Electron Capture Dissociation as Structural Probe for Noncovalent Gas-Phase Protein Assemblies

Abstract

Electron capture dissociation (ECD) of proteins in Fourier transform ion cyclotron resonance mass spectrometry usually leads to charge reduction and backbone-bond cleavage, thereby mostly retaining labile, intramolecular noncovalent interactions. In this report, we evaluate ECD of the 84-kDa noncovalent heptameric gp31 complex and compare this with sustained off-resonance irradiation collisionally activated dissociation (SORI-CAD) of the same protein. Unexpectedly, the 21+ charge state of the gp31 oligomer exhibits a main ECD pathway resulting in a hexamer and monomer, disrupting labile, intermolecular noncovalent bonds and leaving the backbone intact. Unexpectedly, the charge separation over the two products is highly proportional to molecular weight. This indicates that a major charge redistribution over the subunits of the complex does not take place during ECD, in contrast to the behavior observed when using SORI-CAD. We speculate that the ejected monomer retains more of its original structure in ECD, when compared to SORI-CAD. ECD of lower charge states of gp31 does not lead to dissociation of noncovalent bonds. We hypothesize that the initial gas-phase structure of the 21+ charge state is significantly different from the lower charge states. These structural differences result in the different reaction pathways when using ECD.Since the late 1990s, electron capture dissociation 1 (ECD) has been available as a tool for structural analysis in Fourier transform ion cyclotron resonance mass spectrometry (FT-ICR-MS). To date, ECD is routinely used for top-down sequencing and identification of the posttranslational modifications, such as sulfide bridges, phosphorylation, and methylation. [2][3][4][5][6][7] During an ECD experiment, low-energy electrons are injected into the ICR cell and captured by multiply protonated ions, resulting in the release of approximately 2-6 eV of recombination energy into the ion. 1,8 Typically, the capture of one or more electrons leads to the rapid dissociation of covalent backbone bonds, resulting in the formation of c and z‚ fragment ions. 9 The ECD process has so far been studied using peptides and proteins with a molecular mass up to 50 kDa 5,10 but not yet for protein complexes or proteins with a higher molecular mass. There is still an active debate about the exact mechanisms of ECD. 8,[11][12][13][14][15][16][17] The early research demonstrated that ECD leads to rapid dissociation of the backbone close to the site of electron capture, not necessarily fragmenting the weakest bonds in the molecule. In line with this observation, ECD has been found to often preserve labile noncovalent bonds, 10 for instance in cytochrome c 18 and vancomycin complexed with diacetyl-L-Lys-D-Ala- This makes ECD a potentially useful technique for examining interaction sites in larger, noncovalent protein complexes.In this report, we use ECD to study the interaction sites in the large gp31 heptameric protein complex from bacteriophage T4. The protein complex has a molecular mass of 84 kDa and co...

Characterization of permethylated β-cyclodextrin-peptide noncovalently bound complexes using electron capture dissociation mass spectrometry (ECD MS)

“…This result was in stark contrast to SORI-CAD experiments in which disassembly of two constituent molecules, Angiotensin II and ␤-CD, was a dominant process. Conservation of noncovalent interactions during ECD peptide backbone dissociation, as observed here, is consistent with well-known ECD characteristics [28][29][30][31][32][33][34]41,42,44]. In addition, a singly reduced species [A:CD+2H] •+ and its ammonia-loss counterpart [A:CD-NH 3 +2H] •+ were observed at m/z 2476.9 and 2459.6, respectively.…”

“…ECD MS has many useful characteristics, including high fragmentation efficiency and superior capability in characterizing post-translational modifications such as phosphorylation and glycosylation [27][28][29][30][31][32][33][34][35][36][37][38][39][40][41][42][43][44][45][46]. Furthermore, it has been shown that noncovalent interactions, both intramolecular and intermolecular, generally remain intact in ECD MS [28][29][30][31][32][33][34]41,42,44]. We utilized this unique property of ECD MS to study the interaction characteristics of permethylated ␤-CD-peptide complexes.…”

Collisional activation of protein complexes: Picking up the pieces

“…Black-body infrared radiative dissociation [15], electron-capture dissociation [17], infrared multiphoton dissociation [14,16], and surface-induced dissociation [18,64] have all been applied to protein assemblies. Of these, the latter is notable for the large amount of energy deposited into the protein complexes in a very short time, resulting in more extensive dissociation than that in CID [65].…”

Extensive Charge Reduction and Dissociation of Intact Protein Complexes Following Electron Transfer on a Quadrupole-Ion Mobility-Time-of-Flight MS

“…In these experiments, charge reduction and the subsequent transmission and detection of the resulting extremely high m/z species typically require instrument modifications [15,18]. Limited charge reduction is also typically observed as a side reaction in electron capture (ECD) and electron transfer dissociation (ETD) experiments, without instrument modification, particularly when working with intact proteins [19][20][21][22][23][24]. In addition, ECD or ETD fragments can remain bound to each other by noncovalent interactions so that they appear as chargereduced precursor ions in the spectra [24][25][26][27].…”