Flow-Through Electron Capture Dissociation in a novel Branched RF Ion Trap

Transcription

1 Flow-Through Electron Capture Dissociation in a novel Branched RF Ion Trap Takashi Baba, J. Larry Campbell, Yves Le Blanc, Jim. W. Hager and Bruce A. Thomson ASMS, June 18 / AB SCIEX

2 Trapping vs. Flow-Through for ECD ECD was discovered in FT-ICR MS (Zubarev et al., 1998) key requirements: low energy electrons ~1 ev interaction time > a few ms ECD in RF ion traps? YES, but low duty cycle because of trapping sequence RF + magnetic field (Baba et al / Silivra et al. 2004) Digital RF field (Ding et al. 2006) Flow through ECD? YES, but low electron capture efficiency Static magnetic ion guide (Voinov et al. 2009) New challenge: efficient flow-through ECD by branched RF ion trap, or Chimera trap AB SCIEX

3 ECD device using Chimera Chimera ion guide multiple ion source coupling Chimera ECD device electron emitter electron gate ions magnetic field electrons magnetic field six-way crossed pseudo potential minimum AB SCIEX ions wall electrodes electrons * B. A. Thomson, I. V. Chernushevich, and A. V. Loboda, Practical Aspects of Trapped Ion Mass Spectrometry, Volume IV, Edit R. E. March, J. F.J. Todd, 2010 by CRC Press

4 Instrument Neodymium magnets 0.17 [T] at the trap center yttria (Y 2 O 3 ) e - emitter* *Kimball Physics, Inc. TripleTOF 5600 system (AB Sciex) UPLC-MS C18 column (2 mm x10mm) Information Dependent Acquisition (IDA) in Analyst Software (AB Sciex) AB SCIEX

5 Advantages of Chimera ECD device Advantage #1: Q-TOF like operation Inline ECD device easy to operate Chimera works as an ion guide for MS and CID Advantage #2: flow through operation Electron cloud can be formed always in the trap precursors products high sample consumption duty cycle (~100%) e AB SCIEX

6 intensity [ions] intensity [ions] Quasi flow through ECD pure flow through [neurotensin+3h] 3+ L2 closed : 0 ms intensity [ions] quasi flow through open : 20 ms L2 closed : 10ms open : 10ms [M+3H] 3+ L2 closed : 19ms open : 1ms Z=2+ CRS : [M+3H] 2+ z 10 z 11 z 12 c 10 c 11 c 12 m/z m/z L2 m/z duty cycle: 95% Q1 isolated precursors e AB SCIEX product TOF

7 e- capture efficiency Electron capture efficiency : z and m/z dependence electron capture efficiency (total products) (total ions in spectrum) Sample: BSA, Lys C digest and tryptic digest Z=2+ Z=3+ Z=4+ precursor m/z precursor m/z precursor m/z Electron capture efficiency of Z=2+ is lower. but ECD are still useful for Z=2+ precursors AB SCIEX

8 dissociation efficiency Dissociation efficiency is high for all charge states dissociation efficiency (dissociated products) (total products) Sample: BSA as Lys C digest and tryptic digest Z=2+ Z=3+ Z=4+ precursor m/z precursor m/z precursor m/z Dissociation efficiency is high for all charge states AB SCIEX

9 ECD on BSA LysC digest BSA LysC 002.wiff 6+ Five ECD spectra / sec injected sample: 30 pmol flow: 300uL/min AcN in water: gradient: 2% 50% / 10 min sequence coverage: 85% AB SCIEX

10 Advantage of electrons (ECD) to reagent anions (ETD) tunable electron energy : ExD family non proteomics applications AB SCIEX

11 ExD Family: Hot ECD ev ETD ECD HotECD EIEIO HotECD (ΔZ = -1) *Leu / Ile identification **glycosylated peptides & proteins Demonstration in Chimera ECD: glycopeptides in Fetuin 15 N glycosylation O glycosylation 20 PEPTIDE-NXS PEPTIDE LC-ECD MS conditions Gal Man GalNAc GlcNAc Neu5Ac Neu5Gc bovine fetuin digest by trypsin, Cys: carboamidomethylated, Injection: 100 pmol LC condition: AcN in water: 2 35% (20 min.), flow rate: 450 ul/min ECD: 7 ev, accumulation: 0.5 sec / spectrum, quasi flow through duration: 49ms AB SCIEX *ref: F. Kjeldsen et al. Chemical Physics Letters 356 (2002) **ref: N. Manri et al. Anal. Chem. 85(2013)

12 intact O and N glycosylated peptides by single LC-Hot ECD(7eV) MS Hot ECD on N-glycosylated peptide Hot ECD on O glycosylated peptide 979(5+) 1151(4+) 1078(4+) 1176(5+) 1138(6+) 1176(5+) 1059(4+) 991(4+) 987(4+) *Glycan 12 subunits 2014 were AB SCIEX verified by CID. 1041(6+) 1089(6+) 1047(7+) 999(8+) 1118(5+)

13 ExD Family: EIEIO ev ETD ECD HotECD EIEIO *EIEIO (ΔZ = 0) Electron Induced Excitation of Ions from Organics * Ref: R. B. Cody and B. S. Freiser, Analytical Chemistry, 51, Mechanism: No electron capturing electric and vibrational excitation of molecules Applicable to singly charged small molecules 20 Demonstration in Chimera ECD double bond identification in singly charged lipids OPPC POPC AB SCIEX 1-oleoyl-2-palmitoyl-sn-glycero-3-phosphocholine 1-palmitoyl-2-oleoyl-sn-glycero-3-phosphocholine

14 EIEIO (5eV): Alkyl chain dissociation x30 radical [OPPC+H] + [M+H] + non-radical c13 alkyl chain fragments -CH 2 - -CH 2 - -CH 2 - -CH -CH CH 2 - -CH 2 - -CH 2 - -CH 2 - -CH 2 - -CH 2 - -CH 2 - -CH 2 - -CH AB SCIEX

15 EIEIO (5eV): double bond location double bond [OPPC+H] + double bond location AB SCIEX

16 EIEIO application: POPC/OPPC identification x40 [POPC+H] [M+H]+ POPC unique m/z [OPPC+H] + OPPC unique [M+H]+ 16 x30 [M+H] AB SCIEX

17 Summary Chimera ExD device Q-TOF friendly Flow-Through Electron Capture Dissociation in a novel Branched RF Ion Trap ions Quasi flow through ExD high sample consumption duty cycle high ExD efficiency e- Wide variety of ExD family on multiply charged precursor ions singly charged precursor ions Special thanks Pablo Dominguez (mechanical design) Deolinda Fernandes (sample preparation) 0 ev 5 10 ECD HotECD EIEIO AB SCIEX

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