1. 1 The world leader in serving science Proprietary & Confidential WiSIM DIA Method Set-up Orbitrap Fusion MS MS Instrument Control Software v.1.1 SP1

2. 2 WiSIM DIA, how does it work? Unique to the Orbitrap Fusion MS: Three HR/AM SIM are used to cover precursors ions from 400 to 1000 m/z MS1 ultra high resolution quantification In parallel with each HR/AM SIM scan, 17 sequential Ion Trap CID MS/MS with 12 amu isolation windows are acquired to cover the associated 200 amu HR/AM SIM mass range MS2 confirmation SIM 400-600 amuSIM 600-800 amuSIM 800-1000 amu 406418430442598 606618630642898 806818830842998 tSIM Orbitrap tMS2 Ion Trap

3. 3 WiSIM DIA, how does it work? SIM 400 – 600 m/z MS2 No. 1 … MS2 No.17 1 st MS1 SIM 600 – 800 m/z Ion Trap OT 240k 3.6 sec SIM 800 – 1000 m/z 2 nd MS1 MS2 No. 1 … MS2 No.17 Ion Trap 3 rd MS1 MS2 No. 1 … MS2 No.17 Ion Trap OT 240k OT 240k 17 Windows of 12 amu each 17 Windows of 12 amu each 17 Windows of 12 amu each

4. 4 WiSIM DIA Method Set-up at a glance: SIM 400-600, 600-800, 800-1000 m/z 200 amu ion trap isolation width Resolution@ 240 000 AGC: 3e4 50 ms Max injection Profile tMS2 12 amu quadrupole isolation 150-1850 m/z mass range Ion Trap detection AGC: 5e4 35 ms Max Injection CID 30 Rapid Scan rate X3 6 experiments: 406514 418526 430538 442550 454562 466574 478586 490598 502 606714 618726 630738 642750 654762 666774 678786 690798 702 806914 818926 830938 842950 854962 866974 878986 890998 902

5. 5 WiSIM DIA Method Set-up: 6 experiments: -Three HR/AM tSIM -Three Ion trap tMS2 CID

6. 6 Experiment 1: SIM 400-600 amu SIM 600-800 amuSIM 800-1000 amu 406418430442598 606618630642898 806818830842998

7. 7 Experiment 2: SIM 400-600 amuSIM 600-800 amuSIM 800-1000 amu 406418430442598 606618630642898 806818830842998

8. 8 Experiment 2: SIM 400-600 amuSIM 600-800 amuSIM 800-1000 amu 406418430442598 606618630642898 806818830842998

9. 9 Experiment 3: SIM 400-600 amu SIM 600-800 amu SIM 800-1000 amu 406418430442598 606618630642898 806818830842998

10. 10 Experiment 4: SIM 400-600 amuSIM 600-800 amuSIM 800-1000 amu 406418430442598 606618630642898 806818830842998

11. 11 Experiment 4: SIM 400-600 amuSIM 600-800 amuSIM 800-1000 amu 406418430442598 606618630642898 806818830842998

12. 12 Experiment 5: SIM 400-600 amuSIM 600-800 amu SIM 800-1000 amu 406418430442598 606618630642898 806818830842998

13. 13 Experiment 6: SIM 400-600 amuSIM 600-800 amuSIM 800-1000 amu 406418430442598 606618630642898 806818830842998

14. 14 Experiment 6: SIM 400-600 amuSIM 600-800 amuSIM 800-1000 amu 406418430442598 606618630642898 806818830842998

15. 15 WiSIM-DIA advantages Low detection limits, high selectivity is achieved using 240,000 SIM scan. High sensitivity Ion Trap CID MS2 is used for confirmation of the low abundant components. Unsurpassed speed with 2 detectors working in parallel allows for 3.6 s cycle. LOD and LOQ down to 10 attomole. CVs are better than 15 % for 85 % of compounds, better than 20% for 95% of compounds. Linear dynamic range up to 4 orders of magnitude.

16. 16 For More details, please see Application Note 600 And: http://planetorbitrap.com/orbitrap-fusion-tips