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Higher-energy C-trap dissociation for peptide modification analysis

Abstract

Peptide sequencing is the basis of mass spectrometry–driven proteomics. Here we show that in the linear ion trap–orbitrap mass spectrometer (LTQ Orbitrap) peptide ions can be efficiently fragmented by high-accuracy and full-mass-range tandem mass spectrometry (MS/MS) via higher-energy C-trap dissociation (HCD). Immonium ions generated via HCD pinpoint modifications such as phosphotyrosine with very high confidence. Additionally we show that an added octopole collision cell facilitates de novo sequencing.

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Figure 1: Schematic of the hybrid linear ion trap–orbitrap instrument indicating the electrostatic potentials used in HCD, and comparison of LTQ and HCD fragmentation.
Figure 2: Detection of the phosphotyrosine-specific reporter ion.
Figure 3: Determination of y ions for de novo sequencing.

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Acknowledgements

This work was supported by the EU research directorate (Interaction Proteome grant LSHG-CT-2003-505520). We thank other members of the department for Proteomics and Signal Transduction and of Thermo Fisher Scientific, especially R. Pesch and K. Strupat, for constructive comments and discussion.

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Correspondence to Stevan Horning or Matthias Mann.

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O.L., A.M. and S.H are employees of Thermo Fisher, the manufacturer of the LTQ Orbitrap instrument used in this research.

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Supplementary Figures 1–3, Supplementary Table 1, Supplementary Methods (PDF 3566 kb)

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Olsen, J., Macek, B., Lange, O. et al. Higher-energy C-trap dissociation for peptide modification analysis. Nat Methods 4, 709–712 (2007). https://doi.org/10.1038/nmeth1060

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