GSAT501: Proteomics Fragmentation: CID & ETD

1. GSAT501: Proteomics Fragmentation: CID & ETD Nichollas Scott

2. The sum of the parts can be more informative then the whole Consider a peptide observed with a mass of 1000.45 Da ±0.02 ~10 elemental compositions i.e Example of nearly mass indistinguishable peptide Bradykinin (C50H73N15O11)- RPPGFSPFR, 1059.5614 Da bradykininisobare (C52H73N11O13)- H-VGPPGFSPFVG-OH, 1059.5389Da Isotopomers can not be identified by mass alone RPPGFSPFR, (C50H73N15O11)- 1059.5614 Da RPPGFFPSR, (C50H73N15O11)- 1059.5614 Da Zubarev, R et alAnal. Chem. 1996, 68, 4060-4063

3. What do we want out of fragmentation? % E T I D P E P m/z • Fragmentation should be predictable • The resulting fragmentation approach should be informative • The fragmentation needs to be efficient for your analyte

4. Fragmentation should be predictable • Proteomics experiments have developed to generate samples which when fragmented lead to predictable and informative fragment ions • Cation: Fragmentation generates predictable sequence ion • Anion: Dominated by neutral losses and internal fragment • Peptides (and proteins) are linearly arranged chains of amino acids Roepstorff, P and Fohlman, J. Biomed Mass Spectrom. 1984, 11, 601

5. Fragmentation terminology H H O H N C C OH R Residue (amino acid -H2O) Amino acid H H O N C C R N-terminus C-terminus Sequence ions Internal Cleavage Ions Immonium Ions

6. Types of fragmentations: • Thermal(vibrational) induced fragmentation • Electron induced fragmentation Fragmentation Electron Thermal Resonance based collision induced dissociation (IT) Beam type collision induced dissociation (Quad) Electron transfer dissociation (IT) Electron capture dissociation (ICR) IT- ion trap Quad- quadrupole ICR- Ion cyclotron resonance

7. Collision induced dissociation • Most commonly used form of fragmentation in proteomics • Produces mainly b and y ions • Exciting of ions by collision with non-reactive particles such as an inert gas • Collisions lead to the translational energy of the ion being converted to internal energy • Ideal for +2, +3, typtic peptides Beam type CID Resonance CID Neuhauser N. et al J. Proteome Res. 2012, 11, 5479-5491

8. Resonance based CID • Used on trapping instruments (ion traps) • Low-energy collision method which activates a specific m/z region by exciting ions in the m/z region to collide with inert gas • Very efficient and leads to product ion not being subjected to further vibrational energy • The bottom lower mass region of the spectrum ion is lost in this process • Slow process (10 to 100 ms) 30ms 15ms % % % m/z m/z m/z Wells J and Mcluckey S. Methods Enzymol. (2005) 402:148-85.

9. Beam type CID • Uses a dedicated collision cell • Low-energy collision method which activates a specific m/z and its product ions with the collision gas of the collision cell • Efficiency is a balance between effectively fragmenting the precursor and maintaining the product ions • Maintains the low mass ions • Fast process (0.1 to 1 ms) NCE: 45 NCE: 15 NCE: 0 NCE: 28 % % % % m/z m/z m/z m/z Wells J and Mcluckey S. Methods Enzymol. (2005) 402:148-85.

10. Proton mobility theory of CID • Provides a model to explain the fragmentation of protonated peptides • Involves the movement of a proton from a site of proton affinity (basic site) to to the nitrogen of the amide bond to be cleaved • The movement of the protein to the nitrogen of the amide allow the nucleophilic attack by the oxygen leading to fragmentation Paizs B, Suhai S. Mass Spectrom Rev. 2005(4):508-48.

11. Proton mobility theory of CID • Charge states (the availability of a mobile proton) effects the fragmentation +2 +3

12. Electron based fragmentation • More recently developed class of fragmentation approaches • Maintains liable modifications such as phosphorylation and glycosylation • High charge density peptides lead to more informative fragmentation • The addition of an electron largely leads to the specific cleavage of the amide bond and generation c’ and z. *Simons J and Ledvina A R. Int J Mass Spectrom. 2012 (in press)

13. Electron transfer dissociation • An increasingly common alterative to CID, electron transfer dissociation (ETD) is now commercially available on Ion traps • ETD occurs by the incubation of a electron carrier (fluoranthene) molecule with the cation(ion-ion interaction) in the gas phase

14. ETD is complementary to CID • Electron based fragmentation approaches work better on higher charge states compared to CID B Swaney D L, McAlister G C, Coon J J. Nature Methods (2008) 5, 959 – 964

15. Not all fragmentation approaches yield the same amount of information: phosphopeptide • The charge state, composition and the present of labile modification all effect which fragmentation approach is most appropriate for your sample Kim, M.-S. and Pandey, A. Proteomics. (2012) 10.1002/pmic.201100517

16. ETD leads to enhance site localization of phosphorylation events • As ETD leads to more uniform (random) fragmentation it has been noted that more fragments flanking modifications can be identified= better site localisation. Blue =ETD, Red = CID Swaney D L et al. PNAS 2009;106:995-1000

17. Not all fragmentation approaches yield the same amount of information: glycopeptide CID Only labile glycan fragmentation Only peptide fragmentation ETD HCD Peptide fragmentation with extensive sugar fragmentation

18. Conclusions • Exact mass alone will not always allow the identification of a peptide • Fragmentation give additional information • Cations fragment predictably are used for most high-throughput proteomic • Multiple types of fragmenation exist • CID is most useful for tryptic peptide with a charge state of +2 and +3 • ETD is better for high charge density peptides