Ion/ion reactions and collisional activation of biomolecules in a linear ion trap time of flight mass spectrometer

Ian K Webb, Purdue University

Abstract

Means for effecting ion trap dipolar DC collision induced dissociation (DDC CID) on a quadrupole/time-of-flight in a mass spectrometer have been implemented for the broadband dissociation of a wide range of analyte ions. The DC dipolar-based fragmentation method provides an ion trap-based means for inducing fragmentation of ions in a large mass-to-charge range simultaneously. It can be effected within an ion storage step in a quadrupole collision cell that is operated as a linear ion trap or as ions are continuously transmitted through the collision cell. A dipolar DC potential is applied across one pair of rods in the quadruple collision cell of a QqTOF hybrid mass spectrometer to effect fragmentation. The application of dipolar DC (DDC) in the RF-only ion guide (Q0) of a hybrid quadrupole/time-of-flight (QqTOF) mass spectrometer for collision-induced declustering of large bio-ions is also described with DDC collisional activation (DDC CA) is employed in the trapping mode. Ions are collisionally cooled by Q0 trapping to prevent radial ion loss before and after DDC CA is applied. As a broadband technique, ion trap DDC CA can decluster a large mass-to-charge range simultaneously. Gas-phase intra-molecular crosslinking of the protein ubiquitin cations has been demonstrated via ion/ion reactions with anions of homobifunctional N-hydroxysulfosuccinimide (sulfo-NHS) ester reagent. The ion/ion reaction between multiply-protonated ubiquitin or melittin and crosslinker monoanions produces a stable, charge reduced complex. Covalent crosslinking is indicated by the consecutive loss of two molecules of sulfo-NHS under ion trap collisional activation conditions. The presence of the covalent modification is verified by the presence of covalently crosslinked sequence ions produced by ion-trap collision-induced dissociation of the ion generated from the losses of sulfo-NHS. Analysis of the crosslinked sequence fragments allows for the localization of crosslinked primary amines, enabling proximity mapping of the gas-phase 3-D structures. The formation of gas-phase intermolecular covalent cross-links has been demonstrated via ion/ion reactions of mutually stored ions of opposing polarities. Specifically, dimers of ubiquitin and cytochrome C were cross-linked with the sulfo-NHS ester-based reagent sulfo-EGS. The sulfo-NHS losses from the dimers were isolated and subjected to additional ion trap CID. Both ubiquitin and cytochrome C homodimers gave CID product ions corresponding to sequence fragments of the monomer cross-linked to intact monomers, indicating that intermolecular crosslinking has taken place. Control of both proton transfer and complex forming ion/ion reaction rates has been demonstrated by applying dipolar DC in both the mutual storage and transmission modes.

Degree

Ph.D.

Advisors

McLuckey, Purdue University.

Subject Area

Analytical chemistry

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