Abstract
Native electron capture dissociation (NECD) is a process during which proteins undergo fragmentation similar to that from radical dissociation methods, but without the addition of exogenous electrons. However, after three initial reports of NECD from the cytochrome c dimer complex, no further evidence of the effect has been published. Here, we report NECD behavior from horse spleen ferritin, a ∼490 kDa protein complex ∼20-fold larger than the previously studied cytochrome c dimer. Application of front-end infrared excitation (FIRE) in conjunction with low- and high-m/z quadrupole isolation and collisionally activated dissociation (CAD) provides new insights into the NECD mechanism. Additionally, activation of the intact complex in either the electrospray droplet or the gas phase produced c-type fragment ions. Similar to the previously reported results on cytochrome c, these fragment ions form near residues known to interact with iron atoms in solution. By mapping the location of backbone cleavages associated with c-type ions onto the crystal structure, we are able to characterize two distinct iron binding channels that facilitate iron ion transport into the core of the complex. The resulting pathways are in good agreement with previously reported results for iron binding sites in mammalian ferritin.
Highlights
In conjunction with fragment ions observed from a spectrum acquired at higher collision energy, products from cleaving 37 L-chain backbone sites were characterized (Figure 1c, matching fragment masses for this and other fragment maps are listed in Table S-1, Supporting Information)
Leveraging information from front-end infrared excitation (FIRE)-assisted dissociation and gas-phase isolation/dissociation, we show that the resulting fragments are predominantly native electron capture dissociation (NECD) type c-ions, undergo asymmetric charge partitioning, and can be formed in the electrospray droplet or in the gas phase upon collisional or infrared activation
When mapped on the crystal structure of the all L-chain ferritin, prevalent NECD fragments indicated significant iron binding at the loop-pocket and 3-fold axis regions. These residues are not implicated in the catalytic process of ferritin but instead correspond to channels which mediate iron ion transport into the protein cage
Summary
Horse spleen ferritin was desalted and characterized by native top down mass spectrometry (nTDMS)[6,31] (Figure 1). As reported previously for NECD14 and ETD39 fragments of cytochrome c dimer, ferritin c-fragments undergo asymmetric charge partitioning, as is evident from the great decrease in m/z between the precursor and its product ions (from >8000 to 1000−2000, respectively, Figure 1a,b). Plotting the average charge of each NECD fragment with respect to cleavage site indicates a relatively high level of charge density in the fragment ions, with discrete steps corresponding to the locations of arginines (Figure 3a); residues known to be charged in nESI.[40] In comparison, CAD fragments from the isolated 14+ ejected monomer (overall weighted-average charge of ejected monomers = 13.93) exhibited a similar distribution of charge states repeating the discrete jumps in charge at arginines 18, 26, and 39 (Figure 3a). These interactions are broken, transferring an electron from complexed iron and cleaving the monomer’s peptide bond
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