Negative-Ion Electron Capture Dissociation: Radical-Driven Fragmentation of Charge-Increased Gaseous Peptide Anions

Abstract

The generation of gaseous polyanions with a Coulomb barrier has attracted attention as exemplified by previous studies of fullerene dianions. However, this phenomenon has not been reported for biological anions. By contrast, electron attachment to multiply charged peptide and protein cations has seen a surge of interest due to the high utility for tandem mass spectrometry (MS/MS). Electron capture dissociation (ECD) and electron transfer dissociation (ETD) involve radical-driven fragmentation of charge-reduced peptide/protein cations to yield N-C(α) backbone bond cleavage, resulting in predictable c'/z(•)-type product ions without loss of labile post-translational modifications (PTMs). However, acidic peptides, e.g., with biologically important PTMs such as phosphorylation and sulfonation, are difficult to multiply charge in positive ion mode and show improved ionization in negative-ion mode. We found that peptide anions ([M - nH](n-), n ≥ 1) can capture electrons within a rather narrow energy range (~3.5-6.5 eV), resulting in charge-increased radical intermediates that undergo dissociation analogous to that in ECD/ETD. Gas-phase zwitterionic structures appear to play an important role in this novel MS/MS technique, negative-ion electron capture dissociation (niECD).

The Negative Mode Proteome with Activated Ion Negative Electron Transfer Dissociation (AI-NETD)

“…Negative mode approaches for the characterization of peptide anions offer a valuable dimension to proteomic analyses (21,24,25,66), especially as biologically relevant posttranslational modifications and other analytes that pose challenges to canonical positive mode techniques continue to emerge (7,53,(67)(68)(69)(70)(71)(72). Platforms for shotgun analysis of complex mixtures of peptide anions have been introduced, most notably using UVPD and NETD; however, these approaches have yet to provide considerable proteomic depth (fewer than ϳ800 total proteins identified in a given experiment), restricting the degree to which peptide anion characterization can benefit the proteomic community.…”

“…Alterna-tively, a number of emerging fragmentation techniques, including electron-based dissociation methods and photodissociation approaches, can generate sequence informative MS/MS spectra from peptide anions (17)(18)(19)(20)(21)(22)(23). Both negative electron transfer dissociation (NETD) and ultraviolet photodissociation (UVPD) have been employed in large-scale proteomic studies, enabling sequencing of thousands of unique peptides in a single experiment (24,25).…”

Top-down proteomics: applications, recent developments and perspectives

“…The same instrument was also utilized to perform collision-induced dissociation (CID) and nozzle-skimmer dissociation (NSD)of ubiquitin, using the high resolving power of the instrument to determine charge state and identity of the fragment ions [62]. An advanced hyphenation to FT-ICR technology, often focused on improving the analysis of intact proteins, included increases in magnetic field [63,64], an accumulation octupole for ion storage before transmission to the ICR cell [65,66], addition of a resolving quadrupole for mass selection [67]. These modifications, allowing for increased sensitivity, dynamic range, and resolution were used on a 9.4 T instrument for the detection and identification of proteins from M. jannaschii and S. cerevisiae [68].…”

Charge Transfer Dissociation (CTD) Mass Spectrometry of Peptide Cations: Study of Charge State Effects and Side-Chain Losses

“…Unlike CID, ExD cleaves peptide backbone N-Cα bonds to produce c/z ions with a more extensive peptide/protein sequence coverage than CID [13]. In addition, ExD retains PTMs to a much greater extent than CID, which facilitates the elucidation of PTM site information [12].…”