Advances in Hybrid Mass Spectrometry

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1 The world leader in serving science Advances in Hybrid Mass Spectrometry ESAC 2008 Claire Dauly Field Marketing Specialist, Proteomics

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3 New hybrids instruments LTQ Orbitrap XL with ETD MALDI LTQ Orbitrap

4 OUTLINE Implementation of ETD in the LTQ Orbitrap XL Analytical Applications: PTM characterization (phosphorylation, O-Sulfonation ) Middle-Down/Top-Down Proteomics: analyses of large peptides or proteins Complex sample analyses with CID and ETD Acquisition strategies Data-Dependant Decision Tree

5 Electron Transfer Dissociation (ETD) Concept Ion / Ion Chemistry [M + 3H] 3+ + A - [M + 3H] 2+ + A [M + 3H] 2+ [C+2H] 1+ + [Z+H] 1+ C Z Made by electron capture ionization in a chemical ionization source Syka et al, PNAS USA 2004, 101,

6 ETD is Fast - Less Than 350 ms Per MS/MS Scan Syka JE, Coon JJ, Schroeder MJ, Shabanowitz J, Hunt DF. Peptide and protein sequence analysis by electron transfer dissociation mass spectrometry. Proc Natl Acad Sci U S A Jun 29;101(26):

7 LTQ XL with ETD Segmented Linear Ion Trap Design ESI Front Center Back + - CI Step 1: Trap and isolate Cations in center, then move them to front section Step 2: Anion Injection Front Lens Back Lens +5 V - 0 V Step 3: Step 4: Ion/Ion Reaction new charge-reduced species created Remove any un-reacted anions; scan V 0 V

8 Nonergodic Fragmentation Pathway CID fragmentation y z y z y z b c b c b c ETD fragmentation ETD fragmentation is indifferent to peptide sequence and PTM presence.

9 Design Objectives for ETD on the LTQ Orbitrap XL As simple to use as CID, PQD and HCD just another dissociation technique fully automatic ETD spectra detection either in the LTQ or in the Orbitrap Robust reagent ion delivery system stable delivery of reagent anions for long durations requires a minimum of maintenance Reproducible and predictable results well controlled and optimized parameters Flexible for future capabilities (Fluoranthene ions as lockmass)

10 Implementation of ETD in the LTQ Orbitap XL Operation principle Accumulation and isolation of precursor ions in the LTQ Introduction of Fluoranthene anions from the back into the LTQ ETD reaction in the LTQ Detection of ETD production ions LTQ (fast, sensitive) bottom-up proteomics Orbitrap (high resolution, high mass accuracy) top-down proteomics, de novo sequencing

11 Implementation of ETD in the LTQ Orbitrap XL Ion accumulationin in linear ion ion trap trap Precursor ion isolation Move precursor ions into first section of linear ion trap Anion injection Isolation of Flouranthene anions Anion cation reaction Axial removal of anions ETD fragment ion detection either in LTQ or in Orbitrap detector V 11

12 Implementation of ETD in the LTQ Orbitrap XL Ion accumulation in linear ion trap Precursor ion isolation Move precursor ionsinto into first first section section of linear ion trap Anion injection Isolation of Flouranthene anions Anion cation reaction Axial removal of anions ETD fragment ion detection either in LTQ or in Orbitrap detector V 12

13 Implementation of ETD in the LTQ Orbitrap XL Ion accumulation in linear ion trap Precursor ion isolation Move precursor ionsinto into first first section section of linear ion trap Anion injection Isolation of Flouranthene anions Anion cation reaction Axial removal of anions ETD fragment ion detection either in LTQ or in Orbitrap detector + 0 V 13

14 Implementation of ETD in the LTQ Orbitrap XL Ion accumulation in linear ion trap Precursor ion isolation Move precursor ions into first section of linear ion trap Anion injection Isolation of of Flouranthene anions Anion cation reaction Axial removal of anions ETD fragment ion detection either in LTQ or in Orbitrap detector V 14

15 Implementation of ETD in the LTQ Orbitrap XL Ion accumulation in linear ion trap Precursor ion isolation Move precursor ions into first section of linear ion trap Anion injection Isolation of of Flouranthene anions Anion cation reaction Axial removal of anions ETD fragment ion detection either in LTQ or in Orbitrap detector + 0 V 15

16 Implementation of ETD in the LTQ Orbitrap XL Ion accumulation in linear ion trap Precursor ion isolation Move precursor ions into first section of linear ion trap Anion injection Isolation of of Flouranthene anions Anion cation reaction Axial removal of anions ETD fragment ion detection either in LTQ or in Orbitrap detector + 0 V 16

17 Implementation of ETD in the LTQ Orbitrap XL Ion accumulation in linear ion trap Precursor ion isolation Move precursor ions into first section of linear ion trap Anion injection Isolation of of Flouranthene anions Anion cation reaction Axial removal of anions ETD fragment ion detection either in LTQ or in Orbitrap detector V 17

18 Implementation of ETD in the LTQ Orbitrap XL Ion accumulation in linear ion trap Precursor ion isolation Move precursor ions into first section of linear ion trap Anion injection Isolation of Flouranthene anions Anion cation reaction Axial removal of anions ETD fragment ion detection either in LTQ or in Orbitrap detector V 18

