Rapidly Alternating Transmission Mode Electron-Transfer Dissociation and Collisional Activation for the Characterization of Polypeptide Ions

Abstract

Cation transmission/electron-transfer reagent anion storage mode electron-transfer ion/ion reactions and beam-type collisional activation of the polypeptide ions are performed in rapid succession in the high-pressure collision cell (Q2) of a quadrupole/time-of-flight tandem mass spectrometer (QqTOF), where the electron-transfer reagent anions are accumulated. Duty cycles for both electron-transfer dissociation (ETD) and collision-induced dissociation (CID) experiments are improved relative to ion trapping approaches since there are no discrete ion storage and reaction steps for ETD experiments and no discrete ion storage step and frequency tuning for CID experiments. For this technique, moderately high resolution and mass accuracy are also obtained due to mass analysis via the TOF analyzer. This relatively simple approach has been demonstrated with a triply charged tryptic peptide, a triply charged tryptic phosphopeptide, and a triply charged tryptic N-linked glycopeptide. For the tryptic peptide, the sequence is identified with more certainty than would be available from a single method alone due to the complementary information provided by these two dissociation methods. Because of the complementary information derived from both ETD and CID dissociation methods, peptide sequence and post-translational modification (PTM) sites for the phosphopeptide are identified. This combined ETD and CID approach is particularly useful for characterizing glycopeptides because ETD generates information about both peptide sequence and locations of the glycosylation sites, whereas CID provides information about the glycan structure.

Simultaneous Glycan-Peptide Characterization Using Hydrophilic Interaction Chromatography and Parallel Fragmentation by CID, Higher Energy Collisional Dissociation, and Electron Transfer Dissociation MS Applied to the N-Linked Glycoproteome of Campylobacter jejuni

“…CID/ETD has a time delay needed for capturing ions for CID and then switching to capture ions for ETD (50) unlike CID/HCD where ions are split between the ion and orbital traps, allowing effectively concurrent ion detection. This delay in CID/ETD lowers the duty cycle and may help to explain the lower number of glycopeptides identified within this data set.…”

Energy-resolved collision-induced dissociation pathways of model N-linked glycopeptides: implications for capturing glycan connectivity and peptide sequence in a single experiment

“…Hakansson et al obtained 68.8% sequence coverage by ECD (nine c ions and two z ions representing cleavage of 11 out of 16 N-C a bonds), 35 while McLuckey and coworkers achieved 75.0% sequence coverage by ETD (11 c ions and 12 z ions representing cleavage of 12 out of 16 N-C a bonds). 38 To more completely delineate the energy-resolved CID behavior of this model glycopeptide, the peak areas of the [M + 3H] 3+ precursor ion and the corresponding product ions (or classes of product ions) were plotted as a function of DU (Fig. 2).…”

Analyzing Glycopeptide Isomers by Combining Differential Mobility Spectrometry with Electron- and Collision-Based Tandem Mass Spectrometry

“…However, alternative MS/ MS methods to CID are generally implemented to retain otherwise labile glycan groups on peptide fragment ions-electroncapture dissociation (ECD) [5,6] and electron-transfer dissociation (ETD) [7,8]. These methods have also been blended in elegant ways to provide CID and ECD/ETD complementary information in back-to-back scans for a given precursor glycopeptide ion [9,10].…”