Q Exactive TM : A True Qual-Quan HR/AM Mass Spectrometer for Routine Proteomics Applications Yi Zhang, Ph.D. ThermoFisher Scientific
Outline Introduction of Q Exactive Performance in Discovery Proteomics Flexible Targeted Quantification Mass Measurement of Intact mab Summary 2
Q Exactive TM - Innovations and Features First Benchtop Quadrupole-Orbitrap Mass Spectrometer HCD/C-Trap Combo Cell QUADRUPOLE MASS FILTER Mass Range: 5 4 amu Prec Isolation:.4 1 amu ORBITRAP Resolution: 14, Mass Accuracy: < 2 ppm ION SOURCE S-Lens 3
High Sensitivity with Quadrupole-based SIM Scan 1 8 6 195.876 N=24842.81 Full MS S/N = 745 6 5 Gain in sensitivity (7x) Relative Abundance 4 2 1 8 6 4 2 195.877 N=2741.58 SIM (1amu) S/N = 54 S/N (spectrum) 4 3 Caffeine 2 1 195.82 195.84 195.86 195.88 195.9 195.92 195.94 S/N (FMS) S/N (SIM1) Sensitivity Gain: 5 1 folds 4
Improved Resolution Advanced Signal Processing Increases resolution by 2X at the same transient length Resolution 16 14 12 1 8 6 4 2 5 1 15 Scan Speed [Hz] Exactive Q-Exactive Resolution at 2 Resolution at 4 Max. scan speed (Hz) 17,5 12,5 12 35, 25, 7 7, 5, 3 14, 1, 1.5 5
Scan Innovations Parallel Filling and Detection Spectrum Multiplexing 6
Outline Introduction of Q Exactive Performance in Discovery Proteomics Peptide/Protein Identification TMT based Quantification Flexible Targeted Quantification Mass Measurement of Intact mab Summary 7
Discovery Proteomics 1Hz HCD Scan Parallel Filling and Detection Fast Orbitrap Scanning pagc 1 HR/AM HCD scans in 1s 256ms 64ms 8
1 Full MS +1 HR/AM HCD Scans in One Second Ecoli_5min_newcolumn_Top1_15cm_reje... 2/17/211 3:39:42 PM RT: 15.8279-51.4773 Relative Abundance 1 8 6 4 2 16.95 17.924 25.91 27.492 22.3863 25.7411 27.1739 3.486 33.433 41.5779 41.6221 3.9771 35.5786 39.185 38.7325 2.3567 23.972 28.4175 49.3818 18.727 34.8784 37.5942 41.1998 21.9561 25.446 43.423 34.7425 41.942 43.5925 46.5441 49.1418 47.5177 49.6742 16 18 2 22 24 26 28 3 32 34 36 38 4 42 44 46 48 5 Time (min) RT: 32.2438-32.271 Relative Abundance.3.2.1. 32.2447.3 32.2464 32.2645 =.181 min = 1.86 sec 32.2512 32.2525 32.2538 32.2551 32.2564 32.2577 32.259 32.263 32.2616 32.2629 32.2694 1 2 3 4 5 6 7 8 9 1.2.1. 32.244 32.246 32.248 32.25 32.252 32.254 32.256 32.258 32.26 32.262 32.264 32.266 32.268 32.27 Time (min) Ecoli_5min_newcolumn_Top1_15cm_reject_1 # 12365 RT: 32.25 AV: 1 NL: 1.58E8 T: FTMS + p NSI Full ms [3.-2.] x2 536.7949 1 x5 Relative Abundance 8 6 4 2 4 329.2184 382.946 z=3 392.7555 6 419.2133 z=3 5 445.7585 484.2459 z=3 55.2733 8 575.8452 3 551.8184 1 581.997 z=3 63.373 664.3292 9 7.3623 711.3712 z=3 7 725.8647 1 35 4 45 5 55 6 65 7 75 8 85 9 95 1 15 11 115 784.537 z=? 835.3868 871.9796 92.8879 95.9638 2 18.5358 z=1 172.5819 z=1 196.3795 z=? 115.6826 z=1 1182.612 z=? 9