19 Implementation of ETD in the LTQ Orbitrap XL Ion accumulation in linear ion trap Precursor ion isolation Move precursor ions into first section of linear ion trap Anion injection Isolation of Flouranthene anions Anion cation reaction Axial removal of of anions ETD fragment ion detection either in LTQ or in Orbitrap detector V 19

20 Implementation of ETD in the LTQ Orbitrap XL Ion accumulation in linear ion trap Precursor ion isolation Move precursor ions into first section of linear ion trap Anion injection Isolation of Flouranthene anions Anion cation reaction Axial removal of of anions ETD fragment ion detection either in LTQ in LTQ or in Orbitrap detector

21 Implementation of ETD in the LTQ Orbitrap XL Ion accumulation in linear ion trap Precursor ion isolation Move precursor ions into first section of linear ion trap Anion injection Isolation of Flouranthene anions Anion cation reaction Axial removal of anions ETD fragment ion detection either in LTQ in LTQ or in Orbitrap detector

22 OUTLINE Implementation of ETD in the LTQ Orbitrap XL Analytical Applications: PTM characterization (phosphorylation, O-Sulfonation ) Middle-Down/Top-Down Proteomics: analyses of large peptides or proteins Complex sample analyses with CID and ETD Acquisition strategies Data-Dependant Decision Tree

23 LTQ Orbitrap XL ETD for Phosphopeptide Analysis RpSRGGKLGpSLGK Mechter_1880_3+_ETD_FT_Recal #1-20 RT: AV: 20 NL: 3.18E5 T: FTMS + p NSI Full ms @etd [ ] [M+3H] z=3 100 Relative Abundance z=1 ΔpS z= z=? z= z= z= z= z=2 [M+3H] z=2 ΔpS z= z=1 z= z= z= z= z= z= z= z= m/z Phosphopeptide standards courtesy of Karl Mechtler, IMP Vienna, Austria

24 CID and ETD provide complimentary phosphoproteome coverage* LGGLRPES*PESLTSVSR, 3+ Data courtesy of S. Gygi and J. Coon CID, Xcorr = 3.6 CID ETD, Xcorr = ETD *unique phosphopeptides, from HeLa cell lysate, enriched by SCX/IMAc and analyzed by LC/LTQ OT ETD

25 OUTLINE Implementation of ETD in the LTQ Orbitrap XL Analytical Applications: PTM characterization (phosphorylation, O-Sulfonation ) Middle-Down/Top-Down Proteomics: analyses of large peptides or proteins Complex sample analyses with CID and ETD Acquisition strategies Data-Dependant Decision Tree

26 α-defensin 5, ETD on 4+ precursor ions with Orbitrap detection alpha_defensin-5_etd_4+_ #1 RT: AV: 1 NL: 4.39E5 T: FTMS + p ESI sa Full ms @etd [ ] z=1 4.5 Relative Abundance z= z= z=2 * * z= z= z= z= z= m/z

27 α-defensin 5, ETD on 6+ precursor ions, example for high resolution c / c z / z z 6 1+

28 Histone H4 ETD spectrum with Orbitrap detection Histone-f3_ETD-810_05_30msec_ETDact-time_Recal # RT: AV: 22 NL: 3.87E4 T: FTMS + p ESI Full ms @etd30.00 [ ] [M+14H] z= Relative Abundance z=? z=1 z=? z= z=? z=1 z= z=? z= z=? z= z=5 [M+14H] z=14 [M+14H] z=12 [M+14H] z= z=? z=? z=? z=? m/z z=?

29 Ubiquitin_ETD_11 #1-106 RT: AV: 106 NL: 1.10E3 T: FTMS + p ESI Full ms @etd30.00 [ ] z=1 100 Relative Abundance ETD spectrum of Ubiquitin, 12+ charge state, with Orbitrap detection z=? z=1 * z= z= z= z= z= z= z= z= z= z= z= z= z= z= z= z= z= m/z *

30 OUTLINE Implementation of ETD in the LTQ Orbitrap XL Analytical Applications: PTM characterization (phosphorylation, O-Sulfonation ) Middle-Down/Top-Down Proteomics: analyses of large peptides or proteins Complex sample analyses with CID and ETD Acquisition strategies Data Dependant Decision Tree

31 Arabidopsis thaliana complex sample analysis using ETD and CID CID: 544 proteins RT: Relative Abundance CID ETD Time (min) NL: 3.25E9 TIC MS arabidopsis _tryp_top7- cid_180min NL: 2.43E9 TIC MS Arabidopsis _tryp_top7- ETD_180mi n ETD: 370 proteins Total: 671 proteins identified Dr. B. Mueller, LMU Muenchen

32 OUTLINE Implementation of ETD in the LTQ Orbitrap XL Analytical Applications: PTM characterization (phosphorylation, O-Sulfonation ) Middle-Down/Top-Down Proteomics: analyses of large peptides or proteins Complex sample analyses with CID and ETD Acquisition strategies Data-Dependant Decision Tree

