the mass spectrometer."

"And because we have ETD at the back, we can now break apart those ions and determine where those exchanges occur on an amino acid residue-basis," he added. "And in addition to that we have the ion mobility information, so we can get information about the collision cross section."

Valentine said that he and his colleagues have recently submitted a paper detailing this structural work.

The WVU researchers are not collaborating with any vendors on their work, though Valentine noted there are efforts ongoing in the field to combine ion mobility with Thermo Fisher's Orbitrap analyzers.

Ion mobility has most commonly been paired with TOF instruments due to certain disadvantages inherent in linking the technology to trapping machines. In particular, Valentine said, ion mobility-trap combinations suffer from losses in sensitivity and from slowed cycle times.

Primarily these disadvantages stem from the fact that the researchers must select ions of specific mobility for mass spec analysis, which requires them to cycle through all the different drift time windows to collect information on the entire sample.

This selection process lowers the number of ions that make it through to the mass spec, reducing sensitivity slightly, Valentine said. More significant, though, he said, is the increased cycle time this approach necessitates.

"So there are disadvantages compared to a time-of-flight in terms of how rapidly you can collect an IMS-MS distribution," he said, noting that ion mobility coupled to a TOF instrument can perform a comparable analysis around an order of magnitude more quickly.

On the other hand, Valentine noted, "You can't … trap and do MSN to identify something or do MSN with [collision-induced dissociation] and ETD."

Valentine and his colleagues have continued to work on the device and aim in the next iteration to improve the resolving power of the ion mobility system by extending the length of the drift tube to around 2 meters, he said.