Charge State Dependent Fragmentation of Gaseous α-Synuclein Cations via Ion Trap and Beam-Type Collisional Activation

Abstract

Ions derived from nano-electrospray ionization (nano-ESI) of α-synuclein, a 14.5 kDa, 140 amino acid residue protein that is a major component of the Lewy bodies associated with Parkinson's disease, have been subjected to ion trap and beam-type collisional activation. The former samples products from fragmentation at rates generally lower than 100 s(-1) whereas the latter samples products from fragmentation at rates generally greater than 10(3) s(-1). A wide range of protein charge states spanning from as high as [M+17H](17+) to as low as [M+4H](4+) have been formed either directly from nano-ESI or via ion/ion proton transfer reactions involving the initially formed protein cations and have been subjected to both forms of collision-induced dissociation (CID). The extent of sequence information (i.e., number of distinct amide bond cleavages) available from either CID method was found to be highly sensitive to protein precursor ion charge state. Furthermore, the relative contributions of the various competing dissociation channels were also dependent upon precursor ion charge state. The qualitative trends in the changes in extent of amide bond cleavages and identities of bonds cleaved with precursor ion charge state were similar for two forms of CID. However, for every charge state examined, roughly twice the primary sequence information resulted from beam-type CID relative to ion trap CID. For example, evidence for cleavage of 86% of the protein amide bonds was observed for the [M+9H](9+) precursor ion using beam-type CID whereas 41% of the bonds were cleaved for the same precursor ion using ion trap CID. The higher energies required to drive fragmentation reactions at rates necessary to observe products in the beam experiment access more of the structurally informative fragmentation channels, which has important implications for whole protein tandem mass spectrometry.

Figure 1

a) Nano-ESI mass spectrum of α-synuclein. b) Post-ion/ion reaction data from a parallel ion parking transmission mode reaction involving α-synuclein cations and PFO anions in Q0.

Figure 2

Ion trap CID post-ion/ion reaction MS/MS spectrum of the [M + 9H]⁹⁺ ion of α-synuclein via activation at 81.06 kHz, 600 mV_p-p, nitrogen as a collision gas at ~ 5 mTorr. The loss of water from the corresponding b or y ions is labeled with an asterisk.

Figure 3

Summed b and y abundances for complementary ion pairs as a function of residue number resulting from cleavages of the α-synuclein backbone with ion trap CID for (a) [M+5H]⁵⁺, (b) [M+9H]⁹⁺, and (c) [M+15H]¹⁵⁺ ions; and with beam-type CID for (d) [M+5H]⁵⁺, (e) [M+9H]⁹⁺, and (f) [M+15H]¹⁵⁺ ions.

Figure 4

The percent sequence coverage of the fragments that result from beam-type CID as a function of ion injection energy for +15 charge state of α-synuclein. Three replicate measurements were performed to establish the error bars.

Figure 5

Post-ion/ion reaction MS/MS spectrum of the [M+9H]⁹⁺ ion of α-synuclein: injection energy, 630 eV; collision gas, nitrogen at ~ 5mTorr. The loss of water from the corresponding b or y ions is labeled with an asterisk.

Figure 6

Summed abundances of b- and y-type complementary product ions derived from beam-type CID are plotted as a function of residue number and parent ion charge state for all parent ion charge states examined. The abundance scale is normalized by dividing the summed b- and y- type ions by the total product ion signal for the relevant charge state.

Figure 7

Percent sequence coverage observed for α-synuclein ions from a representative of each charge state group ([M+5H]⁵⁺, [M+9H]⁹⁺, and [M+15H]¹⁵⁺) under ion trap CID and beam-type CID conditions as well as the sequence coverage associated with combining information from both activation conditions.

Figure 8

Combined backbone cleavages resulting from (a) the ion trap CID of α-synuclein ions and from (b) the beam-type CID of α-synuclein ions. The specific cleavages (major peaks observed in spectra) are indicated by bold line for emphasis.