High Quality HCD Spectra Extracted from: C:\Xcalibur\data\Kai\Ecoli_5min_newcolumn_Top1_15cm_reject_1.raw #1241 RT: 32.33 FTMS, HCD, z=+2, Mono =467.75339 Da, MH+=934.4995 Da, Match Tol.=2 mmu Extracted from: C:\Xcalibur\data\Kai\Ecoli_5min_newcolumn_Top1_15cm_reject_1.raw #12415 RT: 32.34 FTMS, HCD, z=+3, Mono =456.5826 Da, MH+=1367.73163 Da, Match Tol.=2 mmu Intensity [counts] (1^6) 12 1 8 6 4 2 a₁+ 12.899 y₂+ a₂+ 288.227 219.1495 y₁+ b₂+ 175.11888 247.1441 y₅+ 589.2915 y₆+ 688.36139 Intensity [counts] (1^6) 4 3 2 1 a₂+ 157.13342 y₂+ 244.16534 a₇²+ 332.69171 y₄+ 43.2316 y₁₁²+ y₉²+ 592.3835 492.26865 y₅+ 577.29816 b₇+ 692.37195 y₆+ 676.36572 y₈+ 846.4783 1 2 3 4 5 6 7 8 9 2 4 6 8 1 12 14 Extracted from: C:\Xcalibur\data\Kai\Ecoli_5min_newcolumn_Top1_15cm_reject_1.raw #12411 RT: 32.33 FTMS, HCD, z=+2, Mono =341.69992 Da, MH+=682.39256 Da, Match Tol.=2 mmu Extracted from: C:\Xcalibur\data\Kai\Ecoli_5min_newcolumn_Top1_15cm_reject_1.raw #12416 RT: 32.34 FTMS, HCD, z=+2, Mono =536.79449 Da, MH+=172.58171 Da, Match Tol.=2 mmu Intensity [counts] (1^6) 4 3 2 1 a₁+ 12.888 y₁+-h₂o 129.1219 y₂+ 26.19653 y₄+ 388.25467 Intensity [counts] (1^6) 4 3 2 1 a₂+ 185.16463 b₂+ 213.15939 b₆²+, b₃+ 342.2215 y₄+ 487.2622 y₅+ 618.317 y₆+ 731.38538 y₇+ 86.4278 1 2 3 4 5 6 7 Extracted from: C:\Xcalibur\data\Kai\Ecoli_5min_newcolumn_Top1_15cm_reject_1.raw #12412 RT: 32.33 FTMS, HCD, z=+2, Mono =658.38232 Da, MH+=1315.75737 Da, Match Tol.=2 mmu 1 2 3 4 5 6 7 8 9 1 11 Extracted from: C:\Xcalibur\data\Kai\Ecoli_5min_newcolumn_Top1_15cm_reject_1.raw #12417 RT: 32.34 FTMS, HCD, z=+2, Mono =72.3452 Da, MH+=1439.67375 Da, Match Tol.=2 mmu 1.4 Intensity [counts] (1^6) 5 4 3 2 1 b₂+ b₃+-h₂o 171.11258 254.14955 a₉²+-h₂o y₇+ b₃+ 398.24179 y₅+ 589.32959 272.15991 475.28632 y₄+ y₆+ 388.25351 532.3197 y₈+ 72.41449 y₉+ 83.45911 y₁₀+ 931.52252 y₁₀+-nh₃ 914.49854 Intensity [counts] (1^6) 1.2 1..8.6.4.2 a₁+ 12.897 y₁+ y₆+ 175.11887 y₅+ y₉+ 77.3461 y₁₁+ 62.31427 934.4732 1132.57153 2 4 6 8 1 12 Extracted from: C:\Xcalibur\data\Kai\Ecoli_5min_newcolumn_Top1_15cm_reject_1.raw #12413 RT: 32.34 FTMS, HCD, z=+3, Mono =384.23242 Da, MH+=115.68271 Da, Match Tol.=2 mmu. 2 4 6 8 1 12 14 Extracted from: C:\Xcalibur\data\Kai\Ecoli_5min_newcolumn_Top1_15cm_reject_1.raw #12418 RT: 32.34 FTMS, HCD, z=+2, Mono =892.97919 Da, MH+=1784.9511 Da, Match Tol.=2 mmu 1. Intensity [counts] (1^6) 3.5 3. 2.5 2. 1.5 1. y₂+ y₁+-h₂o 246.1897 b₄+ b₅+ 129.122 367.23297 a₇³+-nh₃ b₃+, a₆²+ 466.32 y₅+ 517.29755 197.12845 268.16522 a₅+ y₆+ 438.3667 588.33417 y₇+ 685.38513 Intensity [counts] (1^6).9.8.7.6.5.4.3.2 a₂+ 25.11826 y₁+ 147.11258 b₃+ 393.13989 b₅+ 62.26434 b₄+ 521.19879 y₇+ 755.46484 y₁₁+ 1165.69214.5.1. 2 4 6 8 1 12 Extracted from: C:\Xcalibur\data\Kai\Ecoli_5min_newcolumn_Top1_15cm_reject_1.raw #12414 RT: 32.34 FTMS, HCD, z=+3, Mono =453.2371 Da, MH+=1357.67759 Da, Match Tol.=2 mmu. 2 4 6 8 1 12 14 16 18 Extracted from: C:\Xcalibur\data\Kai\Ecoli_5min_newcolumn_Top1_15cm_reject_1.raw #12419 RT: 32.35 FTMS, HCD, z=+3, Mono =73.7172 Da, MH+=219.961 Da, Match Tol.=2 mmu Intensity [counts] (1^6) 2.5 2. 1.5 1..5 a₂+ 173.12833 a₆³+, b₂+, a₄²+ 21.12317 y₆+ 598.332 y₅+ 527.29431 y₄+ 44.2659 y₇+ 729.36896 y₈+ 8.4826 a₁₀+-h₂o, y₉+ 929.44843 y₁₀+ 144.47485 Intensity [counts] (1^3) 3 25 2 15 1 5 y₃³+ 129.1216 b₂+-h₂o 241.8124 y₂+ 272.1718 a₂+ 231.9689 b₃+-h₂o 328.11536 b₄+-h₂o 457.1557 y₁₀²+ 568.84778 b₅+-h₂o 528.19189 y₁₂²+ 653.933 y₁₃²+ 717.9292 y₁₄²+ 782.4526 y₈+ 99.54944 y₁₇²+ 926.58. 2 4 6 8 1 12 14 2 4 6 8 1 12 1
