Q Exactive TM : A True Qual-Quan HR/AM Mass Spectrometer for Routine Proteomics Applications. Yi Zhang, Ph.D. ThermoFisher Scientific
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1 Q Exactive TM : A True Qual-Quan HR/AM Mass Spectrometer for Routine Proteomics Applications Yi Zhang, Ph.D. ThermoFisher Scientific
2 Outline Introduction of Q Exactive Performance in Discovery Proteomics Flexible Targeted Quantification Mass Measurement of Intact mab Summary 2
3 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
4 High Sensitivity with Quadrupole-based SIM Scan N= Full MS S/N = Gain in sensitivity (7x) Relative Abundance N= SIM (1amu) S/N = 54 S/N (spectrum) 4 3 Caffeine S/N (FMS) S/N (SIM1) Sensitivity Gain: 5 1 folds 4
5 Improved Resolution Advanced Signal Processing Increases resolution by 2X at the same transient length Resolution Scan Speed [Hz] Exactive Q-Exactive Resolution at 2 Resolution at 4 Max. scan speed (Hz) 17,5 12, , 25, 7 7, 5, 3 14, 1, 1.5 5
6 Scan Innovations Parallel Filling and Detection Spectrum Multiplexing 6
7 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
8 Discovery Proteomics 1Hz HCD Scan Parallel Filling and Detection Fast Orbitrap Scanning pagc 1 HR/AM HCD scans in 1s 256ms 64ms 8
9 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: Relative Abundance Time (min) RT: Relative Abundance =.181 min = 1.86 sec Time (min) Ecoli_5min_newcolumn_Top1_15cm_reject_1 # RT: AV: 1 NL: 1.58E8 T: FTMS + p NSI Full ms [3.-2.] x x5 Relative Abundance z= z= z= z= z= z=? z= z= z=? z= z=? 9
10 High Quality HCD Spectra Extracted from: C:\Xcalibur\data\Kai\Ecoli_5min_newcolumn_Top1_15cm_reject_1.raw #1241 RT: FTMS, HCD, z=+2, Mono = Da, MH+= Da, Match Tol.=2 mmu Extracted from: C:\Xcalibur\data\Kai\Ecoli_5min_newcolumn_Top1_15cm_reject_1.raw #12415 RT: FTMS, HCD, z=+3, Mono = Da, MH+= Da, Match Tol.=2 mmu Intensity [counts] (1^6) a₁ y₂+ a₂ y₁+ b₂ y₅ y₆ Intensity [counts] (1^6) a₂ y₂ a₇² y₄ y₁₁²+ y₉² y₅ b₇ y₆ y₈ Extracted from: C:\Xcalibur\data\Kai\Ecoli_5min_newcolumn_Top1_15cm_reject_1.raw #12411 RT: FTMS, HCD, z=+2, Mono = Da, MH+= Da, Match Tol.=2 mmu Extracted from: C:\Xcalibur\data\Kai\Ecoli_5min_newcolumn_Top1_15cm_reject_1.raw #12416 RT: FTMS, HCD, z=+2, Mono = Da, MH+= Da, Match Tol.=2 mmu Intensity [counts] (1^6) a₁ y₁+-h₂o y₂ y₄ Intensity [counts] (1^6) a₂ b₂ b₆²+, b₃ y₄ y₅ y₆ y₇ Extracted from: C:\Xcalibur\data\Kai\Ecoli_5min_newcolumn_Top1_15cm_reject_1.raw #12412 RT: FTMS, HCD, z=+2, Mono = Da, MH+= Da, Match Tol.=2 mmu Extracted from: C:\Xcalibur\data\Kai\Ecoli_5min_newcolumn_Top1_15cm_reject_1.raw #12417 RT: FTMS, HCD, z=+2, Mono = Da, MH+= Da, Match Tol.=2 mmu 1.4 Intensity [counts] (1^6) b₂+ b₃+-h₂o a₉²+-h₂o y₇+ b₃ y₅ y₄+ y₆ y₈ y₉ y₁₀ y₁₀+-nh₃ Intensity [counts] (1^6) a₁ y₁+ y₆ y₅+ y₉ y₁₁ Extracted from: C:\Xcalibur\data\Kai\Ecoli_5min_newcolumn_Top1_15cm_reject_1.raw #12413 RT: FTMS, HCD, z=+3, Mono = Da, MH+= Da, Match Tol.=2 mmu Extracted from: C:\Xcalibur\data\Kai\Ecoli_5min_newcolumn_Top1_15cm_reject_1.raw #12418 RT: FTMS, HCD, z=+2, Mono = Da, MH+= Da, Match Tol.