Novel Implementations and Insights for Improved Gas Phase Ion-Electron Reactions in Biomolecular Tandem Mass Spectrometry

Abstract

Ion-electron reactions are powerful tandem mass spectrometry (MS/MS) tools for structural analysis and characterization of biomolecules containing labile acidic moieties. Such chemical groups, including phosphorylation, sulfation, and sialylation, are integral to biomolecular function and regulate critical biomolecular interactions; however, they are difficult to characterize with conventional MS/MS techniques. Ion-electron reactions can provide enhanced and/or complementary structural information, including sequencing and site determination of labile moieties. On the other hand such reactions can follow a variety of dissociation mechanisms depending on precursor ion charge state/polarity and electron number/energy. This dissertation describes novel implementations and in-depth characterization of ion-electron reactions in both positive and negative ion mode on a modern Fourier transform ion cyclotron resonance (FT-ICR) instrument. Chapter two describes the combination of matrix assisted laser desorption/ionization (MALDI) with negative ion electron capture dissociation (niECD). While both techniques favor singly charged ions, the feasibility of this pairing was uncertain as niECD requires gaseous zwitterionic structures, typically not associated with MALDI. However, with an equivalent amount of material, MALDI demonstrated 20-fold more abundant fragment ion signal compared with electrospray ionization (ESI) for an niECD sulfopeptide standard. MALDI was also shown to improve the mass range of niECD from m/z ~1800 to ~4300 due to superior ionization of larger polypeptides into singly deprotonated anions than possible with ESI. Intriguingly, multiple electron captures were observed for the first time, indicating that multiple positive charges may be present within net singly deprotonated precursor anions. Chapter three is an investigation into the cathode bias voltage (CBV), electron extraction lens voltage (LV), and irradiation time parameters that govern cation-electron reactions. We found that low (0.1 V) CBV and high (>50 V) LV generated unexpected tandem ionization and complex tandem mass spectra that include all possible peptide backbone fragment types. This result suggested that the LV has a direct effect on electron energy, a hypothesis that was further supported by electron energy measurements. Further experiments decoupled the effects of electron energy and flux, two elements which are typically interconnected. This work provides an explanation to controversial previous reports of internal fragments in intact protein ECD, which used a high LV. Moreover, in-depth analysis and visualization of the interplay between reaction parameters yielded novel insights into the boundaries between different reactions such as electron capture dissociation (ECD) and electron induced dissociation (EInD). Chapter four continues the investigation of ion-electron reaction parameters in negative ion mode with particular focus on comparing results from different FT-ICR cell designs. A modern ICR cell with improved charge capacity demonstrates improved niECD and negative ion EInD (nEInD) for hirudin at high (>50 V) LV without excessive fragmentation, unlike in positive ion mode. This divergence is explained by the lower efficiency of anion-electron reactions. Systematic increase of CBV clearly demonstrates differences in optimum niECD and nEInD conditions as well as in the fragments generated from each reaction for glycans and gangliosides. While niECD was previously described to yield abundant cross ring fragments, this work indicates that such fragments, which can provide important linkage information, are more abundant in nEInD; niECD only produced 0-22% of the summed nEInD cross ring fragment abundance. Gangliosides showed a bimodal distribution for niECD optimization, suggesting that multiple gaseous structures are present. Overall, these findings provides improved implementations and insights for ion-electron reactions for advancing mass spectrometry based labile biomolecular characterization.