New DIA Workflows for Ultimate Flexibility in LCMS Proteomics
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The world leader in serving science Data-Independent Analysis on Thermo Scientific Orbitrap MS Systems
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Transcript of New DIA Workflows for Ultimate Flexibility in LCMS Proteomics
1 The world leader in serving science
Data-Independent Analysis on Thermo Scientific Orbitrap MS Systems
2
Main Topics
Introduction: Why Do DIA?
• Advantages of data independent acquisition, where, when, and why its useful
Where does DIA fit in?
• Positioning of DIA methods with other workflows like DDA and PRM
Data Independent Analysis
• Prerequisites, hardware and software
Challenges in DIA
• The three characteristics of the ideal DIA experiment and the compromises among them
Flexibility in DIA Methods
• Multiple varieties of MS2 and MS1+MS2 methods to suit experimental needs
Conclusion: If you Are Going to Make an Archive…
• The new Thermo ScientificTM Q ExactiveTM HF MS as a DIA workhorse
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Introduction: Traditional Quantitation Tools
Relative Quantification (lower accuracy and precision)
Absolute Quantification (highest accuracy and precision)
Discovery Routine
MS1 level quan., DDA methods, 1000’s analytes DDA or SRM
up to 100 analytes
SRM, MS2 level quan., 1-10 analytes
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Introduction: Quantitation Transformed
Relative Quantification (lower accuracy and precision)
Absolute Quantification (highest accuracy and precision)
Discovery Routine
MS1 level quan., DDA methods, 1000’s analytes DIA or PRM
several 100 analytes
SRM, MS2 level quan., 1-10 analytes
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Introduction: A Time and Place for DIA
Typical Samples Published On:
Best Suited When
Protein Complexes
Enriched Samples (e.g. phosphopeptides)
Cellular Fractions
IP Pulldowns
GLOBAL QUANTITA
TION PHASE
Data Dependent Acquisition
Data Independe
nt Acquisition
wiSIM
Methods for a variety of sample conditions:
pSMART
basicDIA
msxDIA
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Introduction: Advantages of DIA
No targeting or timing
Retrospectively find new targets of interest
Run unmodified sample
Why perform Data Independent Analysis?
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HIGH QUALITY SPECTRAL LIBRARIES are critical to a successful DIA experiment
Generating Spectral Libraries
Validated High Quality Library
SEQUEST Search
y₁₆⁺1815.73828
y₁₅⁺1684.69775
y₂⁺248.16026
b₁₄⁺1497.80652
b₁₀²⁺564.29541
b₁₃²⁺-H₂O683.85907
y₈⁺907.41931
b₁₁⁺1226.65344
y₁₀⁺1035.47791
b₇⁺754.38391
y₁₁⁺1195.50806
500 1000 1500 2000
m/z
0.0
0.5
1.0
1.5
Inte
nsity
[cou
nts]
(10^
6)
Extracted from: D:\Yeast_Smart\W303_Yeast_50cm_140min_120k30k_2.raw #67524 RT: 120.83 FTMS, [email protected], z=+3, Mono m/z=1104.85144 Da, MH+=3312.53977 Da, Match Tol.=0.02 Da
Matches VALIDATED at 1% FDR
High resolution accurate mass archive of all identified peptides
• Peptide Intact Mass
• Fragment Masses
• Fragment Intensities
• Retention Time
• Isotope Ratios
under actual experimental conditions
1 µg HeLa
60 min
The Q Exactive HF MS generates a more comprehensive spectral library
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Using Libraries to Validate Results
DIA Experimental Data Wide Window MS2 Fragment Ion Spectra
Validated Spectral Library
y₁₆⁺1815.73828
y₁₅⁺1684.69775
y₂⁺248.16026
b₁₄⁺1497.80652
b₁₀²⁺564.29541
b₁₃²⁺-H₂O683.85907
y₈⁺907.41931
b₁₁⁺1226.65344
y₁₀⁺1035.47791
b₇⁺754.38391
y₁₁⁺1195.50806
500 1000 1500 2000
m/z
0.0
0.5
1.0
1.5
Inte
nsity
[cou
nts]
(10^
6)
Extracted from: D:\Yeast_Smart\W303_Yeast_50cm_140min_120k30k_2.raw #67524 RT: 120.83 FTMS, [email protected], z=+3, Mono m/z=1104.85144 Da, MH+=3312.53977 Da, Match Tol.=0.02 Da
y11 y10 y9 Y8 y7
• Number of Coeluting Library Transitions
• Retention Time Correlation
• Dot Product with Library Spectra
• Mass Accuracy
How many library fragments are in the integrated peak