Peptide/Protein Identification Yeast Tryptic Digest (1, 1, 1ng) Column: C18, 75µmx15cm LC: 35nL/min, 9min Q Exactive: Top1 TripleTOF 56: Top2 Number of Unique Peptides Number of Unique Protein Groups 1 8 6 4 2 Q Exactive TripTOF 56 2452 1361 6975 3539 8353 4767 15 125 1 75 5 25 691 357 1321 724 1395 117 1ng 1ng 1ng 1ng 1ng 1ng *About 45 proteins are expressed in yeast during log-phase growth 11
Peptide/Protein Identification Protein identified from 1 ng yeast digest Number of Proteins Identified 16 14 12 1 8 6 4 2 4x more ID Q Exactive TripleTOF 56 Cellular copy number Same sample, Same LC column, Same gradient, 1% FDR 12 *Yeast cellular protein copy numbers are from Weissman and co-workers, Nature, 23, 16, 737-7341.
Peptide/Protein Identification - Larger Dynamic Range on Q Exactive Most Abundant Least Abundant 1 ng load 1 ng load TripleTof 56 Q Exactive Precursor intensity of identified peptides (normalized to the most intense peak) 13 Same Sample, Same LC column, Same Gradient, 1% FDR
Peptide/Protein Identification High Quality HCD Spectrum High confidence identification from 1 ng of Yeast Digest Extracted from: C:\Xcalibur\data\Zhiqi\ID_QE\211321_1ngYeastDigest_Top1_14min_1.raw #2348 RT: 77.42 FTMS, HCD, z=+2, Mono =645.83948 Da, MH+=129.67168 Da, Match Tol.=2 mmu L G N D D E V I L F R MASCOT IonScore: 9 Exp Value: 3.8 E-9 Intensity [counts] (1^3) 5 4 3 2 1 y₁+ 175.11826 y₂+ 322.18576 y₃+ 435.2699 b₅+-nh₃ 498.17874 y₄+ 548.35284 y₅+ 647.42157 y₆+-h₂o 758.4541 y₆+ 776.46594 y₇+ 891.48932 y₈+ 16.51752 y₉+ 112.55273 y₁₀+ 1177.57886 2 4 6 8 1 12 Peptide of YOR2C, 149 copy number, identified from 1 ng yeast digest 14
Discovery Quan (TMT) TMT 6-plex labeled E. coli digest 1 9298 Number of peptides at 1% FDR 9 8 7 6 5 4 3 2 1 Quantification Rate: >97% 718 5537 5252 485 4376 3888 3245 2736 6527 unique peptide total IDed MSMS spectra quantifiable MSMS spectra 2ng 4ng 8ng 2ng 5ng Amount of E. coli digest 15
Discovery Quan (TMT) Accuracy and Precision Quantification Error <5%, CV<13% 1.4 TMT reporter ion ratio 1.2 1.8.6.4.2 1.4 1.1 1.2 1.5 1.3 observed expected 127/126 128/126 129/126 13/126 131/126 Result from 8 ng E. Coli digest 16
Outline Introduction of Q Exactive Performance in Discovery Proteomics Flexible Targeted Quantification Targeted msx SIM Targeted HCD Mass Measurement of Intact mab Summary 17
HR/AM Targeted Quantification on the Q Exactive HR/AM Targeted Quantification Instrument method Targeted msx SIM Targeted HCD Quantification LC Peak Area of Precursor LC Peak Area of MS/MS Fragments Selectivity, Speed, Sensitivity 18
