Thermo Scientific Velos Pro Ion Trap and Orbitrap Elite Hybrid Mass Spectrometer – Creating New Possibilities in Research and Discovery

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Part 2 – Introducing the Orbitrap Elite

Martin Zeller, PhD Thermo Fisher Scientific Bremen, Germany

October 18, 2011 Hybrid Orbitrap Technology: Thermo Scientific Orbitrap Velos Pro

New: • Generation II ion optics • Novel detection system

Software platform: • LTQ Tune 2.7 • Xcalibur 2.1 SP1 • Found ati on 1 102SP2.0.2 SP2 • LC Devices 2.5 • Windows 7 (32 bit)

2 Hybrid Orbitrap Technology: Thermo Scientific Orbitrap Elite

New: • Generation II ion optics • Novel detection system • High-field Orbitrap analyzer • New pre-amplifier • Advanced Sigggnal Processing

Software platform: • LTQ Tune 2.7 • Xcalibur 2.1 SP1 • Found ati on 1 102SP2.0.2 SP2 • LC Devices 2.5 • Windows 7 (32 bit)

3 Orbitrap Elite: Key Application Areas

• Key application: Proteomics • Orbitrap Elite provides a new standard of performance for comprehensive proteomics • Applications: • Quantitative cellular proteomics (SILAC, iso bar ic la be ling exper iments (TMT), la be l free ) • Cell signaling applications (intact proteins, PTM analysis, protein ID)

• Key application: Metabolomics • Orbitrap Elite opens a new analytical frontier by providing ultra high resolu tion ana lys is a t high sensiti v ity w ithou t comprom is ing spee d or MSn performance • Applications: • Infusion based lipidomics, fluxomics (isotope tracing, pathway analysis), MSn de novo structural elucidation with UHPLC compatibility

4 Hardware Changes – High-field Orbitrap Analyzer & Advanced Signal Processing High-field Orbitrap Analyzer 1.5 x 1.2 x

Standard Orbitrap High-field Orbitrap • Smaller size- 1.8x frequency at the same voltage → 118.8 x higher resolution over standard Orbitrap • New lenses for focusing ions onto Orbitrap entrance • Change of Orbitrap ggyeometry for the first time since the introduction of Orbitrap technology in 2005!!! Prof. Neil Kelleher, Kelleher Lab ,Northwestern University: “This is not an incremental improvement…!”

6 Advanced Signal Processing

A) Initial phase 0 degrees B) Initial phase -45 degrees • Fourier transformations provide a Magnitude spectra complex value consisting of magnitude and phase component • On all our previous FT mass Absorption Mixed- spectrum mode spectrometers magnitude mass spectrum spectra were generated • Mass spectrum doesn‘t depend on Dispersion Mixed-mode spectrum spectrum „initial phase“ (but broader peak Figure courtesy of R. Malek shape)

• To increase resolution „absorption-spectra“ are used:

This requires an initial phase of 0 degrees (a) for all ions

→ Synchronization of ion injection in the Orbitrap and start of transient acquisition

(determination of t0) is required See aslo ASMS Poster MP093 → Hardware optimization Oliver L ange et al .

7 Advanced Signal Processing - Working Principle

Injection of ions into the Orbitrap:

• At time t0 all the ions are injected at (nearly) identical phase φ0 and start oscillating

• Recorded transient starts close to this point t0 but not exactily (ToF, electronics delay and jitter)

OE-1 Deflector C-trap Lenses CEC

OE-2

• In order to make use of the „inner information“ these starting conditions must be known with high accuracy (tens of nanoseconds)

• If multiple ions are present, the parameters t0 and φ0 can be found out by matching

8 See ASMS poster: M093 Lange et al., Enhanced FT for Orbitrap Mass Spectrometry Advanced Signal Processing on the Orbitrap Elite

993. 97701 R=110221 100 993.89200 R=108377 994.08274 Advanced 80 R=107785 993.82838 60 994.14601 Signal R=113310 R=107537 Processing 40 993.63666 R=108853 994.23048 992.0 992.5 993.0 993.5 994.0 994.5 995.0 995.5 996.0 996.5 R=103203 m/z ON 20

