Introduction to the Thermo Scientific Q Exactive HF-X MS for Proteomics

Transcription

1 Introduction to the Thermo Scientific Q Exactive HF-X for Proteomics October 217 Josh Nicklay 1 The world leader in serving science

2 Thermo Scientific Q Exactive HF-X Hybrid Quadrupole-Orbitrap Pushing the leading edge in protein analysis Revolutionizing insights, from discovery to verification Enhance your productivity Achieve faster than ever scan speed Confirm with greater confidence Superior consistency in quantitative accuracy, sensitivity and reproducibility 2

3 Thermo Scientific Q Exactive HF-X Hybrid Quadrupole-Orbitrap Novel architecture with a high capacity transfer tube and electrodynamic ion funnel Ultra-high field Orbitrap analyzer 24, resolution at m/z 2 4 Hz data acquisition 7,5 resolution Advanced Peak Determination (APD) BioPharma Option for intact proteins 3

4 Thermo Scientific Q Exactive HF-X New Architecture Optimized Scan Matrix with accelerated HCD 4 Hz / Advanced DDA (APD) for bottom-up and top-down HyperQuad Mass Filter with Advanced Quadrupole Technology (AQT) Advanced Active Beam Guide (AABG) Electrodynamic Ion Funnel C-Trap HCD Cell Dedicated transient for TMT 1plex Ultra-High Field Orbitrap Mass Analyzer High Capacity Transfer Tube (HCTT) Brighter ion beam More sensitive 4

5 Key Technologies of Thermo Scientific Q Exactive HF-X High Capacity Transfer Tube Electrodynamic Ion Funnel 28 Hz 32 ms maxit 32 ms 4 Hz 16 ms 16 ms Ultra-High Field Orbitrap Optimized Scan Matrix and new Transient lengths 5

6 Optimized Scan Matrix Q Exactive HF Hz 32 ms 15k maxit Q Exactive HF-X 28 Hz 32 ms 15k 4 Hz maxit 32 ms / 32 ms Longer maxit Reduced scan overhead ahcd Brighter ion beam, reduced scan overhead, and accelerated HCD (ahcd) is boosting acquisition speed Advantage for both and / mode Fast and high quality / acquisition up the 4 Hz with new 16 msec transient (7,5 resolution setting) 7.5k 16 ms 16 ms Similar maxit Reduced scan overhead ahdc Orbitrap detection Maximum fill time with precursor ions Inter and intra scan overheads 6

7 / Scan Rate 37 Scan time [ms] Inject time [ms] Scan rate [Hz] / scan rate at resolution setting m/z 2 for Q Exactive HF-X with respect to the ion injection time is displayed. Sample: Calmix 4 Hz / scan rate is reached with a maximum injection time (maxit) of 1 ms AS 17: TP 389, T.N. Arrey et al. New innovations implemented on the Thermo Scientific Q Exactive HF. 7

8 Ultra Fast / Scan Speed > 4 Hz Relative Abundance Duty cycle: 1.5 s Scan rate (1 FS + 4 2): 38.1Hz 4 2: 45.1 Hz Duty cycle: 1.3 s Scan rate(1 FS + 4 2): 38.8 Hz 4 2: 45.8 Hz Duty cycle: 1.2 s Scan rate (1 FS + 4 2): 39.2 Hz 4 2: 46.1 Hz Duty cycle: 1.9 s Scan rate (1 FS + 4 2): 36.6 Hz 4 2: 42.8 Hz Time (min) full scan m/z 2) and 4 2 scans 7,5@ m/z 2) at LC time scale in 1 second. 3 min gradient, 2 max IT: 11 ms AS 17: TP 389, T.N. Arrey et al. New innovations implemented on the Thermo Scientific Q Exactive HF. 8

9 Calculated Scan Rate Across the LC Gradient for 1 µg HeLa Digest 5 45 Scan Frequency [Hz] 3 min_7.5k, 3min_7.5k, 11 11ms, Top4 3 min gradients: Scan Frequency [Hz] min_7.5k, 3min_7.5k, 35 35ms, Top2 3 min_7.5k, 25 ms, Top2 3min_7.5k, 25ms, Top Top4 method: Scan rates of 38 Hz are obtainable at high ion flux with 11 ms max. inj. times applied Top2 method: Scan rates of > 25 Hz are obtained with longer max. inj. times (25 ms and 35 ms, resp.) Retention Time [min] AS 17: TP 389, T.N. Arrey et al. New innovations implemented on the Thermo Scientific Q Exactive HF. 9

