a Quadrupole Mass Filter

Transcription

1 Performance Investigation of an Orbitrap Mass Analyzer Combined with a Quadrupole Mass Filter Jan-Peter Hauschild Ulf Fröhlich Oliver Lange Alexander Makarov Eugen Damoc Sebastian Kanngiesser Frank Czemper Catharina Crone Yue Xuan Markus Kellmann Andreas Wieghaus

2 Instrumentomics in action Detect speed Instrument polymerasa Speed of isolation Image from: Thermo Scientific Q Exactive LC-MS/MS 2

3 What are new interesting features? Quadrupole mass filter Enhanced Fourier transformation (eft ) for Orbitrap data processing Predictive automatic gain control (pagc) and parallel filling & detection Possibility of multiple fills for spectrum multiplexing S-lens for higher transmission (like in Thermo Scientific LTQ Orbitrap Velos ) with rugged optics C-trap directly interfaced to HCD (like in LTQ Orbitrap Velos instrument) 3

4 It is a simple instrument! C-trap Quadrupole mass filter Bent Flatapol HCD collision cell Outer electrodes Injection Flatapole Central electrode Z-lens S-Lens assembly Figures and design courtesy J. Rykl, Thermo Fisher Scientific, 211 Capillary 4

5 Layout of the new instrument 5

6 Transmission curves for quadrupole mass filter # R3 ance Relative Abunda 2 # # # amu IT=15 ms NL=1e8 1 amu IT=7.8 ms NL=9e7 2 amu IT=5.7 ms NL=8e7 3 amu IT=3.6 ms NL=8.5e7 Abundance Relative A # # # amu IT=2.7 ms NL=8.6e7 7 amu IT=2.4 ms NL=8.9e7 1 amu IT=1.2 ms NL=9e7 Ion current in a peak (NL) is sustained at the level of a perfectly transmitting (no resolving DC) quadrupole for all filter windows Ion fill time is adjusted to account for transmission of quadrupole and appearance of additional mass peaks in the window. 6

7 Enhanced FT as a tool for increasing resolving power MS analysis of two peptides (Val5-Angiotensin II and Lys-des-Arg9-Bradikynin) C:\Xcalibur\...\EDA\doublets_eFTon_256ms 5/9/211 9:52:16 AM differing in mass by 12.1 mmu (triply charged ions) RT: Relative Ab bundance Hz 3.7Hz Hz 3.7Hz Time (min) NL: 2.84E8 R= R=168 NL: 5.73E9 TIC MS doublets_efto ff_256ms 5 NL: E9 TIC MS doublets_ef Ton_256ms doublets_eftoff_256ms# 38 RT:.17 AV: 1 T: FTMS + p ESI Full ms [15.-2.] R= R= NL: 3.78E8 R=654 doublets_efton_256m 1 Val5 Angiotensin II Lys des Arg9 Bradykinin s#5 RT:.2 AV: 1 T: C H 71 N 13 O C 12 5 H 75 N 13 O 11 FTMS + p ESI Full ms R=61 [15.-2.] ppm -.87ppm R= R= eft off, 256ms eft on, 256ms eft off, 256ms eft on, 256ms Please see: O. Lange; E. Damoc; A. Wieghaus; A. Makarov. Enhanced FT for Orbitrap Mass Spectrometry. Proc. 59th Conf. Amer. Soc. Mass Spectrom., Denver June 5-9, 211, Poster MP93. 7

8 Resolving power and mass accuracy of the Orbitrap analyzer Calmix_14kRes_1.8Hz # 1 RT:.1 AV:1 NL:2.73E R= R= R= R= R= R=5872 R= R=5652 R= R= R= R= R= R= Res Transient Max. scan length, speed, 2 ms Hz 14, , , , Calmix_ 17.5kRes_ 13.2Hz # 82RT:.1 AV:1 NL:3.1E8 e R= R= R= R= R=772 R= R= R= R= R= R=652 R= R= R= Ion Score nanolc of E.coli: Sample of 7775 points r.m.s ppm Mass Error, ppm 8

