Electron-Based Dissociation Techniques in Mass Spectrometry for the Structural Characterisation of Small Molecules and Synthetic and Natural Peptides

Abstract

The work detailed herein describes the development of electron-based tandem mass spectrometric techniques for the structural elucidation of small pharmaceutical molecules and peptides. Electron-induced dissociation (EID), electron-capture dissociation (ECD) and, for the first time, electron-transfer dissociation (ETD) have been shown to generate complementary information to collision-induced dissociation (CID), for the analysis of small pharmaceutical molecules and peptides.

For EID of phosphorylated small molecules, selecting precursor ions with varying numbers of sodium atoms associated with the ion allowed for targeted bond dissociation. When no sodium atoms were associated with the precursor ion, EID induced bond dissociation at the non- phosphorylated end of the precursor ion. When precursor ions with one or two sodium atoms were selected for EID, the product ions formed were the result of bond dissociation closer to, and on, the phosphate moiety.

For the first time, ETD has been successfully performed on small organic molecules. Either proton-transfer or electron-transfer occurred following interaction of a multiply protonated precursor molecule and an ETD reagent radical anion. When electron-transfer occurred, product ions were formed unique to ETD, providing complementary information to CID. Following proton-transfer, limited bond dissociation was observed. By the
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use of supercharging reagents (SCRs), small molecules added to the ESI solution to aid in ionisation, the abundance of doubly protonated small organic molecules was increased with respect to singly protonated small organic molecules. The increased abundance of doubly protonated molecules allowed for the use of ETD, where the ratio of multiply protonated precursor molecules to ETD reagent radical anions is a key factor in generating efficient ETD. The efficiency of ETD for analytes over an ultra-high-performance liquid chromatography (UHPLC) gradient was significantly improved by dynamically controlling the ion abundance of multiply protonated precursor molecules, as the molecules eluted from UHPLC column. The controlled precursor ion abundance enabled the ratio of analyte to ETD reagent to remain relatively constant, resulting in more efficient ETD across the entire UHPLC peak.