=2 mmu 1. Intensity [counts] (1^6) y₂+ y₁+-h₂o b₄+ b₅ a₇³+-nh₃ b₃+, a₆² y₅ a₅+ y₆ y₇ Intensity [counts] (1^6) a₂ y₁ b₃ b₅ b₄ y₇ y₁₁ Extracted from: C:\Xcalibur\data\Kai\Ecoli_5min_newcolumn_Top1_15cm_reject_1.raw #12414 RT: FTMS, HCD, z=+3, Mono = Da, MH+= Da, Match Tol.=2 mmu Extracted from: C:\Xcalibur\data\Kai\Ecoli_5min_newcolumn_Top1_15cm_reject_1.raw #12419 RT: FTMS, HCD, z=+3, Mono = Da, MH+= Da, Match Tol.=2 mmu Intensity [counts] (1^6) a₂ a₆³+, b₂+, a₄² y₆ y₅ y₄ y₇ y₈ a₁₀+-h₂o, y₉ y₁₀ Intensity [counts] (1^3) y₃³ b₂+-h₂o y₂ a₂ b₃+-h₂o b₄+-h₂o y₁₀² b₅+-h₂o y₁₂² y₁₃² y₁₄² y₈ y₁₇²
11 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 Q Exactive TripTOF ng 1ng 1ng 1ng 1ng 1ng *About 45 proteins are expressed in yeast during log-phase growth 11
12 Peptide/Protein Identification Protein identified from 1 ng yeast digest Number of Proteins Identified x 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,
13 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
14 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: FTMS, HCD, z=+2, Mono = Da, MH+= 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) y₁ y₂ y₃ b₅+-nh₃ y₄ y₅ y₆+-h₂o y₆ y₇ y₈ y₉ y₁₀ Peptide of YOR2C, 149 copy number, identified from 1 ng yeast digest 14
15 Discovery Quan (TMT) TMT 6-plex labeled E. coli digest Number of peptides at 1% FDR Quantification Rate: >97% unique peptide total IDed MSMS spectra quantifiable MSMS spectra 2ng 4ng 8ng 2ng 5ng Amount of E. coli digest 15
16 Discovery Quan (TMT) Accuracy and Precision Quantification Error <5%, CV<13% 1.4 TMT reporter ion ratio observed expected 127/ / /126 13/ /126 Result from 8 ng E. Coli digest 16
17 Outline Introduction of Q Exactive Performance in Discovery Proteomics Flexible Targeted Quantification Targeted msx SIM Targeted HCD Mass Measurement of Intact mab Summary 17
18 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
19 Quantification with Targeted msx SIM High Resolution (14K) Accurate Mass (<5ppm) Selectivity R: 35K R: 7K R: 14K ppm Spectrum Multiplexing Speed 19
20 Quantification with Targeted msx SIM Quadrupole-Based SIM Sensitivity 2
21 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 GISNEGQNASIK 5ppm XIC Time (min) fmol 1fmol 1amol 4.E+9 R 2 : 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
22 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
23 Quantification with Targeted HCD in Complex Matrix 11 Heavy Peptides (1amol 1fmol) In 25ng CSF Digest RT: Relative Abundance Relative Abundance VAHTVAYLGK GPGEDFR amol thcd Resolution: 17,5 Relative Abundance YFQGYAR Time (min) 1E LOD: 1-1amol Linear Dynamic Range: 3-4 Orders Peak area y = 382.1x R² =.9999 YFQGYAR Sample amount (amole) 23
24 Mass Measurement of Intact Monoclonal Antibody Relative Abundance R: 17.5K Protein Deconvolution -.7 ppm 5. ppm G+GF 2xMan5 G+G GF+GF -7 ppm GF+G1F Relative Abundance ppm GF+G2F (or 2G1F) -.9 ppm G1F+G2F G2F+G2F G1F+G2F+SA
25 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
26 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
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