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High Resolution MS/MS by CID or HCD in an LTQ Orbitrap

ESI Ion source Linear Ion Trap C-Trap. Orbitrap. High Resolution MS/MS by CID or HCD in an LTQ Orbitrap. Precursor Isolation for either. Fragmentation by CID. Fragmentation by HCD.

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High Resolution MS/MS by CID or HCD in an LTQ Orbitrap

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  1. ESI Ion source Linear Ion Trap C-Trap Orbitrap High Resolution MS/MS by CID or HCDin an LTQ Orbitrap Precursor Isolation for either Fragmentation by CID Fragmentation by HCD Separation in space of precursor isolation and fragmentation Results in no low mass cutoff for HCD. Mass Analyzer for either HCD is caused by the increased potential difference between LTQ and C-trap (over 25V). Ions are fragmented by collisions with nitrogen. Normal transfer is at ~8 volts.

  2. HCD uses higher CE and lower Q than CID

  3. Electron Transfer Dissociation (ETD) on an LTQ • Injection of multiply protonated peptide molecules (precursor ions) generated by ESI. • Application of a dc offset to move the precursor ions to the front section of the linear trap. • Injection of negatively charged reagent ions from the CI source into the center section of the linear trap. • Application of a supplementary dipolar broadband ac field to eject all ions except those within 3 mass-unit windows centered around the positively charged precursor ions and the negatively charged electron-donor reagent ions. • Removal of the dc potential well and application of a secondary RF voltage (100 V zero to peak, 600 kHz) to the end lens plates of the linear trap to allow positive and negative ion populations to mix and react. • Termination of ion ion reactions by axial ejection of negatively charged reagent ions while retaining positive ions in the center section of the trap. This is followed by mass-selective, radial ejection of positively charged fragment ions to record the resulting MSMS spectrum. Syka et al, PNAS, 101, 9528–9533, 2004.

  4. ETD Acquistion Parameters

  5. ETD Charge Reduction Electron Gain or Proton Loss (M+H)+ (M+3H+2e)+ (K)L/A/D/L/R/G/Q|N/E|D|Q|N|V G\I\K(V) (M+3H+2e)+ (R)F/L/R P\G|D|D|S/S\H|D|L|M|L|L\R(L)

  6. Asp-N Precursor Charge +5 (Y) D/R/E/K L Q E/R V\A K\L A\G\G V|A V I K V\G A A/T E\V\E\M\K\E\K/K\A\R V\E (D) ETD (Y) D R E K L Q E R V A K L A\G G V\A\V\I|K/V/G A A/T/E/V/E/M/K/E/K/K/A R V E (D) HCD (Y) D R E K L Q E R V A K L A G G V A\V|I|K|V|G\A A T E\V E/M K E K\K\A R V E (D) CID

  7. Asp-N Precursor Charge +6, +5, +4 ETD 50 msec (Y) D R\E|K\L Q E|R V\A K\L A\G G|V|A V\I K V G A A T|E V E|M K E\K/K/A/R V E (D) ETD +6 (Y) D/R/E/K L Q E/R V\A K\L A\G\G V|A V I K V\G A A/T E\V\E\M\K\E\K/K\A\R V\E (D) ETD +5 (Y) D/R/E/K L Q/E|R V/A K L A G G V A V I K V G/A A T/E\V\E\M\K\E\K K\A\R\V\E (D) +4 ETD

  8. Asp-N Precursor Charge +6, +5, +4 ETD 30 msec (Y)D/R\E|K\L/Q E|R V\A\K L A\G\G\V|A V I K V G A A/T|E V\E|M K/E\K/K|A/R V\E(D) ETD +6 (Y)D/R/E/K/L/Q E/R V A K/L A/G\G|V|A V I K V|G A\A/T\E\V\E\M\K\E\K\K\A\R V\E(D) ETD +5 (Y)D/R/E/K/L/Q/E/R/V A K L\A G G V A\V I K/V G A A/T/E V\E\M\K\E\K\K\A\R V\E(D) +4 ETD

  9. Lys-C Precursor Charge +5 (K) H K|I P I|K|Y\L|E|F/I S|D|A I I H/V/L H/S\K (H) ETD (K)H K\I\P I K Y L E\F\I/S D/A I/I/H/V/L/H/S K(H) HCD CID (K)H K I P I K Y L E|F|I|S|D/A I I H V L H S K(H)