10 Protein Identification Faster than Ever Protein groups Q Exactive HF, 6 min Q Exactive HF-X, 3 min Unique Peptides Q Exactive HF, 6 min Q Exactive HF-X, 3 min Maximizing protein identifications Same protein identifications in half the analysis time Faster, with same high quality results Similar data, half time Similar data, half time Q Exactive HF, 6 min Q Exactive HF-X, 3 min Q Exactive HF, 6 min Q Exactive HF-X, 3 min 1 µg HeLa, nano LC, 12k res 1, DDA Top 2/4, minimum injection time 45/25 msec AS 17: TP 389, T.N. Arrey et al. New innovations implemented on the Thermo Scientific Q Exactive HF. 1

11 Deeper Dive into Proteome - More Productivity with Thermo Scientific Q Exactive HF-X Q Exactive Plus Q Exactive HF Q Exactive HF-X Q Exactive Plus 12 min Q Exactive HF 6 min Q Exactive HF-X 3 min Maximizing peptide identifications 3478 increased peptide ID efficiency Hz 18Hz 4Hz / speed Highest peptide coverage Deep proteome analysis Spectral library building 2x productivity increase vs. Q Exactive HF 4x productivity increase vs. Q Exactive Plus Peptide Groups - 6min Peptide Groups Sample: 1 ug Pierce HeLa digest AS 17: TP 389, T.N. Arrey et al. New innovations implemented on the Thermo Scientific Q Exactive HF. 11

12 Dilution Series Pierce HeLa Digest Reproducible and consistent identifications across a large range of sample load on column 5 4 Average no of protein groups ng 1ng 5ng 1ng 5ng 1ng 2ng 5ng 1ng 2ng Average no of unique peptides ng 1ng 5ng 1ng 5ng 1ng 2ng 5ng 1ng 2ng min gradients each. Method set-up adapted to according to different sample load on column from.5 ng 2 ng 12

13 Rapid Proteomics - More than 1, Peptides Identified per Minute 12 Q Exactive HF (15k) 5% increased rate of peptide identifications when ion flux is max Up to ~ 11 peptides identified per minute Sample: 1 µg HeLa number of unique peptides per minute Q Exactive HF - X (7.5k) Short transient time of 16 ms combined with accelerated HCD allows for scan rates up to 4 Hz in data dependent and targeted analyses Benefit is increased productivity, expressed by more peptide IDs per time gradient length (min, log scale) Data with courtesy from J. Olsen, Novo Nordisk Foundation, Center for Protein Research, University of Copenhagen. 13

14 Proteome Perspectives: Rapid and Deep Proteome Sequencing in Less than Half the Acquisition Time New data from Thermo Scientific Q Exactive HF-X overlaid with published data 1 µg fractionated into 12 concentrated fractions 3 min gradient, 15, res. method on Q Exactive HF-X J. Proteome Res. 214, 13(12), pp Data with courtesy from J. Olsen, Novo Nordisk Foundation, Center for Protein Research, University of Copenhagen 14

15 New 96 msec Transient Ideal for TMT 1plex R=66,72 at m/z R=87,72 R=66,22 at m/z R=86,92 A dedicated resolution setting of 45, at m/z 2 (FWHM) Optimally resolves reporter ions of the Thermo Scientific TMT1plex Isobaric Mass Tag Labeling Kit Frees up scan time to quantify 1 2% more peptides in discovery experiments m/z Optimal separation of TMT reporter ions 15

16 Scan Cycle Comparison Relative Abundance scan cycle: 3. s Scan freq (1FS+22): 6.8Hz 6k Scan cycle: 2.3 s Scan freq (1FS+22): 8.7Hz 45k Scan cycle: 2.3 s Scan freq (1FS+22): 8.7Hz Scan cycle:2.9s Scan freq (1FS+22): 6.8Hz 6k scan cycle: 2.3 s Scan freq (1FS+22): 8.7Hz 45k Scan cycle: 3. s Scan freq (1FS+22): 6.6Hz Duty cycle: 2.3 s Scan freq (1FS+22): 8.7Hz Scan cycle comparison between 6k resolution setting, Thermo Scientific Q Exactive HF (top) Novel 45k resolution setting Thermo Scientific Q Exactive HF-X (bottom) Time (min) Acquisition speed increase by ca. 25% allowing quantitation of more peptides per unit time with TMT 1plex. Method: Full (12k res), dd Top 2 / (6k or 45k) scans 16