How close is the RT of the integrated peak to the library peptide (blue line)
What is the relative intensity of integrated peak relative to the real fragment masses
What is the mass error of the integrated peak relative to the real fragment masses
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Achieving the Utmost DIA Performance
Shorter Injection
Longer Injection More Resolution Narrower Windows
Less Resolution
Wider Windows
Reduced Mass Range
Greater Mass Range
Speed
Sensitivity
Selectivity
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basicDIA: 25 Da Segments
Quantify and confirm on the MS2 level using wide isolation windows
400 – 1000 m/z
MS2 No. 1 … … … … … … … … … … … MS2 No.30
120 Windows of 6 Da Each with 1 Da Overlap
MS1 No.1 m/z 400
400 – 1000 m/z MS2 No.2
m/z 1000
60k
120k
4 se
c
120k
MS2 No. 1 … … … … … … … … … … … MS2 No.30
60k
DIA • 26 amu isolation • Variable first mass • 60 000 resolution • AGC: 2e5 • 105ms Injection • NCE 28% • Loop Count 32
Windows 412.5 612.5 812.5 1012.5 437.5 637.5 837.5 1037.5 462.5 662.5 862.5 1062.5 487.5 687.5 887.5 1087.5 512.5 712.5 912.5 1112.5 537.5 737.5 937.5 1137.5 562.5 762.5 962.5 1162.5 587.5 787.5 987.5 1187.5 412.5 612.5 812.5 437.5 637.5 837.5 462.5 662.5 862.5 487.5 687.5 887.5
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60k
basicDIA at 60K FWHM
25 Da windows covering the mass range from 400 to 1200 (optional MS1)
MS2 No. 1 … … … … … … … … … … … … … MS2 No.32
32 Windows of 26 Da Each with 1 Da Overlap m/z 400 m/z 1200
60k
4 se
c
MS2 No. 1 … … … … … … … … … … … … … MS2 No.32
basicDIA provides good reproducibility due to short cycle times
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msxDIA
Speed / Cycle Time of using 20 Da windows with the selectivity of 10 Da windows
400 – 1000 m/z
MS2 No. 1 … … … … … … … … … … … MS2 No.30
120 Windows of 6 Da Each with 1 Da Overlap
MS1 No.1 m/z 400
400 – 1000 m/z MS2 No.2
m/z 1000
60k
120k
20 s
ec
4 se
c
120k
MS2 No. 1 … … … … … … … … … … … MS2 No.30
60k
Full MS • 400-1000 m/z • Resolution: 120 000 • AGC: 3e6 • 60 ms Injection Profile
DIA • 10 amu isolation • Variable first mass • 60 000 resolution • AGC: 1e5 • 54 ms Injection • NCE 28% • MSX Count 2 • Loop Count 16
Windows
835.6297 985.6979 635.5388 615.5297 775.6024 765.5979 475.466 925.6706 715.5751 605.5251 915.6661 745.5888 785.607 445.4524 575.5115 685.5615 655.5479 855.6388 555.5024 845.6343 515.4842 795.6115 995.7025 425.4433 885.6525 465.4615 875.6479 625.5342 905.6615 565.5069 705.5706 665.5524 585.516 595.5206 945.6797 505.4796 495.4751 645.5433 805.6161 675.557 955.6843 455.4569 725.5797 405.4342 695.5661 525.4887 825.6252 935.6752 535.4933 435.4478 815.6206 415.4387 965.6888 485.4706 865.6434 545.4978 975.6934 755.5933 895.657 735.5842
Randomized List Generated by Skyline
………... continues to 5000 targets
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msxDIA
Speed / Cycle Time of using 20 Da windows with the selectivity of 10 Da windows
400 – 1000 m/z
MS2 No. 1 … … … … … … … … … … … MS2 No.30
120 Windows of 6 Da Each with 1 Da Overlap
MS1 No.1 m/z 400
400 – 1000 m/z MS2 No.2
m/z 1000
60k
120k
20 s
ec
4 se
c
120k
MS2 No. 1 … … … … … … … … … … … MS2 No.30
60k
Full MS • 400-1000 m/z • Resolution: 120 000 • AGC: 3e6 • 60 ms Injection Profile
DIA • 10 amu isolation • Variable first mass • 60 000 resolution • AGC: 1e5 • 54 ms Injection • NCE 28% • MSX Count 2 • Loop Count 16
………... continues to 5000 targets
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DIA on the Q Exactive HF MS: msxDIA
Speed / Cycle Time of using 20 Da windows with the selectivity of 10 Da windows
400 – 1000 m/z
MS2 No. 3 … … … … … … … … … … … … … … … MS2 No.30
60 Windows of 10 Da Each
Full Coverage of 400–1000 m/z
Based upon: Egertson et al. (2013) Nature Methods 10, 744-766
Multiplex 2 x 10 Da Windows Simultaneously
MS1 No.1 m/z 400
400 – 1000 m/z MS1 No.2
MS2 No. 1 MS2 No. 2
m/z 1000
MS2 No. 1
Ran
dom
ly D
istri
bute
d W
indo
ws
60k
120k
12
0k
60k
5 se
c 2.
