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 beamtype 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.

Modulation of Protein Fragmentation Through Carbamylation of Primary Amines

“…In the context of activation of proteins, performance metrics tend to decrease with increasing mass, and charge state plays a major role [17, 18]. Upon electrospray ionization, protein ions are generated in a wide array of charge states, thus making it especially important to more extensively evaluate and understand the impact of charge state on the fragmentation of proteins [5, 19]. There have been several systematic studies of protein dissociation using collisional activation, including ones that have examined the influence of charge state and other factors on fragmentation pathways [19–23].…”

“…Upon electrospray ionization, protein ions are generated in a wide array of charge states, thus making it especially important to more extensively evaluate and understand the impact of charge state on the fragmentation of proteins [5, 19]. There have been several systematic studies of protein dissociation using collisional activation, including ones that have examined the influence of charge state and other factors on fragmentation pathways [19–23]. CAD of intact proteins depends on proton mobility for fragmentation, as also well-recognized for peptide fragmentation induced by collisional activation [24].…”

Increasing fragmentation of disulfide-bonded proteins for top–down mass spectrometry by supercharging

“…The effect of precursor ion charge state on protein and peptide fragmentation has been observed previously with various activation techniques including CAD [46–50], ECD [51], and ETD [52]. The charge state effect is especially prominent for electron based fragmentation techniques such as ECD [51] and ETD [52], with more sequence ions being produced for higher charge states.…”

Nano-LC FTICR Tandem Mass Spectrometry for Top-Down Proteomics: Routine Baseline Unit Mass Resolution of Whole Cell Lysate Proteins up to 72 kDa

“…35 "Hybrid" instruments generate fragment ions in a quadrupole ion trap through collision-induced dissociation, source-induced dissociation, or high-energy collisional dissociation (HCD) prior to transfer to a high resolution mass analyzer. 14, 17, 24, 27, 48–51 In this report, we apply source-induced dissociation to identify whole cell lysate proteins between 25 and 50 kDa in molecular weight.…”