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Manual MS/MS spectra interpretation

Manual MS/MS spectra interpretation. Practical course 18 .03.2014. Overview of fragmentation modes. CID: collision induced dissociation HCD: higher energy collision induced dissociation ETD: electron transfer dissociation. CID, ETD. HCD. Typical scan cycle.

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Manual MS/MS spectra interpretation

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  1. Manual MS/MS spectra interpretation Practical course 18.03.2014

  2. Overview of fragmentation modes • CID: collision induced dissociation • HCD: higher energy collision induced dissociation • ETD: electron transfer dissociation CID, ETD HCD

  3. Typical scan cycle • Accumulation of 1e6 intact peptide ions in linear ion trap • Transfer of intact peptide ions into orbitrap and acquisition • Overfilling of linear ion trap and isolation of ~3e4 precursor ions in linear ion trap • Fragmentation of precursor ions and acquisition of fragment spectrum (MS/MS)

  4. Characteristics of CID and HCD • CID • Peptides are fragmented in an ion trap • “low energy” fragmentation • Peptides undergo multiple collisions with inert gas molecules • Additional excitation (RF pulse) required • HCD • Peptides are fragmented in dedicated fragmentation cell (quadrupole, octopole) • “high energy” fragmentation • Peptides are accelerated and collide with inert gas molecules • Peptides fragment instantaneously

  5. Characteristics of CID and HCD • CID • Works best for 2+/3+charge states • Efficient fragmentation • Sensitive detector (1 ion -> 1 detector count) • Produces predominantly b and y ions • Low mass cutoff • Low resolution/mass accuracy • Fragment charge state determination (>1+) impossible • HCD • Efficient fragmentation • Detector less sensitive (~5 ions required for orbi signal) • Produces mainly y ions, but also internal ions • No low mass cutoff • High resolution/mass accuracy • Fragment charge states

  6. Peptide fragmentation nomenclature

  7. Peptide fragmentation • MS/MS spectra y-ions: C  N B-ions: N  C

  8. y1 ions • If you have performed a trypsin digest then one of these ions should be present in the MS/MS fragmentation spectrum and this will reveal the C-terminal residue.  • The N-terminal b1 ion is never observed (often the b1 ion can be found after acetylation of the peptide) 1xOH = 17,00274 1xH = 1,00783 1xH+ = 1,00728

  9. y1 ions

  10. MS/MS interpretation • Good MS/MS spectrum

  11. MS/MS interpretation • Example exercise m/z 448.22 z =2

  12. MS/MS interpretation • Example exercise m/z 448.22 z =2

  13. MS/MS interpretation • Example exercise m/z 448.22 z =2

  14. MS/MS interpretation • Example exercise m/z 448.22 z =2

  15. MS/MS interpretation • Example exercise m/z 448.22 z =2

  16. MS/MS interpretation • Example exercise m/z 448.22 z =2

  17. MS/MS interpretation • Example exercise m/z 448.22 z =2

  18. MS/MS interpretation m/z 448.22 (2+) [M+H] = 895.43 • Example exercise

  19. MS/MS interpretation m/z 448.22 (2+) [M+H] = 895.43 • Example exercise

  20. MS/MS interpretation m/z 448.22 (2+) [M+H] = 895.43 • Example exercise C  N

  21. MS/MS interpretation m/z 448.22 (2+) [M+H] = 895.43 • Example exercise

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