The Waters Synapt system in combination with the e-MSion ExD cell provides some unique capabilities to perform combined IMS /ECD experiments which unleashes the true power of the technology.

Electron-capture dissociation is a technique ideally suited to fragment intact proteins. The reason for this is the nature of the fragmentation mechanism. First, the interaction between highly positively charged intact proteins and negative electrons is intense. Second, ECD is a process involving charge reduction. Thus when an electron is absorbed by the precursor, one charge is taken away. If this happens with a singly-charged precursor it results in two neutral fragments which cannot be detected. If a doubly-charged precursor is fragmented, it results in one singly charged and one neutral fragment, thus statistically half of the fragment ions are lost.

Only when we do the fragmentation on triply-charged precursors we will, on statistic average, get two charged fragments. And here the magic starts… While a doubly-charged precursor results in a pair of a neutral and a charged fragment, the two may still stick together by electrostatic forces (ECnoD) and to separate and detect them properly, additional vibrational activation is required. When a triply-charged precursor is fragmented resulting in two charged fragments, these are driven apart by electrostatic repulsion and the fragmentation becomes much more efficient.

So, high precursor charges are favourable. But when starting with a highly-charged precursor and fragmenting it to large number of highly-charged fragments with mixed charge states, spectra will get very crowded making deconvolution potentially difficult. This has been observed as a major bottleneck for the implementation of ECD or ETD (electron-transfer dissociation) on QTOF systems in the past. The way to overcome this is to either increase resolution (e.g. by going from QTOF to Orbitrap or FT-ICR based systems) or to use an additional separation step to simplify the fragmentation spectra. Here, the combination of IMS and ECD is an extremely powerful combination and we will highlight in this article some aspects of this.

ECD/IMS allows for spectra simplification

As mentioned, the IMS based separation of differently charged ECD fragments allows to simplify the spectra, to assist in deconvolution and to assign more fragment ions which are otherwise hidden. In the TWAVE-based IMS implementation it is easily possible to separate 1+ – 4+ charge states and to simplify higher charges state spectra by removing the lower charge states. An example is given with the fragmentation of the 7+ charged precursor of Ubiquitin. It produces fragment ions with charges between 1+ and 5+, all mixed and overlapping in the MS/MS spectrum.

Ion mobility-baesd charge separation of Ubiquitine electron capture dissociation (ECD) fragments simplifying fragmentation spectra

IMS data were processed with mineXpert software (Rusconi et al, JASM 2021,32, 1138-1141)

The IMS-based charge separation allows for the separation of the 2+ charged z29 ion from the 4+ charged c59 ion resulting in clear assignment of both ions. Another example is given below, where two overlaps of 1+/3+ and 4+/5+ could be resolved.

The IMS based charge separation of the ECD fragments thus dramatically increases the separation spaces, adds clarity in assignments and reveals fragment ions otherwise hidden due to low ion abundance. The additional possibilities of IMS make ECD much more powerful in this case than an ECD implementation on a conventional QTOF without IMS capability.

IMS/ECD in combination with CID before IMS

Another application is to combine classical CID fragmentation in the Synapt’s trap region (before IMS) with ECD after the IMS region. This requires the ECD cell behind the IMS cell. This combination allows for unique types of experiments where both types of fragmentation may be applied in parallel:

Electron capture dissociation (ECD) and collision induced dissocitation of Substance P with subsequent ion mobility separation

IMS data were processed with mineXpert software (Rusconi et al, JASM 2021,32, 1138-1141)

While the singly charged CID fragments can be separated by IMS, the singly charged fragments generated by ECD show a nice and clean spectrum without interference of the CID generated ions. This can help to focus on labile modifications which are lost in CID fragmentation such as Lys, His or Arg phosphorylation or sulfatation due to differences between CID and ECD fragments being revealed in a single analysis.

IMS/ECD for activated precursor fragmentation – collision induced unfolding (CIU)

Another very powerful approach is to activate ions either in the source or the trap region of the Synapt instrument by collisional activation. This opens up the structure of globular proteins and gives insights into protein assembly and packaging of native proteins:

Collision induced unfolding of the 16 kDa two-domain protein Calmodulin with subsequent ECD fragmentation. Data courtesy of Carolina Rojas Ramirez and Brandon Ruotolo, University of Michigan.

The activated proteins are then separated by IMS to analyze structural differences and subsequently fragmented by ECD. Performing ECD after collisional activation turned out to be more efficient as the structural integrity of the globular proteins have already been disturbed which improves the fragmentation effiency due to lower electrostatic interactions within the proteins structure. This approach has been described by V.V. Gadkari et al.  in a paper from 2020. As the starting point for this type of analysis is native proteins and protein complexes, this application is particularly powerful on dedicated MSVision high mass systems  which extend the accessible mass range in excess to 32kDa for precursor isolation and also enables the use of higher activation energies in the source region and the collision region.

The collisional unfolding basically creates some sort of “melting curve” of the native structure, “unfolding” the structural details of native proteins and protein complexes. The easiest way to facilitate this type of analysis is with an ECD cell behind the IMS cell. Yet it is also possible to record the “melting” curve and to perform a separate ECD fragmentation analysis at a given activation energy. This has the additional benefit of being able to use the additional performance gain by separating ECD fragments by IMS to improve sequence coverage and simplify spectra as outlined in the first section.

Learn more about the power of IMS and ECD on dedicated high mass instrumentation!

If you are working in the area of biopharma and biologics characterization or the characterization of non-covalent complexes, don’t miss to contact us! By teaming up with e-MSion, MSVision became even more powerful as a provider of dedicated high mass systems.