5 se
c
60k
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DIA on the Q Exactive HF MS: msxDIA
Speed / Cycle Time of using 20 Da windows with the selectivity of 10 Da windows
400 – 1000 m/z
MS2 No. 3 … … … … … … … … … … … … … … … MS2 No.30
60 Windows of 10 Da Each
Full Coverage of 400–1000 m/z
Based upon: Egertson et al. (2013) Nature Methods 10, 744-766
Multiplex 2 x 10 Da Windows Simultaneously
MS1 No.1 m/z 400
400 – 1000 m/z MS1 No.2
MS2 No. 1 MS2 No. 2
m/z 1000
MS2 No. 1
Ran
dom
ly D
istri
bute
d W
indo
ws
60k
120k
12
0k
60k
5 se
c 2.
5 se
c
An approach providing a good balance of selectivity, speed, and sensitivity suited for complex mixtures
60k
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DIA on the Thermo Scientific™ Orbitrap Fusion™ Tribrid™ MS: WiSIM
The power of two detectors working in parallel: unsurpassed speed and sensitivity
400 – 1000 m/z
MS2 No. 1 … … … … … … … … … … … MS2 No.30
120 Windows of 6 Da Each with 1 Da Overlap
MS1 No.1 m/z 400
400 – 1000 m/z MS2 No.2
m/z 1000
60k
120k
20 s
ec
4 se
c
120k
MS2 No. 1 … … … … … … … … … … … MS2 No.30
60k
SIM • 400-600, 600-800, 800-
1000 m/z • 200 amu isolation • Resolution: 240 000 • AGC: 3e4 • 50 ms injection • Profile
tMS2 • 12 amu isolation • 150-1850 m/z • Ion Trap • AGC: 5e4 • 47 ms Injection • CID 30 • Rapid Scan
Windows
406 514 606 714 806 914 418 526 618 726 818 926 430 538 630 738 830 938 442 550 642 750 842 950 454 562 654 762 854 962 466 574 666 774 866 974 478 586 678 786 878 986 490 598 690 798 890 998 502 702 902
Large-Scale Targeted Protein Quantification Using WiSIM-DIA on an Orbitrap Fusion Tribrid Mass Spectrometer Kiyonami R, Senko M, Zabrouskov V, Huhmer A, Egertson J, Ting S, and MacCoss M.
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DIA on the Thermo Scientific™ Orbitrap Fusion™ Tribrid™ MS: WiSIM
The power of two detectors working in parallel: unsurpassed speed and sensitivity
400 – 1000 m/z
MS2 No. 1 … … … … … … … … … … … MS2 No.30
120 Windows of 6 Da Each with 1 Da Overlap
MS1 No.1 m/z 400
400 – 1000 m/z MS2 No.2
m/z 1000
60k
120k
20 s
ec
4 se
c
120k
MS2 No. 1 … … … … … … … … … … … MS2 No.30
60k
SIM • 400-600, 600-800, 800-
1000 m/z • 200 amu isolation • Resolution: 240 000 • AGC: 3e4 • 50 ms injection • Profile
tMS2 • 12 amu isolation • 150-1850 m/z • Ion Trap • AGC: 5e4 • 47 ms Injection • CID 30 • Rapid Scan
Large-Scale Targeted Protein Quantification Using WiSIM-DIA on an Orbitrap Fusion Tribrid Mass Spectrometer Kiyonami R, Senko M, Zabrouskov V, Huhmer A, Egertson J, Ting S, and MacCoss M.