  10. Lys-C Precursor Charge +5, +4, +3 ETD 50 msec (K) K/G/H/H/E|A|E|L|K P|L A/Q\S\H|A T\K (H) ETD +5 (K) K/G|H|H/E|A\E|L|KP\L|A Q|S|H|A\T\K (H) ETD +4 (K) K/G/H|H|E|A|E|L|KP|L|A\Q|S|H\A T\K (H) +3 ETD

  11. Lys-C Precursor Charge +5, +4, +3 ETD 30 msec ETD +5 Upper range not colleced from CSS z =2 ETD +4 Upper range not colleced from CSS z =3 +3 ETD

  12. Lys-C Precursor Charge +4 (K) K/G|H|H/E|A\E|L|KP\L|A Q|S|H|A\T\K (H) ETD (K) K\G H H\E/A E\L\K/P/L/A/Q/S H A T K (H) HCD (K) K/G/H/H|E|A/E/L K|P L A Q/S H A\T\K (H) CID

  13. CNBr Precursor Charge +4 (F) T G/H P E|T L|E|K|F|D|K|F|K|H|L\K\T\E\A\E\m (K) ETD (F) T G H|P E/T/L E\K/F D/K/F/K/H/L/K T E A/E m (K) HCD (F) T/G/H|P E/T L E|K/F D|K F K|H/L/K T E A\E\m (K) y20+4 603.3 CID

  14. CNBr Precursor Charge +4 (E) T/L|E|K|F|D|K\F K|H|L|K\T\E\A\E\m (K) ETD (E) T/L\E K/F D/K F K/H/L/K T E A E\m (K) HCD (E) T/L/E/K F D|K/F K|H L\K T E A E\m (K) CID

  15. Trypsin Precursor Charge +3 (K) V/E/A/D/I|A|G|H|G|Q|E|V|L|I\R (L) ETD (K) V/E|A|D/I/A/G/H/G/Q/E/V/L I/R (L) HCD (K) V/E|A|D/I/A|G/H/G Q|E|V\L I R (L) CID

  16. Glu-C Precursor Charge +3 (E) V/L/I|R|L/F|T|G|H P\E (T) ETD (E) V/L|I/R L F/T G\H/P E (T) HCD (E) V/L|I/R L F|T/G H|P E (T) CID

  17. Glu-C Precursor Charge +3 (E) V/L/I|R|L/F|T|G|H P\E (T) 50 msec ETD (E) V/L/I|R|L/F|T|G|H P\E (T) 30 msec ETD 30 msec Rxn tends to diminsh +3 fragmentation, more residual reduced precursor.

  18. Chymotrypsin Precursor Charge +3 (L) N/V/W/G|K|V|E|A|D|I|A|G|H G Q\E\V\L (I) ETD (L)N V\W\G K V/E A D\I A G H G Q\E\V L(I) HCD (L)N V/W G K V E A D I\A|G H G Q\E\V\L(I) CID

  19. Glu-C Precursor Charge +3 (E)L F/R|N|D|I|A|A|K|Y|K\E(L) ETD (E)L/F/R N D\I|A|A|K|Y|K/E(L) HCD (E)L/F/R N D|I|A|A|K/Y\K\E(L) CID

  20. Lys-C Precursor Charge +4 (K)Y/L|E|F|I/S|D|A I I|H|V|L|H|S\K(H) ETD (K)Y L\E\F\I/S/D/A I/I/H/V/L/H/S K(H) HCD (K)Y L E F\I|S/D/A I I H V L H S K(H) CID

  21. Precursor Charge +6 (E)A E\M K A S E D\L K K H G T V V\L/T/A/L/G/G I/L/K/K/K/G H/H E A E(L) HCD (E)A E/M K A S E/D|L K K H G T V V L|T\A|L|G G I L K K K G H H E\A\E(L) CID

  22. Precursor Charge +4 (R)L F/T/G/H/P E/T L/E/K/F/D/K/F K(H) HCD (R)L/F|T/G/H/P E T L E K/F D K F K(H) CID

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