Data-independent acquisition (DIA) is becoming increasingly important for quantitative proteomics, especially for large-scale targeted protein quantification. DIA provides better reproducibility, acquires fragment ions of all precursors, and breaks through the limit of quantification throughput. Learn more here: www.thermoscientific.com/DIAwebinar

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The world leader in serving science
Data-Independent Analysis on Thermo Scientific Orbitrap MS Systems

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Main Topics
Introduction: Why Do DIA?
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Advantages of data independent acquisition, where, when, and why its useful
Where does DIA fit in?
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Positioning of DIA methods with other workflows like DDA and PRM
Data Independent Analysis
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Prerequisites, hardware and software
Challenges in DIA
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The three characteristics of the ideal DIA experiment and the compromises among them
Flexibility in DIA Methods
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Multiple varieties of MS2 and MS1+MS2 methods to suit experimental needs
Conclusion: If you Are Going to Make an Archive…
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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

4. 4
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 QUANTITATION PHASE
Data Dependent Acquisition
Data Independent 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⁺1815.73828y₁₅⁺1684.69775y₂⁺248.16026b₁₄⁺1497.80652b₁₀²⁺564.29541b₁₃²⁺-H₂O683.85907y₈⁺907.41931b₁₁⁺1226.65344y₁₀⁺1035.47791b₇⁺754.38391y₁₁⁺1195.50806500100015002000m/z0.00.51.01.5 Intensity [counts] (10^6) Extracted from: D:Yeast_SmartW303_Yeast_50cm_140min_120k30k_2.raw #67524 RT: 120.83 FTMS, HCD@26.00, 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
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Fragment Masses
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Fragment Intensities
•
Retention Time
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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⁺1815.73828y₁₅⁺1684.69775y₂⁺248.16026b₁₄⁺1497.80652b₁₀²⁺564.29541b₁₃²⁺-H₂O683.85907y₈⁺907.41931b₁₁⁺1226.65344y₁₀⁺1035.47791b₇⁺754.38391y₁₁⁺1195.50806500100015002000m/z0.00.51.01.5 Intensity [counts] (10^6) Extracted from: D:Yeast_SmartW303_Yeast_50cm_140min_120k30k_2.raw #67524 RT: 120.83 FTMS, HCD@26.00, z=+3, Mono m/z=1104.85144 Da, MH+=3312.53977 Da, Match Tol.=0.02 Da
y11 y10 y9 Y8 y7
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Number of Coeluting Library Transitions
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Retention Time Correlation
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Dot Product with Library Spectra
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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
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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 sec
120k
MS2 No. 1 … … … … … … … … … … … MS2 No.30
60k
DIA
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26 amu isolation
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Variable first mass
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60 000 resolution
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AGC: 2e5
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105ms Injection
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NCE 28%
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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
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MS2 No.32
32 Windows of 26 Da Each with 1 Da Overlap
m/z 400
m/z 1200
60k
4 sec
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
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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 sec
4 sec
120k
MS2 No. 1
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MS2 No.30
60k
Full MS
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400-1000 m/z
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Resolution: 120 000
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AGC: 3e6
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60 ms Injection Profile
DIA
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10 amu isolation
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Variable first mass
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60 000 resolution
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AGC: 1e5
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54 ms Injection
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NCE 28%
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MSX Count 2
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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 sec
4 sec
120k
MS2 No. 1
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MS2 No.30
60k
Full MS
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400-1000 m/z
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Resolution: 120 000
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AGC: 3e6
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60 ms Injection Profile
DIA
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10 amu isolation
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Variable first mass
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60 000 resolution
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AGC: 1e5
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54 ms Injection
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NCE 28%
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MSX Count 2
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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
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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
Randomly Distributed Windows
60k
120k
120k
60k
5 sec
2.5 sec
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
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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
Randomly Distributed Windows
60k
120k
120k
60k
5 sec
2.5 sec
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
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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 sec
4 sec
120k
MS2 No. 1
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MS2 No.30
60k
SIM
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400-600, 600-800, 800- 1000 m/z
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200 amu isolation
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Resolution: 240 000
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AGC: 3e4
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50 ms injection
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Profile
tMS2
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12 amu isolation
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150-1850 m/z
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Ion Trap
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AGC: 5e4
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47 ms Injection
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CID 30
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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
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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 sec
4 sec
120k
MS2 No. 1
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MS2 No.30
60k
SIM
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400-600, 600-800, 800- 1000 m/z
•
200 amu isolation
•
Resolution: 240 000
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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
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MS2 No.17
17 Windows of 12 Da Each
MS1 No.1
SIM 600 – 800 m/z
Ion Trap
240k
3.6 sec
SIM 800 – 1000 m/z
MS1 No.2
240k
MS2 No. 1
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MS2 No.17
Ion Trap
MS1 No.3
240k
MS2 No. 1
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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
240k
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
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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⁺1815.73828y₁₅⁺1684.69775y₂⁺248.16026b₁₄⁺1497.80652b₁₀²⁺564.29541b₁₃²⁺-H₂O683.85907y₈⁺907.41931b₁₁⁺1226.65344y₁₀⁺1035.47791b₇⁺754.38391y₁₁⁺1195.50806500100015002000m/z0.00.51.01.5 Intensity [counts] (10^6) Extracted from: D:Yeast_SmartW303_Yeast_50cm_140min_120k30k_2.raw #67524 RT: 120.83 FTMS, HCD@26.00, 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.

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Access the highest resolutions and mass accuracy.
DIA: In Summary

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DIA: In Summary
Have the flexibility to quantify on either the MS1 or MS2 level at high resolution

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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
Resolution
m/z
Resolution at 7 Hz Analyzer Setting
Over Typical Fragment Range
35k
27k
19k

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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
Resolution
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