9 Long-term mass accuracy with external calibration ation [ppm] 1-1 Devi ] Tempera ature Change [K] C/5.5 F Time [h] Time [h] Realistic conditions of an average lab Air cooling only! Experiment was carried out with temperature variations up to 3 C peakto-peak, up to 1 C/hour 9

10 Mass accuracy with polarity switching and external calibration 1 positive + 1 negative scan in 1 second 1

11 Spectral acquisition speed with predictive AGC and parallel filling/detection Scan speed does not change until ion fill time reaches 5 ms Scan ns per second, Hz % 8% 7% 6% 5% 4% 3% 2% 1% % Duty cy ycle of ion filli ing, % Scan speed Duty cycle Up to 65% of the total time could be spent meanwhile on accumulating ions! Ion fill time, ms Resolving power setting: 17,5 (min.) At higher resolving powers, ion fill times and duty cycles are even longer! 11

12 HCD performance 524 (MRFA peptide) 1522 (Ultramark) _HCD_524 #142 RT: 1.27 AV: 1 NL: 6.84E7 T: FTMS + p ESI Full ms @hcd1. [7.-14.] _HCD_1522 #15 RT: 1.35 AV: 1 NL: 1.63E7 T: FTMS + p ESI Full ms @hcd15. [12.-2.] TIC=8.3e TIC=1.9e Relative Abundan nce Relative Abundanc ce _HCD_524 #229 RT: 2.11 AV: 1 NL: 1.14E7 T: FTMS + p ESI Full ms @hcd35. [7.-14.] _HCD_1522 #244 RT: 2.28 AV: 1 NL: 1.39E6 T: FTMS + p ESI Full ms @hcd4. [12.-2.] TIC=6.7e7 7 NCE= TIC=1.3e7 NCE= Fill times are similar to those in LTQ Orbitrap Velos instrument Direct interfacing of the C-trap to HCD cell minimizes losses during fragmentation 12

13 Q Exactive LC-MS/MS in action: E.coli digest Unique peptides 843 <1% FDR 5ug 1ng 2ng E. Coli 584 digest 2h gradient, Proxeon NanoUPLC 3 search engines Unique proteins ug 1ng 2ng 7, 256 ms 17,5 64 ms ddtop1 HCD acquisition method Fill times HCD HCD HCD HCD Full MS HCD HCD HCD HCD HCD HCD Data dependent acquisition cycle time [sec] 1 sec 13

14 Q Exactive LC-MS/MS in action: HeLa digest 25 Unique Peptide IDs at <1% FDR ug HeLa 1h grad 5ug HeLa 2h grad ug HeLa 1h grad Protein IDs ug HeLa 2h grad Total: run I run II Sample courtesy K. Mechtler Please see also: C. Paschke; Y. Xuan; E. Damoc; T. Ueckert; U. Comberg; H. Grensemann; M. Kellmann; B. Delanghe. Breaking the 2 proteins barrier in a standard LC run using a new benchtop Orbitrap instrument and multiple search engines.. Proc. 59th Conf. Amer. Soc. Mass Spectrom., Denver June 5-9, 211, Poster MP

15 What do we gain by selected ion monitoring? 8 In Full MS, total C-trap charge 6 capacity is shared between 4 2 multiple signals of different intensity 1 Signal-to-noise ratio becomes 8 6 dependent on the ratio of compound of interest to other 2 analytes- much less so in SIM! In Orbitrap instruments, SIM could become MRM without any additional overhead! N= NL: 1.94E8 [15.-2.] N= Full MS S/N = 745 IT=.245 ms SIM (1amu) For the same target: S/N = 54 4 IT= ms S/N (spectrum m) Gain in sensitivity (7x) Sensitivity gain 5 1 x with SIM mode 2 The gain will be higher in more 1 complex matrices 3 Lowest signal 2533 NL: 1.12E8 [ ] Lowest signal 2824 Caffeine S/N (FMS) S/N (SIM1) 15

