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Robert Malek, Wolfgang Metelmann-Strupat and Martin Zeller
During the last several years, Electron Capture Dissociation (ECD) has rapidly evolved as an ideal alternative activation method for peptide and protein sequencing in Fourier-transform ion cyclotron resonance-mass spectrometry (FTICR-MS). In the ECD method, multiply charged ions are irradiated with low energy electrons produced by an emitter cathode located behind the ICR cell. Upon capture of electrons, reduced radical cations [M+nH](n-1)+· are generated which dissociate by fast and facile fragmentation of the N-Ca bond of the peptide chain, producing mainly c and z· fragment ions [1]. Due to the nature of the ECD mechanism, co- and post-translational modifications (PTMs) such as phosphorylation, O- and N-linked glycosylation, and sulfation are preserved, allowing site-specific analyses.
However, the cleavage efficiency of ECD can be low. Therefore, this technique used to require long ion accumulation, activation, and detection times in the ICR cell. Together with the need for spectrum averaging, its use for on-line separation of complex peptide or protein mixtures has been difficult.
The LTQ FT hybrid linear ion trap ICR mass spectrometer for the first time provides a means to perform ECD analysis on complex mixtures of peptides and proteins on an LC timescale. High cleavage efficiency is achieved by the use of a cathode with a large electron emitting surface. Ion accumulation and isolation in the linear ion trap prior to transferring to the ICR cell for ECD and detection allows shorter cycle times (less than one second). This allows, if necessary, averaging of multiple ECD scans for each peak eluting during the on-line chromatography.
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Figure 1 shows a single scan ECD MS/MS spectrum of substance P at high resolution (RP = 100,000). The spectrum exhibits high intensities of the ECD fragment ion peaks. Bearing in mind that no fragment ions are generated N-terminally of proline due to its cyclic structure, complete sequence coverage is achieved. This would allow even complete de novo sequencing of that peptide if the amino acid sequence is unknown.
As mentioned above, labile PTMs are preserved in ECD. This is due to the fact that the amide carbonyl has a higher H? affinity than the amino acid side chains and, therefore, the backbone N-C? bonds are cleaved preferentially. |
| Figure 2 shows the ECD MS/MS spectrum of an O-glycosylated peptide carrying one N-acetylglycosamine (GlcNAc) moiety attached to one of its serines.
ECD gives rise to mainly z-type fragment ion peaks in addition to the c-type fragment ion peak. Here, the mass difference between the z6- and z7-fragment ion peak corresponds to the mass of a serine carrying a GlcNAc moiety. The mass difference between the z4- and z5-fragment ion peak reflects an unmodified serine residue. Although no peak for a z11-fragment ion peak can be seen in this spectrum, the mass difference between the z10-fragment ion peak and the mass of the intact peptide corresponds to unmodified serine and proline. Thus, the glycosylation site can be determined unambiguously, demonstrating the potential of ECD for the determination of PTMs. |
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ECD holds much promise to be a supplementary dissociation technique to collision-induced dissociation (CID). In addition, ECD also permits you to distinguish between leucine and isoleucine residues [2] and even between D- and L-amino acids [3]. Therefore, ECD in combination with CID can be used for unambiguous protein identification, de novo sequencing and complete protein characterization.
References:
1. Zubarev, R. A., Kelleher, N. L., and McLafferty, F. W., Electron capture dissociation of multiply charged protein cations. A nonergodic process, J. Am. Chem. Soc. 1998, 120, 3265-3266.
2. Kjeldsen, F., Haselmann, K. F., Sorensen, E. S., and Zubarev, R. A., Distinguishing of Ile/Leu amino acid residues in the PP3 protein by (hot) electron capture dissociation in Fourier transform ion cyclotron resonance mass spectrometry, Anal. Chem. 2003, 75, 1267-1274.
3. Adams, C. M., Kjeldsen, F., Zubarev, R. A., Budnik, B. A., and Haselmann, K. F., Electron capture dissociation distinguishes a single D-amino acid in a protein and probes the tertiary structure, J. Am. Soc. Mass Spectrom. 2004, 15, 1087-1098.
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