0 993.96967 R=61888 100 993.88335 994.07713 R=64816 80 R=62566 Advanced 993.81876 994.14134 Signal 60 R=69298 R=65804 993.73254 994.22684 Processing 40 R=70251 R=66506 992.0 992.5 993.0 993.5 994.0 994.5 995.0 995.5 996.0 996.5 OFF 20 m/z

0 993.6 993.8 994.0 994.2 994.4 m/z Intact Yeast Enolase (46. 64 kDa), 47+ ion, 768 ms transients

9 (P<1*10-10 Torr) Intact Protein Analysis: Yeast Enolase (46.64 kDa)

Yeast Enolase, 47+ is baseline Orbitrap Elite 993.9770 R=110221 resolved on Orbitrap Elite. 100 993.8706 At 47+ c harge s ta te, the mass R=107582 994.1036 80 R=108475 difference between isotopic peaks is only 0.021 Th! 60 undance 994.1885 bb R=109018 40 993.6367 993.0619 R=108853 993.4244 994.4433 R=109083 R=99350 R=105946 994.7200 994.9536

Relative A 20 R=109283 R=98949 0 994.0309 R=44604 100 993.9030 R=42104 Orbitrap Velos Pro 80 993.7112 994.1802 R=35704 R=42804 60 993.5618 994.4540 R=47304 R=52804 40 993.1357 994.6257 R=42304 R=37604 994.8610 20 R=32404

0 993.0 993.5 994.0 994.5 995.0 m/z

ASMS 2011, MP092: Increased Analytical Performance on a Hybrid Linear Ion Trap-FTMS Mass Spectrometer 10 with a High-Field Orbitrap Mass Analyzer, Martin Zeller, Catharina Crone, Mathias Mueller, Eugen Damoc, Eduard Denisov, Alexander Makarov, Dirk Nolting, and Thomas Moehring Resolution of Orbitrap Elite vs. Competition

Orbitrap Elite AB Sciex Triple Tof 5600 Bruker Maxis Agilent 6540 400,000

350, 000

300,000 M)

HH 250, 000

200,000 lution (FW

oo 150, 000 Res 100,000

50,000

0 0 200 400 600 800 1000 1200 1400 1600 1800 2000 m/z

 Outperforming high resolution Q-ToF’s by a factor > 5

11 MSn Capabilities & Multiple Fragmentation Techniques CID – HCD – ETD Benefits of Multiple Fragmentation Methods & MSn Capabilities

- MSn capabilities - sensitivity Freedom of fragg,mentation, freedom of detection & MSn level

13 Proof Statement: Resolution vs. Scan Speed Scan Speed at Constant Resolution (60,000 FHWM)

\\debre-fs01\nike\...\192ms_pcon_agcon 6/22/2010 4:37:09 PM

2 4 5 8 10 13 16 17 19 21 23 26 27 0.0174 0.0450 0.0589 0.1005 0.1284 0.1700 0.2117 0.2255 0.2534 0.2810 0.3089 0.3504 0.3644 60

LTQ Orbitrap undance bb 40 121.2 Hz Velos 20 0

Relative A 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 Time (min) 3 13 17 22 37 40 48 52 64 71 76 83 94 ee 0. 0095 0. 0500 0. 0662 0. 0863 0. 1468 0. 1590 0. 1913 0. 2075 0. 2560 0. 2843 0. 3045 0. 3327 0. 3772 60 Orbitrap Elite 40 20 4.1 Hz 0 elative Abundanc

RR 0.00 0.05 0.10 0.15 0.20 0.25 0.30 0.35 Time (min) 393.22412 R=68501 100 R 68,000 undance

LTQ Orbitrap bb 50 394.22696 392.28671 R=64104 Velos R=67904 0

Relative A 392.0 392.5 393.0 393.5 394.0 394.5 395.0 395.5 396.0 m/z 393.22427 ee R= 63401 100 R 64, 000

Orbitrap Elite 50 394.22751 392.37296 392.94470 393.29700 R=62004 394.94144 395.16736 R=64204 R=67704 R=65504 R=69104 R=66704 0 elative Abundanc

