Electron Capture, Collision-Induced, and Electron Capture-Collision Induced Dissociation in Q-TOF
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Recently, we demonstrated that a radio-frequency-free electromagnetostatic (rf-free EMS) cell could be retrofitted into a triple quad mass spectrometer to allow electron-capture dissociation (ECD) without the aid of cooling gas or phase-specific electron injection into the cell (Voinov et al., Rapid Commun Mass Spectrom 22, 3087–3088, 2008; Voinov et al., Anal Chem 81, 1238–1243, 2009). Subsequently, we used our rf-free EMS cell in the same instrument platform to demonstrate ECD occurring in the same space and at the same time with collision-induced dissociation (CID) to produce golden pairs and even triplets from peptides (Voinov et al., Rapid Commun Mass Spectrom 23, 3028–3030, 2009). In this report, we demonstrate that ECD and CID product-ion mass spectra can be recorded at high resolution with flexible control of fragmentation processes using a newly designed cell installed in a hybrid Q-TOF tandem mass spectrometer.
Key wordsElectron capture dissociation A radio-frequency-free electromagnetostatic cell
The EMS ECD/CID cell was designed to be operated in various dissociation modes. Using Substance P as a model compound (American Peptide Co., Sunnyvale, CA, USA), examples of mass spectra recorded in five of the possible combinations are presented: low energy CID, high energy CID, ECD, ECD with collisional excitation in the collisional quadrupole, and ECD in combination with electron ionization decomposition (EID) [5, 6].
Mounting the electron filament inside the EMS cell between the magnets resulted in an increase in ECD efficiency by at least an order of magnitude relative to earlier cells [1, 2, 3] where the filament was mounted just outside the ion-entry end of the cell. Specifically, the intensity of Substance P’s c5 fragment was consistently observed in this study to be about 1% of the precursor-ion intensity (inserts in Figure 3a and c).
Performing ECD with the new cell simultaneously with CID at 7 eV collisional energy in the remaining segments of the original rf quadrupole collision/cooling cell (CQ2 and CQ3 in Figure 1a) produced product-ion spectra with golden pairs , where mass spectra exhibited both c-type (ECD fragments) and b-type CID fragments (Figure 3b). Similar results were obtained in our earlier experiments wherein ECD and CID were carried out simultaneously (in time and space) in an EMS cell mounted in a triple quadrupole instrument . This capability could prove useful in top-down analyses of oligopeptides and proteins. At present, CID or IR radiation is used to partially denature such molecules prior to ECD (“activated-ion ECD” [11, 12, 13]. An alternative might be to use ECD/CID in an EMS cell with the following multiple low energy collision activation in rf quadrupole cell as analogous of ECD with following IRMPD .
Regions of very high magnetic flux density inside the EMS cell (marked with stars in Figure 1b) dynamically compress large numbers of electrons into a small volume through which the precursor ion beam moves. The electrons in each of these regions propagate in spirals at near thermal energies along directions both opposite to and the same as the direction of the ions through the cell. Consequently, setting the filament at negative potential relative to the grid and magnets results in electrons with energies ranging from 0 eV to a maximum that is determined by the potential difference between the filament and the grid (Figure 1b). Product-ion spectrum shown in Figure 3c was recorded with the filament potential set 50 V lower than the grid potential, confining electrons with energies from 0 eV to about 50 eV in the cell’s reaction spaces. Interactions between doubly protonated Substance P selected as the precursor ion and the low-energy electrons resulted in the appearance of regular ECD c-type fragments in the spectrum. Interactions between doubly protonated Substance P and electrons having energies higher than the precursor’s ionization energy resulted in the formation of [M + 2 H]3+• radical ions. These triply charged radical precursor ions can fragment into ions that are not present in the conventional ECD product-ion spectrum of Substance P. For example, prominent monoisotopic peaks corresponding to [M + 2 H – SC3H7]2+ (theoretical monoisotopic mass = 636.8587) and [M + 2 H – SC3H7 – NH3]2+ (theoretical monoisotopic mass = 628.3458) appear in the spectrum, respectively, at m/z = 636.8580 and m/z = 628.3454 (insert, Figure 3c). These fragments, which have been previously reported , are the result of monomolecular decomposition of a [M + 2 H]3+• radical precursor.
In summary, an EMS ECD cell with an internal electron filament retrofitted into a high performance hybrid quadrupole time-of-flight Bruker ultrOTOF mass spectrometer yielded high mass-resolution, high mass-accuracy ECD product-ion mass spectra that enabled unambiguous identification of fragment ions. This accomplishment supports an earlier assertion  that an EMS ECD cell can be operated in various mass spectrometers regardless of analyzer type. In addition, incorporation of an internal electron filament in the cell has made it possible to achieve an ECD efficiency of about 1% with Substance P. Finally, operating the EMS ECD cell in conjunction with the original rf collision cell enabled analyses in either a simultaneous ECD/low energy CID mode or an electron ionization/ECD mode. The ECD/CID mode should complement existing mass spectrometric approaches to top-down analyses of oligopeptides and proteins.
This research was supported directly by grants from the NSF (CHE-0924027), the Oregon Nanoscience and Microtechnologies Institute (#09-31 #3.5), NIH (R01RR026275–National Center For Research Resources), and the NIEHS (ES00210–Environmental Health Sciences Center). The authors are indebted to Elsworth T. Hinke for his assistance in fabricating the EMS cell used in this study. V.V. thanks Yury Tsybin for helpful discussion. The authors also thank Mel Park and Thomas Knudsen of Bruker Daltonics for their contribution to the work.