Advances in Hybrid Mass Spectrometry

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

New hybrids instruments LTQ Orbitrap XL with ETD MALDI LTQ Orbitrap

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

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, 9528-33

ETD is Fast - Less Than 350 ms Per MS/MS Scan 0.1-10 2 10-100 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. 2004 Jun 29;101(26):9528-33.

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 + - - + - 0 V 0 V

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.

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)

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

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 + + + + 0 V 11

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 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 + 0 V 13

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 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 + 0 V 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 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 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 Flouranthene anions Anion cation reaction Axial removal of anions ETD fragment ion detection either in LTQ or in Orbitrap detector - + - 0 V 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 of anions ETD fragment ion detection either in LTQ or in Orbitrap detector - - + + + + + + - - 0 V 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 in LTQ or in Orbitrap detector + + + 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 anions ETD fragment ion detection either in LTQ in LTQ or in Orbitrap detector + + + 21

LTQ Orbitrap XL ETD for Phosphopeptide Analysis RpSRGGKLGpSLGK Mechter_1880_3+_ETD_FT_Recal #1-20 RT: 0.01-0.31 AV: 20 NL: 3.18E5 T: FTMS + p NSI Full ms2 459.22@etd100.00 [120.00-1500.00] [M+3H] 459.2254 3+ z=3 100 Relative Abundance 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0 174.1356 z=1 ΔpS 301.2004 z=1 258.1456 z=? 341.1339 z=1 426.5608 z=3 497.2346 z=1 554.2560 z=1 623.7946 z=2 [M+3H] 2+ 688.8367 z=2 ΔpS 739.3722 z=1 908.4700 798.3886 z=1 z=1 1036.5426 z=1 938.5614 z=1 1076.4759 z=1 1150.5386 z=1 1247.5928 z=1 1343.6226 z=1 1360.6494 z=1 200 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 m/z Phosphopeptide standards courtesy of Karl Mechtler, IMP Vienna, Austria

CID and ETD provide complimentary phosphoproteome coverage* LGGLRPES*PESLTSVSR, 3+ Data courtesy of S. Gygi and J. Coon CID, Xcorr = 3.6 CID 2822 1162 ETD, Xcorr = 5.6 2452 ETD *unique phosphopeptides, from HeLa cell lysate, enriched by SCX/IMAc and analyzed by LC/LTQ OT ETD

α-defensin 5, ETD on 4+ precursor ions with Orbitrap detection alpha_defensin-5_etd_4+_245-4000 #1 RT: 243.51 AV: 1 NL: 4.39E5 T: FTMS + p ESI sa Full ms2 897.00@etd100.00 [245.00-4000.00] 6.0 5.5 5.0 942.41772 z=1 4.5 Relative Abundance 4.0 3.5 3.0 2.5 2.0 1.5 713.28473 z=1 1435.68079 z=2 1893.86353 z=2 * * 1.0 0.5 0.0 633.29504 z=1 1997.96533 z=1 2384.15430 z=1 2871.36401 z=1 3396.53052 z=1 500 1000 1500 2000 2500 3000 3500 m/z http://upload.wikimedia.org/wikipedia/en/b/b6/pbb_protein_defa5_image.jpg

α-defensin 5, ETD on 6+ precursor ions, example for high resolution c 14 2+ / c 14 2+ z 21 3+ / z 21 3+ z 6 1+

Histone H4 ETD spectrum with Orbitrap detection Histone-f3_ETD-810_05_30msec_ETDact-time_Recal #88-109 RT: 4.66-5.33 AV: 22 NL: 3.87E4 T: FTMS + p ESI Full ms2 810.00@etd30.00 [220.00-2000.00] [M+14H] 13+ 870.7291 z=13 100 Relative Abundance 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0 235.4328 z=? 319.2215 z=1 z=? 1 537.9878 z=3 376.0665 486.9042 z=? z=1 z=5 437.7562 z=? 600.3513 z=2 641.5435 z=? 664.3805 z=2 717.4309 z=5 [M+14H] 14+ 808.4632 z=14 [M+14H] 12+ 943.1226 z=12 [M+14H] 11+ 1027.4040 z=11 1078.2836 z=? 1153.6371 z=? 1269.1373 z=? 1393.1512 z=? 300 400 500 600 700 800 900 1000 1100 1200 1300 1400 1500 m/z 1546.2773 z=?