R 392.0 392.5 393.0 393.5 394.0 394.5 395.0 395.5 396.0 m/z

15 MS Scan Speed at Different Resolutions

9 8 7 (Hz) 6 5 Orbitrap Elite 4 n speed aa 3 Orbitrap Velos Pro 2

MS sc 1 0 7.5 15 30 60 120 240k Resolution @ m/z 400  240,000 at 1 Hz: Exploring new possibilities  60,000 at 4 Hz: Exploiting the faster acquisition rate  15, 000 at 8 Hz: Increasing HCD scan speed

16 Orbitrap Elite vs. LTQ Orbitrap Velos: Cycle Times for TOP10 HCD Experiment

192_ 48_e FTon_ 10ms_ de lay_ 100812172536 8/12/2010 5: 25: 37 PM

1 NL: 45 0.00695 12 23 34 1.62E8 100 0.06968 0.13252 0.19518 0.25802 TIC MS LTQ Orbitrap Velos calmix_ddHCDto 3.76 s p10_MRFA_1500 MS @60k bundance 50 0_2ms_MS_1ms AA 2 6 9 16 19 26 33 37 42 _MS2 HCD@15k 0.02310 0.03939 0.05355 0.09745 0.11161 0.15550 0.18853 0.21819 0.24179 0

Relative Relative 0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 0.20 0.22 0.24 0.26 Time (min) 1 NL: 1.62E8 23 e 0.00696 12 34 45 56 TIC MS cc 100 0.05518 0. 10335 0.15152 0. 19985 0.24802 calmix_ddHCDtop10 LTQ Orbitrap Velos _MRFA_7500_2ms_ 2.9 s MS_1ms _MS2_1010 50 08173929 MS @60k 3 11 14 18 25 29 37 50 55 [email protected] 0.02525 0.05009 0.07348 0.08590 0.12165 0.13408 0.17301 0.22746 0.24300 0 Relative Abundan 0000.00 0020.02 0040.04 0060.06 0080.08 0100.10 0120.12 0140.14 0160.16 0180.18 0200.20 0220.22 0240.24 0260.26 Time (min) NL: 1.50E8 1 12 23 45 34 56 67 TIC MS 0.00695 0.04819 0.08935 0.17202 LTQ Orbitrap Velos 100 0.13069 0.21335 0.25468 calmix_ddHCDtop1 0_MRFA_7500_2m 2.48 s s_MS_1ms_MS2_3 MS @30k bundance 50 0k_FTMS AA 2 7 17 26 39 43 48 59 [email protected] 0.01611 0.03101 0.06885 0.10385 0.15142 0.16385 0.18652 0.22785 0

Relative Relative 0.00 0.02 0.04 0.06 0.08 0.10 0.12 0.14 0.16 0.18 0.20 0.22 0.24 0.26 Time (min) 1 NL:

e 0.00282 12 23 34 45 56 67 78 89 100 111 5.23E8 cc 100 0.02885 0.05389 0.07896 0.10506 0.13135 0.15641 0.18252 0.20756 0.23258 0.25762 TIC MS Orbitrap Elite 192_48_eFTo n_10ms_delay MS @60k 50 1.51 s _10081217253 3 17 32 39 55 60 71 84 91 107 6 HCD@15k 0.01094 0.04275 0.07546 0.09395 0.12876 0.14336 0.16843 0.19834 0.21572 0.25031 0 Relative Abundan 0000.00 0020.02 0040.04 0060.06 0080.08 0100.10 0120.12 0140.14 0160.16 0180.18 0200.20 0220.22 0240.24 0260.26 Time (min)

17 *Calmix, MS: 1e6 ions, IT~2 ms; MS2: 2e4 ions, IT~1 ms with pAGC Orbitrap Elite vs. LTQ Orbitrap Velos: Acquisition Speed for TOP10 HCD Experiment

8 CD

HH 7 LTQ Orbitrap Velos 6 (MS@60k, HCD@15k) (Hz) d MS & 5 LTQQp Orbitrap Velos dd (MS@60k, HCD@7500) 4 LTQ Orbitrap Velos 3 (MS@30k, HCD@7500) ombine ion spee tt cc 2 Orbitrap Elite (MS@60k, HCD@15k) 1 Acquisi 0 1 25 50 100 150 200 HCD IT (()ms) *