Quantification with Targeted msx SIM High Resolution (14K) Accurate Mass (<5ppm) Selectivity R: 35K R: 7K R: 14K 492.2634 492.2665 492.2495 492.2511 492.2661 3ppm Spectrum Multiplexing Speed 19
Quantification with Targeted msx SIM Quadrupole-Based SIM Sensitivity 2
Quantification with Targeted msx SIM in Medium Complex Background 12 Heavy Peptides (1amol 1fmol) In 1ng Yeast Digest msx tsim Resolution: 14K SIM: 4amu msx: 4 1 Relative Abundance 1 8 6 4 2 8 6 4 2 1 8 6 4 2 GISNEGQNASIK 5ppm XIC 17. 17.2 17.4 17.6 17.8 18. 18.2 18.4 18.6 Time (min) 18.12 18.11 18.1 1fmol 1fmol 1amol 4.E+9 R 2 :.9991-.9999 LOD: 1amol LOQ: 5amol (CV <1%) Linear Dynamic Range: 4 orders Peak Area 3.E+9 2.E+9 SSAAPPPPPR GISNEGQNASIK 1.E+9 DIPVPKPK.E+ 5 1 Sample Amount (fmole) 21
Quantification with Targeted HCD Full MS/MS Spectra All Transitions Selectivity MS/MS High Resolution: 17,5 Accurate Mass: <5ppm Speed 12 Hz Sensitivity High quality HCD scan at low amol level 22
Quantification with Targeted HCD in Complex Matrix 11 Heavy Peptides (1amol 1fmol) In 25ng CSF Digest RT: 14.97-21.9 Relative Abundance Relative Abundance 1 8 6 4 2 1 8 6 4 2 15.72 15.67 15.74 15.82 15.85 15.88 15.96 VAHTVAYLGK 355.877-324.235 GPGEDFR 394.193-633.351 18.6 18.57 18.55 18.62 18.67 18.69 1amol thcd Resolution: 17,5 Relative Abundance 1 8 6 4 2 YFQGYAR 457.732-594.2989 2.61 2.58 2.57 2.54 2.64 2.65 2.67 15. 15.5 16. 16.5 17. 17.5 18. 18.5 19. 19.5 2. 2.5 21. Time (min) 1E+9 45 1 LOD: 1-1amol Linear Dynamic Range: 3-4 Orders Peak area 4 35 3 25 2 15 1 1 1 1 1 1 1 1 1 1 1 1 y = 382.1x + 578349 R² =.9999 YFQGYAR 5 2 4 6 8 1 12 Sample amount (amole) 23
Mass Measurement of Intact Monoclonal Antibody Relative Abundance 1 95 9 85 8 75 7 65 6 55 5 45 4 35 3 25 2 15 R: 17.5K 2471.345 2391.6288 2556.4844 2745.772 2851.3544 297.2595 2965.3764 325.8632 1 5 16 18 2 22 24 26 28 3 32 34 36 38 4 Protein Deconvolution -.7 ppm 5. ppm G+GF 2xMan5 G+G GF+GF -7 ppm GF+G1F Relative Abundance 1 95 9 85 8 75 7 65 6 55 5 45 4 35 3 25 2 15 1-8.5 ppm 5 2695.8919 268 27 272 274 276 278 28 282 GF+G2F (or 2G1F) -.9 ppm G1F+G2F 2745.772 G2F+G2F 2797.5697 24 G1F+G2F+SA
Summary Q Exactive is the most competitive instrument for routine discovery proteomics analysis, with its 1Hz duty cycle, high dynamic range and high quality HCD scans. Q Exactive offers flexible HR/AM quantification approaches, targeted msx SIM and targeted HCD, with improved throughput, higher sensitivity and 4-order linear dynamic range. Q Exactive enables smooth transition from discovery proteomics to targeted quantification. Q Exactive accurately and reproducibly measures the mass of intact antibody to ~7ppm, and provides a robust QC solution for biopharmaceutical products. 25
Acknowledgement 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 S. Daniels Y. Xun D. Cho X. He Y. Huang A. Kholomeev K. Persson 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. Ghosh C. Yang M. Blackburn 26