16 Alprazolam, Full-Scan Experiment Alprazolam Y = *X R^2 =.9967 W: 1/X Area ppt 1 ppb 25 fg oc - 5 pg oc Area 2 Zoom in 5 ppt- 1ppt fg/ul fg/ul 16

17 Alprazolam SIM Experiment Alprazolam Y = *X R^2 =.9982 W: 1/X ppt 1 ppb 5 fg oc - 5 pg oc Area Area Zoom 1 ppt- 1ppt fg/ul fg/ul See also: X. He; M. Kozak. Evaluation of quantitative performance for testosterone analysis in plasma on a novel quadrupole Orbitrap mass spectrometer. Proc. 59th Conf. Amer. Soc. Mass Spectrom., Denver June 5-9, 211, Poster WP77. 17

18 Spectrum multiplexing: principle of operation Standard operation mode vs Spectrum multiplexing 5.x x1 8 4.x x1 8 t (arb.) Ion count 3.x x1 8 2.x x1 8 1.x C-Trap storage: No ion loss over a broad range of storage times! (St.Steel, fused silica, ceramics) 5.x Inject time [ms] 18

19 Spectrum multiplexing: example of 4-plex SIM Experimental Setup: 1 Cycle 12 Hz Acquisition Rate 48 Precursors / second 1s 4-plex injection Collecting 4 isolation windows 64ms detection Please see: O. Lange; J.-P. Hauschild; A. Makarov; U. Fröhlich; C. Crone; Y. Xuan; M. Kellmann; A. Wieghaus. Multiple C- Trap Fills as a Tool for Massive Parallelization of Orbitrap Mass Spectrometry- a new Concept for Targeted Mass Analysis. Poster MP13 19

20 Example Fluoxastrobin, [M+H]+ calc RT: RT: Re elative Abundance All 4-plex acquisitions Following just one target out of that 4-plex ance Relative Abunda Time (min) All 4-plex acquisitions Following just one target out of that 4-plex.99s zoom Time (min) Iso1 Iso3 NL: 8.83E8 83E8 TIC MS 11418_tSIM_48pest_4plex_1min_3ppb _ ppm (ext.) C 21 H 16 ClFN 4 O 5 NL: 8.49E7 = F: FTMS + ESI SIM msx ms [ , , , Iso ] MS 11418_tSIM_48pest_4plex_1min_3ppb _ Iso Isolation window NL: 4.1E8 TIC MS 11418_tSIM_48pest_4plex_1min_3ppb _1 Iso4 If we were doing this by conventional SIM, we would have had 4 times less data points across the peak! NL: 8.49E7 = F: FTMS + ESI SIM msx ms [ , , , ] MS 11418_tSIM_48pest_4plex_1min_3ppb _1 At high resolution and mass accuracy, fragmentation is frequently not needed! 2

21 Conclusion: one more step to Mass spec sapiens! Q Exactive instrument combines Orbitrap and quadrupole analyzers to form an innovative instrument with a unique combination of analytical features. It appears to be especially suited for: Screening Quantitation Heavy-duty targeted analysis Ultra-high performance LC/MS Tom Schmal, 1999 Metabolite and peptide identification =File:I%27m_a_mammal!.jpg&oldid=

22 Acknowledgements S. Horning I. Mylchreest A.Guiller R.A.Purrmann F. Grosse-Coosmann A. Kuehn T. Rietpietsch E. Denisov R. Malek M. Biel C. Henrich M. Mueller A. Venckus F. Gebrehewit F. Afroz O. Hengelbrock M. Antonczak S. Moehring S. Nimkar M. Kosak K.Scheffler X. He Y. Huang A. Kholomeev F. Paffen B. Rose A. Boegehold J. Grote W. Huels A. Schumbera S. Simmel P. Bennett K. Cook A. Huhmer Z. Hao S. Peterman Y. Zhang K. Comstock T. Stratton D. Ghoshh C. Yang M. Blackburn 22