Acquisition speed MS & MS2 combined (Hz) = 11/cycle time

18 Proof Statement: Peptide Identification Peptide ID: E.Coli Digest 1000 ng, 7 Hz HCD

EColi_HCD_1000ng_192msec_2uscan_90min... 10/15/2010 10:35:18 AM

RT: 34.27595 - 34.35649 34.28376 34.34726 NL: 5 1.29E9 TIC MS

nce 4 EColi_HCD_1 aa 1x FTMS (2 µscans, 60.000 resolution) + 20x ddHCD 000ng_192ms 3 ec_2uscan_90 min-test 2 3.81 s

1 34.29646 34.30106 Relative Abund Relative 34.30565 34.31749 34.27799 34.32230 34.32950 34.33717 34.34250 0 34.28 34.29 34.30 34.31 34.32 34.33 34.34 34.35 Time (min) RT: 34.29354 - 34.34516 34.29646 34.3010634.3056534.31509 34.31749 34.32230 34.32950 34.33214 34.33717 34.34250 NL: 5 7.50E6 TIC MS

nce 4 EColi_HCD_1 aa 000ng_192ms 3 ec_2uscan_90 6.87 Hz min-test 2

1 Relative Abund Relative 0 34.295 34.300 34.305 34.310 34.315 34.320 34.325 34.330 34.335 34.340 34.345 Time (min) EColi_HCD_1000ng_192msec_2uscan_90min-test #4452 RT: 34.28 AV: 1 NL: 5.22E7 T: FTMS + p NSI Full ms [250.00-2000.00] 442.9521 R=59201

ee z=4 100 380. 7213 681. 3310 R=64401 590.6013 R=47701 z=2 R=51400 z=2 z=3 50 337.2042 525.7828 766.3226 888.4681 1050.5581 1137.5276 1304.6638 R=67201 R=54201 R=44801 R=41501 R=38701 R=36501 R=34601 z=4 z=2 z=1 z=1 z=1 z=1 z=1 elative Abundancelative 0 RR 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 m/z

20 Performance Increase for Bottom-up Proteomics

Top20 HCD method 1 µg E.Coli tryptic digest

Improved duty cycle results in more Peptide Spectrum Matches

21 Proof Statement: TMT Quantitation on the Example of EcoliE.coli Orbitrap Elite vs. LTQ Orbitrap Velos

Orbitrap Elite LTQ Orbitrap Velos Sample load # proteins # peptides % quan. # proteins # peptides % quan.

20 ng 703 4032 86 563 3495 93

80 ng 873 5078 93 694 4309 97

200 ng 933 5696 95 713 4322 99

*Identification data shown for 1%FDR, mean of 2 runs ** #id# peptides = #i# unique peptides

Increased number of ID’s 20-30%

23 Proof Statement: Intact Protein Analysis Intact Protein Analysis: Yeast Enolase (46.64 kDa)

0 993.0 993.5 994.0 994.5 995.0 m/z

ASMS 2011, MP092: Increased Analytical Performance on a Hybrid Linear Ion Trap-FTMS Mass 25 Spectrometer with a High-Field Orbitrap Mass Analyzer, Martin Zeller, Catharina Crone, Mathias Mueller, Eugen Damoc, Eduard Denisov, Alexander Makarov, Dirk Nolting, and Thomas Moehring Analysis of Intact Yeast Enolase (46.64 kDa) on the Orbitrap Elite Hybrid MS

47+ charge state of yeast enolase showing deconvoluted spectrum resolution of > 100,000 FWHM at m/z 1,000 (monoisotopicT: FTMS + p ESI mass Full ms 46642.214 [150.00-20 ... Da)

Enolase_994_transients_1e5_v2-qb #1 RT: 0.00 AV: 1 NL: 1.00E6 46642.586 T: FTMS + p ESI Full ms [150.00-2000.00] 100 993.9770 R=110221 100 90 993.9134 994.0611 90 R= 109564 R= 109627 80

80 993.8706 70 R=107582 994.1249 70 R=106063 60 ce dance nn 60 nn 50 50 993.8066 R=106902 994.1885 40 40 R=109018

993.6367 Abu Relative Relative Abunda R=108853 30 30 994.2531 993.5087 R=115076 R=109212 994.4433 20 R=105946 20 994.7200 46662.425 R=109283 10 10 46679.517