33 CID vs ETD Fragmentation Efficiency ETD is more efficient at low m/z and high z values

34 Intelligent peptide sequencing using the Data Dependent Decision Tree

35 LTQ Orbitrap XL ETD: Combination of three different and complementary fragmentation techniques CID, HCD, and ETD Most comprehensive solution for complex PTM analysis intelligent sequencing of peptides top-down and middle-down analysis protein quantitation via stable isotope labelling such as itraq or label-free quantitation High-throughput sequencing applications with parallel acquisition capabilities Identification of unexpected PTMs using high resolution and accurate mass

36 New hybrids instruments LTQ Orbitrap XL with ETD MALDI LTQ Orbitrap

37 MALDI technique Analyte molecules are imbedded / dissolved in a large molar excess of small organic molecules absorbing the laser light electronically, UV-MALDI. Matrix-analyte solution is dried to solid. A short, (nanosecond) laser pulse desorbs / ionizes matrix and analyte molecules. MALDI is known to be tolerant against common salts, buffers and detergents and allows fast, straightforward analyses. Sample analysis can be carried out without tedious sample purification. Even for low femtomole deposits on sample plates only a friction of the sample is used for full scan analyses. Typically, > 95 99% of the sample remains on the sample plate. Enough material remains to perform MS n analyses or to come back to the sample the next day.

38 Combined with high mass resolution and accurate mass ppm R= m/z Inset into m/z 2846 from full scan m/z 200 m/z 4000, acquired at resolving power m/z 400

39 Stable Mass accuracy Check Mass Calibration, m/z , Normal Mass Range Calibration Mixture for a 1 day old mass calibration, test duration is 20 min.

40 1 fmol Angiotensin, HCCA matrix, 5 scans, 5 laser shots each , ppm FTMS + p MALDI Full ms [ ] Relative Abundance m/z m/z Note: only a fraction of the sample is used for this analysis

41 Mass range 4000 FTMS + p MALDI Full ms [ ] Relative Abundance ppm ppm m/z ppm m/z ppm RP m/z m/z ppm m/z

42 Mellitin (honey bee), m/z 2845 at RP 50,000 Inset into given full scan , RP m/z ppm R= Average of 10 FTMS + p MALDI Full ms [ ] C 131 H 230 N 39 O R= simulation Sequence: GIGAVLKVLTTGLPALISWIKRKRQQ Sum Formula: C 131 H 230 N 39 O 31 Monoisotopic Mass MH + : N-Terminus: H C-Terminus: Amidation FTMS + p MALDI Full ms [ ] ppm R= Single scan m/z

43 2800 : 1 Bradykinin vs A+1 isotope of 392 FTMS + p MALDI Full ms [ ] C 24 H 39 O ppm Bradykinin ppm NL: 1.83 e8 Relative Abundance C C H 39 O ppm m/z NL: 6.53 e / 1 Bradykinin / A+1 (392) 1,83 e8 / 6,5 e4 single scan 8 Laser shots 1 e6 charges m/z RP m/z 400

44 PMF, 5 fmol BSA 5 scans, 20 laser shots each, RP m/z 400; HCCA matrix. FTMS + p MALDI Full ms [ ] Relative Abundance m/z

45 Insets into FTMS + p MALDI Full ms [ ] R= ppm R= ppm R= ppm HLVDEPQNLIK RHPEYAVSVLLR KVPQVSTPTLVEVSR , ppm R= , ppm R= , ppm R= m/z VHKEC@C@HGDLLEC@ADDR m/z ETYGDMADC@C@EKQEPERNEC@FLSHK m/z DAIPENLPPLTADFAEDKDVC@K

46 Sequence Coverage is 50 % Mascot Result, monoisotopic mass obtained by Xtract

47 PMF, BSA plus data dependent MSMS FTMS + p MALDI Full ms [ ] Relative Abundance m/z

48 Data dependent MSMS on M monoisotopic H+ = fmol load , RP 35,000 BSA_DD_23_ #11 RT: 1.15 AV: 1 NL: 3.13E5 T: FTMS + p MALDI d Full ms @cid45.00 [ ] Inset into precursor Orbitrap detection RP m/z 400 Relative Abundance CID, Orbitrap detection m/z BSA_DD_23 #11 RT: 0.55 AV: 1 NL: 3.02E3 T: ITMS + p MALDI d Full ms @cid45.00 [ ] Relative Abundance CID, Linear Ion trap detection m/z BSA_DD_23_ #11 RT: 1.17 AV: 1 NL: 6.70E4 T: FTMS + p MALDI d Full ms @hcd33.00 [ ] Relative Abundance m/z HCD, Orbitrap detection m/z

49 ID, CID m/z Orbitrap detection

50 ID, HCD m/z

51 Summary Benefits of MALDI Technique Robust laser optics Direct beam Nitrogen laser Crystal Positioning System (CPS) Tissue Imaging enabled MALDI/ESI transfer enabled Combined with the Benefits of Orbitrap Technology Routine FT mass accuracy 60,000 resolution at 1 Hz scan cycle Wide dynamic range Parallel MS and MSn analysis Multiple dissociation techniques: CID, PQD and HCD