Ubiquitin_ETD_11 #1-106 RT: 0.01-6.13 AV: 106 NL: 1.10E3 T: FTMS + p ESI Full ms2 778.50@etd30.00 [210.00-2000.00] 277.1328 z=1 100 Relative Abundance ETD spectrum of Ubiquitin, 12+ charge state, with Orbitrap detection 95 90 85 80 75 70 65 60 55 50 45 40 35 30 25 20 15 10 5 0 305.1328 z=? 1 390.2168 z=1 * 869.7391 z=4 433.2516 z=2 537.2850 z=1 636.3535 z=1 764.4484 z=1 669.3921 z=2 790.4653 z=2 948.1839 z=3 1108.7627 z=6 1012.2238 z=3 1136.6501 z=1 1273.6870 z=4 1159.3174 z=3 1347.2301 z=2 1421.7778 z=6 1486.8083 z=5 1702.7334 z=5 400 600 800 1000 1200 1400 1600 1800 m/z * http://upload.wikimedia.org/wikipedia/commons/a/ac/ubiquitin_cartoon.png

Arabidopsis thaliana complex sample analysis using ETD and CID CID: 544 proteins RT: 0.00-179.99 Relative Abundance 100 90 80 70 60 50 40 30 20 10 0 100 90 80 70 60 50 40 30 20 10 0 CID 127.17 65.64 69.56 127.08 64.52 73.62 64.40 126.83 74.39 90.97 128.23 58.76 101.14 126.40 81.44 58.24 125.63 130.73 51.64 125.09 131.99 1.02 51.44 135.39 13.19 28.14 43.36 139.24 27.70 43.01 141.12 12.73 29.15 143.74 12.40 150.30 178.08 161.62 ETD 127.02 127.33 128.14 127.46 127.69 128.02 128.24 63.59 128.08 50.12 51.04 63.90 40.10 49.82 62.85 78.04 79.61 40.31 127.97 128.64 40.02 82.17 128.68 82.42 39.78 16.29 89.84 96.35 127.82 130.74 16.02 29.27 96.55 108.76 131.09 16.90 131.48 15.93 139.30 15.83 148.00 0 20 40 60 80 100 120 140 160 Time (min) 177.53 NL: 3.25E9 TIC MS arabidopsis _tryp_top7- cid_180min NL: 2.43E9 TIC MS Arabidopsis _tryp_top7- ETD_180mi n 297 247 123 ETD: 370 proteins Total: 671 proteins identified Dr. B. Mueller, LMU Muenchen

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

Intelligent peptide sequencing using the Data Dependent Decision Tree

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

New hybrids instruments LTQ Orbitrap XL with ETD MALDI LTQ Orbitrap

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.

Combined with high mass resolution and accurate mass 2846.76416-0.10 ppm 2845.76172 2847.76636 R=52600 2848.76978 2848.76978 2844 2846 2848 2850 2852 m/z Inset into Melittin @ m/z 2846 from full scan m/z 200 m/z 4000, acquired at resolving power 100,000 @ m/z 400

Stable Mass accuracy Check Mass Calibration, m/z 150 2000, Normal Mass Range Calibration Mixture for a 1 day old mass calibration, test duration is 20 min.

1 fmol Angiotensin, HCCA matrix, 5 scans, 5 laser shots each 100 1046.54114, - 0.62 ppm FTMS + p MALDI Full ms [850.00-2000.00] 80 2.0 Relative Abundance 60 40 20 1.0 0.8 0.6 0.4 0.2 0.0 1042 1046 1050 1054 1058 m/z 0 900 1000 1100 1200 1300 1400 m/z Note: only a fraction of the sample is used for this analysis

Mass range 4000 FTMS + p MALDI Full ms [200.00-4000.00] Relative Abundance 100 90 80 70 60 50 40 30 20-1.87 ppm 524.26398 1060.56824-0.33 ppm 1936.9862 1937.9902 1936 1938 1940 1942 m/z - 0.60 ppm 1938.9923 1939.9971 1936.98621 2845.7649 2846.7671 2846 2848 2850 m/z 2845.76489 + 1.21 ppm 2847.7710 2848.7717 2849.7783 3657.9302 3658.9280 3657.93018 RP 30,000 @ m/z 400 3659.9397 3660.9409 3661.9343 3658 3660 3662 3664 m/z 10 0-0.30 ppm 500 1000 1500 2000 2500 3000 3500 4000 m/z

Mellitin (honey bee), m/z 2845 at RP 50,000 Inset into given full scan 200 4000, RP 100,000 @ m/z 400 2846.76352-0.12 ppm 2845.76112 R=48700 2847.76641 2848.76872 Average of 10 FTMS + p MALDI Full ms [200.00-4000.00] C 131 H 230 N 39 O 31 2846.76429 2847.76706 2845.76145 R=49950 2846.76416 2848.76977 2849.77244 simulation Sequence: GIGAVLKVLTTGLPALISWIKRKRQQ Sum Formula: C 131 H 230 N 39 O 31 Monoisotopic Mass MH + : 2845.761447 N-Terminus: H C-Terminus: Amidation FTMS + p MALDI Full ms [200.00-4000.00] - 0.10 ppm 2845.76172 R=52600 2847.76636 2848.76978 2848.76978 Single scan 2844 2846 2848 2850 2852 m/z

