Electron Capture Dissociation of Multiply Charged Protein Cations. A Nonergodic Process

Neutralization-reionization mass spectrometry (MS)1 is of unique value for preparing and characterizing highly reactive and unstable neutral species, such as the intermediate in the dissociative-recombination reaction H3O + ef H2O + H + 6.4 eV.2 Following an earlier suggestion,3 using neutralization accompanying surface-induced dissociation (SID)4 to form an unstable site did not yield new cleavage reactions5 in multiply charged protein cations from electrospray ionization (ESI) with Fourier transform (FT) MS.6 Serendipitously, we now find that one or more charges on such protein cations can be neutralized with low-energy electrons to cause specific cleavage of the amine bond to form c, z products,7 in contrast to the amide cleavage b, y products formed by collisionally activated dissociation (CAD),8 infrared multiphoton (IRMPD)9 and UV10 photodissociation, 70 eV electron impact excitation,11 and SID.5 The b, y products are formed by the lowest energy backbone cleavage of ESI protein ions.6-9 An attempt to cleave stronger bonds using high-energy (6.4 eV) 193 nm photons gave mainly b, y products for 2 kDa protein ions,10 but for 2.8 and 8.6 kDa protein ions12 gave small yields of c, z amine bond cleavage products not previously observed. In this further investigation, extra electrodes were placed outside the ion cell electrodes that trap the positively charged ions. With the outside electrodes at +9 V,13 extensive 193 nm laser irradiation of SWIFT-selected14 (M + 11H)11+ ubiquitin ions (8.6 kDa) only produces b, y, not c, z, ions. However, with the outside electrodes at -1 V, the c, z products are formed along with 10+ molecular ions; unexpectedly, these are mainly (M + 11H)10+• ions (Figure 1d), 1 Da heavier than the (M + 10H)10+ ions formed by ESI (Figure 1c). The 4+ mellitin ion spectrum measured under the same conditions (Figure 1b) similarly contains (M + 4H)3+•, consistent with capture of secondary electrons formed by the 193 nm photons impinging on metal surfaces and trapped by the -1 V electrodes: (M + 4H)4+ + ef (M + 4H)3+•.15 Electrons were produced instead (no laser) by a conventional heated filament source outside the FTMS magnet opposite the ESI source.11 With a 10-5 Torr Ar pulse for ecooling (energy < 0.2 eV; an SF6 pulse lowered the efficiency), the 11+ ions of ubiquitin gave a spectrum that showed c, z cleavage of 50 out of 75 backbone positions; CAD8/IRMPD9 gave b, y cleavage of eight of these positions plus seven others. Cooled electrons plus the 15+ ions of FeIII equine cytochrome c16 produced (Figure 2) c, z fragment ions from cleavages at all but 40 of the 103 possible backbone sites (e.g., N-terminal side of Pro, none; of Ile, Leu, Val, few); CAD produces b, y cleavages (total 19) at eight additional sites. The 21+ apomyoglobin ions (17 kDa) yielded 33 c, z cleavages, but the 34+ ions of bovine carbonic anhydrase (29 kDa) as yet has given only 33+, 32+, and 31+ molecular ions. Electron capture dissociation (ECD)1-3 rationalizes these results. The capture cross section should be proportional to the ionic charge squared, consistent with the minimal secondary fragmentations to produce internal ions and the predominance of cleavages in the central∼70% of the protein chain. Charge values and masses17 of the complementary product ions are consistent with dissociation after ecapture, such as c39/z37 from the 76-residue ubiquitin 11+ ions and c69/z35 from the 104residue cytochrome c 15+ ions. The most favored protonation sites are the side chains of Lys, Arg, and His;18 neutralization to form hypervalent species1-3 at Lys and Arg would account for ions (Figure 2) representing losses of 17, 44, and 59 Da from (M + nH)(n-x)+ (eq 1; neutralization of protonated His gives a more stable radical site).