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DIA on the Orbitrap Fusion MS: WiSIM
Quantify using ultra-high resolution MS1 with MS2 confirmation and IT sensitivity
SIM 400 – 600 m/z MS2 No. 1 … … … … … … MS2 No.17
17 Windows of 12 Da Each MS1 No.1
SIM 600 – 800 m/z
Ion
Trap
24
0k
3.6
sec
SIM 800 – 1000 m/z
MS1 No.2 240k
MS2 No. 1 … … … … … … MS2 No.17
Ion
Trap
MS1 No.3 240k
MS2 No. 1 … … … … … … MS2 No.17
Ion
Trap
Parallelized
Parallelized
Parallelized
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DIA on the Orbitrap Fusion MS: WiSIM
Quantify using ultra-high resolution MS1 with MS2 confirmation and IT sensitivity
SIM 400 – 600 m/z MS2 No. 1 … … … … … … MS2 No.17
17 Windows of 12 Da Each MS1 No.1
SIM 600 – 800 m/z
Ion
Trap
24
0k
3.6
sec
SIM 800 – 1000 m/z
MS1 No.2 240k
MS2 No. 1 … … … … … … MS2 No.17
Ion
Trap
MS1 No.3 240k
MS2 No. 1 … … … … … … MS2 No.17
Ion
Trap
Parallelized
Parallelized
Parallelized wiSIM provides fully parallelized speed, IT sensitivity, and ultimate resolution
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Achieving the Utmost DIA Performance
Short cycle times, good reproducibility
Excellent selectivity and sensitivity
Balance of speed, sensitivity, selectivity
Fully parallelized speed, IT sensitivity, ultimate resolution
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DIA: In Summary
y₁₆⁺1815.73828
y₁₅⁺1684.69775
y₂⁺248.16026
b₁₄⁺1497.80652
b₁₀²⁺564.29541
b₁₃²⁺-H₂O683.85907
y₈⁺907.41931
b₁₁⁺1226.65344
y₁₀⁺1035.47791
b₇⁺754.38391
y₁₁⁺1195.50806
500 1000 1500 2000
m/z
0.0
0.5
1.0
1.5
Inte
nsity
[cou
nts]
(10^
6)
Extracted from: D:\Yeast_Smart\W303_Yeast_50cm_140min_120k30k_2.raw #67524 RT: 120.83 FTMS, [email protected], z=+3, Mono m/z=1104.85144 Da, MH+=3312.53977 Da, Match Tol.=0.02 Da
Obtain super deep proteomic profiling using high resolution / accurate mass in under an hour.
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DIA: In Summary
Access the highest resolutions and mass accuracy.
23
Access the highest resolutions and mass accuracy.
DIA: In Summary
24
DIA: In Summary
Have the flexibility to quantify on either the MS1 or MS2 level at high resolution
25
DIA: In Summary
Have the selectivity of narrow windows AND the speed of wide windows thanks to precursor multiplexing
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DIA: In Summary
Ensure reproducible quantitation with scan speeds up to 18 Hz !
10 points 3 points
RSD 8-15% RSD >25%
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DIA: In Summary
Next generation analyzers provide twice the resolution at the same speed
0
20000
40000
60000
80000
100000
120000
400 600 800 1000 1200 1400 1600 1800 2000
Res
olut
ion
m/z
Resolution at 7 Hz Analyzer Setting Over Typical Fragment Range
35k 27k
19k
28
DIA: In Summary
dotp 0.99
The capabilities of the Orbitrap platform make it the ideal DIA instrument
Next generation analyzers provide twice the resolution at the same speed
0
20000
40000
60000
80000
100000
120000
400 600 800 1000 1200 1400 1600 1800 2000
Res
olut
ion
m/z
Resolution at 7 Hz Analyzer Setting Over Typical Fragment Range
35k 27k
19k
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Concluding Remarks
Transform your DIA experiment with the latest Orbitrap instruments
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Transform Your Science Learn more here:
www.thermoscientific.com/DIAwebinar