2800 : 1 Bradykinin vs A+1 isotope of Phthalate @ 392 FTMS + p MALDI Full ms [150.00-2000.00] 100 391.28406 C 24 H 39 O 4-0.58 ppm 1060.56946 Bradykinin + 0.8 ppm NL: 1.83 e8 Relative Abundance 80 60 40 20 391.20868 392.28754 C 23 13 C H 39 O 4 392.21246-0.27 ppm 391.2 391.4 391.6 391.8 392.0 392.2 392.4 m/z NL: 6.53 e4 2800 / 1 Bradykinin / A+1 (392) 1,83 e8 / 6,5 e4 single scan 8 Laser shots 1 e6 charges 0 200 400 600 800 1000 1200 m/z RP 100,000 @ m/z 400

PMF, 5 fmol BSA 5 scans, 20 laser shots each, RP 30,000 @ m/z 400; HCCA matrix. FTMS + p MALDI Full ms [500.00-4000.00] 100 1882.9097 90 80 Relative Abundance 70 60 50 40 30 20 1639.9388 805.4644 1420.6789 1170.6277 927.4942 2117.8415 2526.1276 10 0 3137.2311 1000 1500 2000 2500 3000 3500 m/z

Insets into FTMS + p MALDI Full ms [500.00-4000.00] 1305.71770 R=18199 + 1.2 ppm 1439.81345 R=17919 + 1.2 ppm 1639.93880 R=16663 + 0.6 ppm 1306.72069 1307.72312 1441.81903 1444.62629 1641.94495 1642.94905 1305 1310 HLVDEPQNLIK 1440 1445 RHPEYAVSVLLR 1640 1645 KVPQVSTPTLVEVSR 2116.83730, + 0.2 ppm 2117.84151 R=14600 2118.84340 2119.85582 3136.22569, - 0.6 ppm 3137.22833 R=12300 3139.23242 3140.22887 2459.16525, + 0.3 ppm 2460.16843 R=14500 2461.16846 2462.18328 2120 m/z VHKEC@C@HGDLLEC@ADDR 3135 3140 m/z ETYGDMADC@C@EKQEPERNEC@FLSHK 2460 2465 m/z DAIPENLPPLTADFAEDKDVC@K

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

PMF, BSA plus data dependent MSMS FTMS + p MALDI Full ms [500.00-4000.00] 100 1882.9097 90 80 Relative Abundance 70 60 50 40 30 20 1639.9388 805.4644 1420.6789 1170.6277 927.4942 2117.8415 2526.1276 10 0 3137.2311 1000 1500 2000 2500 3000 3500 m/z

Data dependent MSMS on 1420.68 SLHTLFGDELC@K, M monoisotopic H+ = 1420.677692 100 fmol load 1420.6787, RP 35,000 BSA_DD_23_080214152101 #11 RT: 1.15 AV: 1 NL: 3.13E5 T: FTMS + p MALDI d Full ms2 1420.68@cid45.00 [380.00-1435.00] 100 1402.66502 Inset into precursor Orbitrap detection RP 60,000 @ m/z 400 Relative Abundance 90 80 70 60 50 40 30 20 10 CID, Orbitrap detection 534.30173 699.38110 722.30080 853.41787 1000.47188 871.42797 1113.55569 1220.56005 1274.57035 1415 1420 1425 1430 1435 1440 m/z BSA_DD_23 #11 RT: 0.55 AV: 1 NL: 3.02E3 T: ITMS + p MALDI d Full ms2 1420.68@cid45.00 [380.00-1435.00] Relative Abundance 100 90 80 70 60 50 40 30 20 10 0 CID, Linear Ion trap detection 869.2 871.3 982.3 1000.2 1113.4 1114.5 1083.4 872.3 722.2 534.1 681.3 1115.4 854.3 671.4 835.4 532.3 653.2 956.5 1274.5 1275.4 1292.3 1293.4 1385.5 1402.5 400 600 800 1000 1200 1400 m/z BSA_DD_23_080214150900 #11 RT: 1.17 AV: 1 NL: 6.70E4 T: FTMS + p MALDI d Full ms2 1420.68@hcd33.00 [100.00-1435.00] 338.18289 100 Relative Abundance 0 90 80 70 60 50 40 30 20 10 0 400 600 800 1000 1200 1400 m/z 110.07093 239.11400 439.23068 534.30408 HCD, Orbitrap detection 552.31628 869.37054 982.46368 722.30304 1083.50989 1203.57007 200 400 600 800 1000 1200 1400 m/z

ID, CID m/z 1420.68 Orbitrap detection

ID, HCD m/z 1420.68

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