Multipurpose Dissociation Cell for Enhanced ETD of Intact Protein Species

Abstract

We describe and characterize an improved implementation of ETD on a modified hybrid linear ion trap-Orbitrap instrument. Instead of performing ETD in the mass-analyzing quadrupole linear ion trap (A-QLT), the instrument collision cell was modified to enable ETD. We partitioned the collision cell into a multi-section rf ion storage and transfer device to enable injection and simultaneous separate storage of precursor and reagent ions. Application of a secondary (axial) confinement voltage to the cell end lens electrodes enables charge-sign independent trapping for ion–ion reactions. The approximately 2-fold higher quadrupole field frequency of this cell relative to that of the A-QLT enables higher reagent ion densities and correspondingly faster ETD reactions, and, with the collision cell’s longer axial dimensions, larger populations of precursor ions may be reacted. The higher ion capacity of the collision cell permits the accumulation and reaction of multiple full loads of precursor ions from the A-QLT followed by FT Orbitrap m/z analysis of the ETD product ions. This extends the intra-scan dynamic range by increasing the maximum number of product ions in a single MS/MS event. For analyses of large peptide/small protein precursor cations, this reduces or eliminates the need for spectral averaging to achieve acceptable ETD product ion signal-to-noise levels. Using larger ion populations, we demonstrate improvements in protein sequence coverage and aggregate protein identifications in LC-MS/MS analysis of intact protein species as compared to the standard ETD implementation.

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Acknowledgments

The authors gratefully acknowledge support from Thermo Fisher Scientific, NSF CAREER grant 0747990, and NIH grant R01 GM080148. C.M.R was funded by an NSF Graduate Research Fellowship and NIH Traineeship (T32GM008505).

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Correspondence to Joshua J. Coon.

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Christopher M. Rose and Jason D. Russell contributed equally to this work.

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Rose, C.M., Russell, J.D., Ledvina, A.R. et al. Multipurpose Dissociation Cell for Enhanced ETD of Intact Protein Species. J. Am. Soc. Mass Spectrom. 24, 816–827 (2013). https://doi.org/10.1007/s13361-013-0622-0

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Key words

  • ETD
  • Instrumentation
  • Intact protein
  • Ion–ion reactions