IMS−IMS and IMS−IMS−IMS/MS for Separating Peptide and Protein Fragment Ions

Merenbloom, Samuel I.; Koeniger, Stormy L.; Valentine, Stephen J.; Plasencia, Manolo; Clemmer, David E.

doi:10.1021/ac052208e

Cited by 188 publications

(211 citation statements)

References 28 publications

(50 reference statements)

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“…Overall, the post-IM fragmentation results are consistent with the presence of a heterogeneous population of a 4 ions (with lifetimes at least of the order of milliseconds), including both linear and cyclic structures, and are in accord with a recent study where cyclic and linear a 4 ions were identified using infra-red spectroscopy [9]. The capability for collision-induced fragmentation of ions after mobility separation provides the general capability of interrogating an ion population through a mobility distribution [13]. This provides the potential for achieving an increase in the effective resolution of the ion mobility separation.…”

Section: Resultssupporting

confidence: 89%

Evidence for structural variants of a- and b-type peptide fragment ions using combined ion mobility/mass spectrometry

Riba-Garcia

Giles

Bateman

et al. 2008

J. Am. Soc. Mass Spectrom.

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Tandem mass spectrometry (MS/MS) of peptides plays a key role in the field of proteomics, and an understanding of the fragmentation mechanisms involved is vital for data interpretation. Not all the fragment ions observed by low-energy collision-induced dissociation of protonated peptides are readily explained by the generally accepted structures for a-and b-ions. The possibility of a macrocyclic structure for b-type ions has been recently proposed. In this study, we have undertaken investigations of linear protonated YAGFL-NH 2 , N-acetylated-YAGFL-NH 2 , and cyclo-(YAGFL) peptides and their fragments using a combination of ion mobility (IM) separation and mass spectrometry. The use of IM in this work both gives insight into relative structural forms of the ion species and crucial separation of isobaric species. Our study provides compelling evidence for the formation of a stable macrocyclic structure for the b 5 ion generated by fragmentation of protonated linear YAGFL-NH 2 . Additionally we demonstrate that the a 4 ion fragment of protonated YAGFL-NH 2 has at least two structures; one of which is attributable to a macrocyclic structure on the basis of its subsequent fragmentation. More generally, this work emphasizes the value of combined IM-MS/MS in probing the detailed fragmentation mechanisms of peptide ions, and illustrates the use of combined ion mobility/ collisional activation/mass spectrometry analysis in achieving an effective enhancement of the resolution of the mobility separator. . Accordingly, it is important to understand the gas-phase ion chemistry that underpins the MS/MS of peptide ions in order to optimize the application of the technique. The structures of the N-terminal a-and b-ions formed from the fragmentation of collisionally activated protonated peptides in the gas phase [2] have been a topic of investigation and discussion for several years. The b-ions have generally been considered to consist of a linear peptide chain terminating in a cyclic oxazolone structure (Scheme 1a) [3]; subsequent formation of a-ions by loss of CO has been presumed to follow opening of the oxazolone ring. These hypotheses alone, however, do not readily explain all the fragment ions observed in low-energy collision-induced dissociation experiments. Yagüe et al.[4] proposed rearrangement of b-ions to give macrocyclic intermediate structures that could fragment further to yield products in more complete agreement with observed data. In earlier work, Tang and Boyd [5] reported evidence of an interaction between the primary amine groups of lysine or ornithine residues with the C-terminus of peptide ions. Recently, Harrison et al.[6] used both computational studies and comparison of the breakdown graphs of the b 5 ion (produced by fragmentation of protonated YAGFL-NH 2 ) and the protonated cyclo-(YAGFL) peptide analogue to provide strong evidence for a cyclic structure for the b 5 -ion moiety (Scheme 1b). Current ideas [4,[7][8][9] suggest a mixture of possible linear and cyclic structures for a-ions.To gain further...

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Section: Resultssupporting

confidence: 89%

Evidence for structural variants of a- and b-type peptide fragment ions using combined ion mobility/mass spectrometry

Riba-Garcia

Giles

Bateman

et al. 2008

J. Am. Soc. Mass Spectrom.

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“…So far, ions in the higher and lower charge states are less prone to these folding changes. Even for these charge states, there may still be subtle changes in the distributions of closely-related conformers hidden by the relatively low IM resolution in our experiments, compared to the much narrower peaks seen in Clemmer's tandem IM work [68,69].…”

Section: Effects Of Drift Tube Injection Conditions On Folding Of Ubimentioning

confidence: 71%

An ion trap-ion mobility-time of flight mass spectrometer with three ion sources for ion/ion reactions

