Most workflows in protein quantification and characterization by mass spectrometry have used collision-induced dissociation (CID). In the CID process, a peptide is selected in the first quadrupole of the instrument (Q1), then accelerated through a collision cell where it collides with nitrogen gas. The collision energy, at which the peptide is moved through this region in the MS, is a user-controlled setting. When the peptide collides with the nitrogen gas molecules, the kinetic energy is converted into internal energy, which results in bond breakage, and the peptide will dissociate into smaller fragments. The typical bond breakages are along the peptide backbone at the amide bond, which produces y and b ions, as shown by the red line in the following figure.

Since 1998, different types of fragmentation have emerged to fragment peptides and proteins and further characterize them. One method, that uses a low-energy electron beam, is called electron capture dissociation (ECD).¹ More recently, dissociation using an electron beam with various kinetic energies was explored, and referred to as electron activated dissociation (EAD). This new technique encompasses a variety of different free electron-based fragmentation mechanisms, which allows various applications complementary to CID.²

Using low electron energy, an electron is captured efficiently by multiply-protonated precursor ions like peptides. One of the protons that gives positive charges to the precursor ions is neutralized by electron capture, so that an unpaired electron is introduced into the molecular ions. This intermediate state, which is often called a charge-reduced species, is a radical, and radical chemistry predominantly dissociates at the peptide backbone between a nitrogen and an alpha carbon (green lines, first figure). The created fragment ions are c-ions (N-term) and z-ions (C-term), more specifically, c’ ions and z^• ions.¹ In a conventional definition of fragments, z^• ions are also referred to z+1 ions.

Because of the radical chemistry, dissociation efficiency is less preferred on the types of two amino acid residues that neighbored the dissociation site. One strict exception is dissociation at the N terminal side of proline residues. ECD never dissociates this position because this N-C_α is in a ring structure of the prolines. Because of this less preferred cleavage, ECD can provide better sequence coverage than CID in peptide analysis. Top-down protein sequencing is one of the EAD applications using this characteristic.

Post-translational modification moieties are often labile—phosphorylation, sulfation, and glycosylation, for example—and such moieties are likely to be detached easily during CID. This results in the difficult localization of the post-translational modification on the backbone. ECD, however, can dissociate the backbone without loss of the moieties because of the radical chemistry process, which does not include a vibration-induced process.

When the dissociation efficiency is low after electron capture, which can be noticed by the charge reduced species with a high intensity, the applied electron beam energy can be elevated. Using a high kinetic energy of electrons (typically 7-12 eV), supplemental activation energy can be introduced into the charge reduced species. The radical dissociation is induced before full redistribution of vibrational energy, allowing dissociation with higher efficiency than ECD. This operation mode is called hot ECD.³ Hot ECD is extremely useful for the analysis of glycopeptides. Hot ECD also induces secondary dissociation of amino acid residues from radical z^• ions. This phenomenon allows differentiation between isomeric leucine and isoleucine.³

You can view the fragment ion matching to your peptide EAD MS/MS using the Bio Tool Kit in Explorer in SCIEX OS software.⁴

References:

1. Zubarev RA, Kelleher NL, McLafferty FW, (1998) Electron capture dissociation of multiply charged protein cations. A nonergodic process, Am. Chem. Soc., 120: 3265–3266.
2. Electron activated dissociation – A new paradigm for mass spectrometry. SCIEX white paper RUO-MKT-19-13372-A
3. Frank Kjeldsen, Kim F. Haselmann, Bogdan A. Budnik, Frank Jensen, Roman A. Zubarev (2002) Dissociative capture of hot (3–13 eV) electrons by polypeptide polycations: an efficient process accompanied by secondary fragmentation, Chemical Physics Letters 356 (2002) 201–206
4. Bio Tool Kit – a complete set of tools for biomolecule characterization. SCIEX technical note, RUO-MKT-02-5248-A.

RUO-MKT-18-13296-A