17 6k on HF vs. 45k on HF-X TMT11-plex labeled HeLa 1 µg on column, ~9 min gradient 12/86 ms max inject on Thermo Scientific Q Exactive HF/HF-X >9% quantified >9% quantified 17

18 Robust DIA Proteome Profiling using CapLC with Thermo Scientific QE HF-X VALUE: Highest depth of coverage and robust quantitative analysis HR-DIA data processed with Spectronaut software, Biognosys AG, Switzerland Similar data on HF-X with 2 µg as HF with 4 µg Greater proportion of peptides with CVs < 1% on HF-X HeLa digest, 3 technical replicates each, 6 mins total run time AS 17: ThP 237 Y. Xuan et al. Revolutionary Proteome Profiling and Quantitation without Compromising Speed, Sensitivity, and Selectivity. 18

19 Top Down Proteomics - A Novel Workflow Brings Intact Proteins Into Focus Base peak chromatogram of E. coli ribosomal proteins separated in a 3 min gradient Relative Abundance RT Time (min) RT min Relative Abundance Thermo Scientific Q Exactive HF-X Full scan acquired in Protein Mode at a resolution setting of 7,5, detecting multiple charge envelopes z= z= z= z= z= z= z= z= z= z=22 On-the-fly deconvolution based on charge envelope to select a single charge state of each dominant proteoform z= z= z= m/z 19

20 Top Down Proteomics - A Novel Workflow Brings Intact Proteins Into Focus On-the-fly deconvolution based on charge envelope to select a single charge state of each dominant proteoform. / analysis of each proteoform fragmented with optimal collision energy and detected at a resolution setting of 12,. Relative Abundance z= z= z= z= z= z= z= z= z= z= z= z= z= m/z post acquisition Reöative Intensity z= Deconvolution with Respect Mass T: FT + p ESI d Full ms @hcd3. [2.-8.] R=6836 z=15 1 Relative Abundance 9 8 / Proteoform R=796 6 z= T: FT + p ESI d Full ms @hcd3. [2.-8.] R= z= R= R= z= z=16 R= R= z= z=3 9 R= R=686 8 z=17 R=7236 z=8 / 1 z=7 7 Proteoform R= z=7 m/z R=746 R= T: FT + p ESI d Full ms2 z= @hcd3. z=8 [2.-8.] R= z=7 R=6446 R= z=8 z=15 R=786 1 R=5996 z=14 1 z= m/z R= z=22 Relative Abundance Relative Abundance R=8346 z= R=7856 z= R=886 z= R=7316 z= R=6516 z= m/z / Proteoform 3 2

21 Thermo Scientific Q Exactive HF-X Hybrid Quadrupole-Orbitrap Pushing the leading edge in protein analysis Revolutionizing insights, from discovery to verification Enhance your productivity Achieve faster than ever scan speed Confirm with greater confidence Superior consistency in quantitative accuracy, sensitivity and reproducibility 21

22 Acknowledgement LS team (Bremen & San Jose): Eric Couzijn Eloy Wouters Oliver Lange Dean Dumitresku Christian Thoeing Franz-Josef Paffen Dirk Nolting Karsten Goepel Jens Grote Tabiwang Arrey Matthias Mueller Martin Zeller Anastassios Christian Klaas Giannakopulos Eugen Damoc Alexander Makarov Markus Kellmann Andreas Boegehold Kerstin Strupat Nicole Zehethofer Yue Xuan Bjorn Rose Thomas Moehring Dennie Kemper Alexander Harder Patrick Huesing Maciej Bromirski Matthias Vollrath Keeley M. Murphy Sebastian Kanngiesser Aaron O. Bailey Sascha Moehring Jonathan Josephs Stefanie Aaron Gajadhar Raffelhueschen Michael Krawitzky Andreas Huhmer Sales Advisory board Brenda Kesler Tony Ziberna Rick Carberry Albar Martucci Claire Dauly Gary Woffendin Glenn Damkroeger Jenny Ho Wilfried Voorhorst Jocelyn Dupuy Kai Scheffler Olaf Scheibner Lin Tang Goh Jing Li Kentaro Takahara Eunyoung Lim Ruby Ong Darren Jones Michael Mariani Collaborators: Jesper V. Olsen, Christian D. Kelstrup, Dorte B. Bekker-Jensen Albert J. R. Heck, Kyle L. Fort, Michiel van de Waterbeemd, Sem Tamara 22