0 993.4 993.6 993.8 994.0 994.2 994.4 994.6 994.8 0 m/z 46600 46700 Mass,m/z Da Precise intact protein characterization

26 Proof Statement: Top-down MS/MS on Intact Histone Proteins LC-MS Analysis of Histone H4

RT: 0.00 - 59.99 32.95 NL: 100 1.19E8 33.35 TIC MS

ee 80 32. 40 Histone_ II- 33.56 32.22 S_1 33.99 60 TIC 30.72 29.96

e Abundanc 34.79

vv 40 35.78 29.64 37.03 Relati 20 0.60 28.16 39.05 50.56 56.64 1.03 6.27 10.40 12.96 17.96 20.86 27.09 41.44 0 30. 10 NL: 100 29.94 5.36E5 m/z= 80 30.15 808.19999- 809.00281 MS 60 XIC 29.83 33.24 Histone_II- 33.35 S_1 40 33.92 28. 62 34. 33 20 35.06 28.37 55.21 55.55 0.63 3.84 8.71 13.27 20.71 26.09 38.39 40.01 49.98 0 0 5 10 15 20 25 30 35 40 45 50 55 Time (min)

28 Full Scan MS Spectrum at RT 30 min

Histone_II-S_1 #1235-1271 RT: 29.69-30.51 AV: 37 NL: 3.73E4 T: FTMS + p ESI Full ms [300.00-2000.00] 870.65445 R=78345 943.12513 zz13=13 R=73398 100 754. 70135 z=12 R=83041 666.03150 z=15 90 R=89115 ETD & HCD z=17 80 1028.86335 629.08567 R=70360 on 14+ charge state R=90943 z=11 70 z=18

ance 60 dd

50 1131.64935 R=67992 1257.16493 40 z=10 R=65990 elative Abun elative zz9=9 RR 30 1414.05873 20 R=61955 1179.74336 z=8 10 R= 71832 z=? 0 600 700 800 900 1000 1100 1200 1300 1400 m/z

29 Extended Top-down Capabilities

843.9602 NL: 4.46E4 R=158115 Histone2_808_HCD_110415 z=2 000415-qb#1 RT: 2.10 AV: 100 HCD 1 T: FTMS + p ESI Full ms2

ee 808. 50@hcd30. 00 80 [120.00-2000.00] 60 762.4287 R=158642 40 z=2

ve Abundanc ve 1001.0440 385. 7177 R=115639 20 R=197410 z=2 Relati z=2 0 600.3526 NL: 2.28E3 R=185866 Histone2__ 808 ETD _ 1104142 z=2 14933-qb#1 RT: 2.96 AV: 1 100 Sequence coverage ETD T: FTMS + p ESI Full ms2 664.3820 [email protected] 80 537.9891 R=171040 Orbitrap [120.00-2000.00]Elite using ETD: 81% R=189201 z=2 60 z=3 LTQ Orbitrap XL using ETD: 43% 787. 4543 1093. 5867 R=154169 R=125517 40 z=1 z=4 1341.7392 R=106010 1620.8704 20 z=5 R=100972 z=3 0 500 1000 1500 2000 m/z Increased coverage due to high resolution, more data points and improved S/N

30 Histone H4 HCD (top trace) and ETD (bottom trace); identification and annotation using ProSight. Proof Statement: MSn Capabilities MS7 is required to assign the structure of 3 co-isolated isomeric permethylated ovalbumin glycans

32 Orbitrap Elite - Summary

Unique feature Benefit Customer value

Higgyh sensitivity S-lens Detection of low abundant Robust and sensitive technology species Generation II ion optics Novel beam blocking Improved robustness technology

Novel Detection System Wider linear dynamic range Robust quantitation and AGC

High-field Orbitrap Higggher resolving power Extended top-down capabilities

Advanced Signal Processing Higher resolving power Extended top-down capabilities

High-field Orbitrap + Improved acquisition speed Improved productivity Advanced Signal Processing 4 Hz at 60,000 Better data in less time 7 Hz at 15,000 HCD MS/MS

New pre-amplifier Improved sensitivity (S/N) Improved data quality

Parallel acquisition Improved duty cycle Improved productivity

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