Zhao

Soyk

Schieffer

et al. 2009

J. Am. Soc. Mass Spectrom.

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This instrument combines the capabilities of ion/ion reactions with ion mobility (IM) and time-of-flight (TOF) measurements for conformation studies and top-down analysis of large biomolecules. Ubiquitin ions from either of two electrospray ionization (ESI) sources are stored in a three dimensional (3D) ion trap (IT) and reacted with negative ions from atmospheric sampling glow discharge ionization (ASGDI). The proton transfer reaction products are then separated by IM and analyzed via a TOF mass analyzer. In this way, ubiquitin ϩ7 ions are converted to lower charge states down to ϩ1; the ions in lower charge states tend to be in compact conformations with cross sections down to ϳ880 Å 2 . The duration and magnitude of the ion ejection pulse on the IT exit and the entrance voltage on the IM drift tube can affect the measured distribution of conformers for ubiquitin ϩ7 and ϩ6. Alternatively, protein ions are fragmented by collision-induced dissociation (CID) in the IT, followed by ion/ion reactions to reduce the charge states of the CID product ions, thus simplifying assignment of charge states and fragments using the mobility-resolved tandem mass spectrum. Instrument characteristics and the use of a new ion trap controller and software modifications to control the entire instrument are described. Ion mobility (IM) [4,5] has become a very useful technique for analysis of biological ions in the gas phase [6]. IM provides information about ion size and structure [7], as it rapidly separates ions based on collision cross-section, rather than just m/z ratio. The use of IM to disperse a mixture of ions in time before analysis via a time-of-flight (TOF) MS, i.e., nested drift (flight) time measurements, is an important recent advance. These experiments were pioneered by Clemmer and coworkers in the mid-1990s [8], and have now been used by several other groups [9 -13]. The recent Synapt ESI-IMS-MS by Waters Corp. provides a commercially available instrument for gas-phase ion conformation studies by IM using a novel traveling wave approach [9].In a series of instrumental designs, the Clemmer group has made various modifications to the initial ESI-IM-TOF, including the insertion of a collision cell between the IM drift tube and the TOF for mobility labeling experiments [14,15], and the addition of an IT before the mobility drift tube to improve the duty cycle from the continuous ESI source [16,17]. One publication demonstrated MS/MS capabilities with an ion trap before IM-TOF [18], but the entire instrument was not under computer control. Therefore, only relatively simple experiments were possible.IM has been used to analyze the products of ionmolecule reactions [19], including proton transfer [20,21], H/D exchange [22], and solvation [23][24][25][26]. In these studies, the desired reactions take place either in the atmospheric pressure ion source interface region or in the drift tube itself. Thus, only short reaction times and certain reagent ions can be used. In addition, performing reactions in the IM cell can make spe...

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“…20 A comparison of the calculated cross-sections to the experimental results shows that the most compact structure (I) could be explained by the cyclic geometry (orange pentagons), whereas the most extended conformer (III) matches the trans imine N-terminal-protonated structure (red triangles). 26 Feature II could be rationalized by the cis imine N-terminal protonated structures (blue dots). It is nonetheless odd that the higher-energy cis imine should be much more abundant than trans imine, the relative contributions of the different structures being estimated at 31% I, 61% II and 8% III.…”

Section: Leu-enkephalin Amentioning

confidence: 98%

On the Dynamics of Fragment Isomerization in Collision-Induced Dissociation of Peptides

et al. 2008

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The structures of peptide collision-induced dissociation (CID) product ions are investigated using ion mobility/ mass spectrometry techniques combined with theoretical methods. The cross-section results are consistent with a mixture of linear and cyclic structures for both b 4 and a 4 fragment ions. Direct evidence for cyclic structures is essential in rationalizing the appearance of fragments with scrambled (i.e., permutated) primary structures, as the cycle may not open up where it was initially formed. It is demonstrated here that cyclic and linear a 4 structures can interconvert freely as a result of collisional activation, implying that isomerization takes place prior to dissociation.

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IMS−IMS and IMS−IMS−IMS/MS for Separating Peptide and Protein Fragment Ions

Cited by 188 publications

References 28 publications

Evidence for structural variants of a- and b-type peptide fragment ions using combined ion mobility/mass spectrometry

Evidence for structural variants of a- and b-type peptide fragment ions using combined ion mobility/mass spectrometry

An ion trap-ion mobility-time of flight mass spectrometer with three ion sources for ion/ion reactions

On the Dynamics of Fragment Isomerization in Collision-Induced Dissociation of Peptides

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