Characterizing the top-down sequencing of protein ions prior to mobility separation in a timsTOF

Mass spectrometry (MS)-based proteomics workflows of intact protein ions have increasingly been utilized to study biological systems. These workflows, however, frequently result in convoluted and difficult to analyze mass spectra. Ion mobility spectrometry (IMS) is a promising tool to overcome these limitations by separating ions by their mass- and size-to-charge ratios. In this work, we further characterize a newly developed method to collisionally dissociate intact protein ions in a trapped ion mobility spectrometry (TIMS) device. Dissociation occurs prior to ion mobility separation and thus, all product ions are distributed throughout the mobility dimension, enabling facile assignment of near isobaric product ions. We demonstrate that collisional activation within a TIMS device is capable of dissociating protein ions up to 66 kDa. We also demonstrate that the ion population size within the TIMS device significantly influences the efficiency of fragmentation. Lastly, we compare CIDtims to the other modes of collisional activation available on the Bruker timsTOF and demonstrate that the mobility resolution in CIDtims enables the annotation of overlapping fragment ions and improves sequence coverage.

. Ubiquitin mass spectrum, CIDtims spectrum, mobility spectrum, and 2D-IMS-MS Mass spectrum, mobility spectrum, and 2D-IMS-MS plot of 2 M ubiquitin generated by CIDtims when tunnel-in pressure is 1.5 mbar, accumulation time is 100 ms, and 6 is A. 30 V and B. 150 V with ladder of resulting fragment ions. Fragments in purple are identified only from the mobility resolved spectra. A. B.

Figure S3. Cytochrome C mass spectrum, CIDtims spectrum, mobility spectrum, and 2D-IMS-MS
Mass spectrum, mobility spectrum, and 2D-IMS-MS plot of 2 M cytochrome C generated by CIDtims when tunnel-in pressure is 1.5 mbar, accumulation time is 100 ms, and 6 is A. 30 V and B. 150 V with ladder of resulting fragment ions. Fragments in purple are identified only from the mobility resolved spectra. A. Figure S4. β-lactoglobulin mass spectrum, mobility spectrum, and 2D-IMS-MS Mass spectrum, mobility spectrum, and 2D-IMS-MS plot of 2 M β-lactoglobulin generated by CIDtims when tunnel-in pressure is 1.5 mbar, accumulation time is 100 ms, and 6 is A. 30 V and B. 150 V with ladder of resulting fragment ions. Fragments in purple are identified only from the mobility resolved spectra.

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B. Figure S5. β-lactoglobulin Survival Plots A. Survival plots of indicated β-lactoglobulin charge states at indicated tunnel-in pressures. B. Ion abundance of select product ions at indicated 6 voltage and tunnel-in pressures.

Figure S6. Cytochrome C Survival Plots
A. Survival plots of indicated cytochrome C charge states at indicated tunnel-in pressures. B. Ion abundance of select product ions at indicated 6 voltage and tunnel-in pressures.    Figure S7. Influence of tunnel-pressure on sequence coverage generation Plotted sequence coverage of ubiquitin, cytochrome C, and β-lactoglobulin generated by CIDtims at tunnel-in pressures of 2.0 mbar, 1.75 mbar, and 1.5 mbar.

Figure S8. Spectra of carbonic anhydrase at decreasing accumulation times
Mass spectra of 2.0 uM carbonic anhydrase as accumulation time is incrementally decreased. tunnel-in pressure was set to 1.5 mbar and Δ6 was set to 30 V.

Figure S9. Carbonic anhydrase CIDtims spectrum, mobility spectrum, and 2D-IMS-MS
Mass spectrum, mobility spectrum, and 2D-IMS-MS plot of 2 M carbonic anhydrase generated by CIDtims when tunnel-in pressure is 1.5 mbar, accumulation time is 20 ms, and 6 is 150 V with ladder of resulting fragment ions. Fragments in purple are identified only from the mobility resolved spectra.

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244 R P A Q P L K N R Q V R G F P K Figure S10. BSA CIDtims spectrum, mobility spectrum, and 2D-IMS-MS Mass spectrum, mobility spectrum, and 2D-IMS-MS plot of 2 M BSA generated by CIDtims when tunnelin pressure is 1.5 mbar, accumulation time is 20 ms, and 6 is 150 V with ladder of resulting fragment ions. Fragments in purple are identified only from the mobility resolved spectra.

Figure S11. Carbonic anhydrase mass spectra at various concentrations
Charge state distribution of intact carbonic anhydrase as concentration is lowered and accumulation time is held constant at 20 ms. Relative abundance of each highlighted charge state is displayed on each spectrum.

Figure S12. Carbonic anhydrase CIDtims fragmentation ladder
Resulting fragmentation ladder of CIDtims of carbonic anhydrase when accumulation time is 20 ms and concentration is 0.5 M. Fragments in purple are identified only from the mobility resolved spectra. Figure S13. Mass spectra of intact cytochrome C at indicated accumulation times Intact CytC distribution when accumulation is 100 ms (top) and 20 ms (bottom).

Figure S14. Mass spectra of intact β-lactoglobulin at indicated accumulation times
Intact BLG distribution when accumulation is 100 ms (top) and 20 ms (bottom).

Figure S15. Cytochrome C CIDtims spectrum, mobility spectrum, and 2D-IMS-MS with reduced accumulation
Cytochrome C mass spectra, mobility resolved spectra, and 2D-IMS-MS when accumulation is 20 ms and Δ6=150 V with ladder of resulting fragment ions. Fragments in purple are identified only from the mobility resolved spectra. Figure S16. β-lactoglobulin CIDtims spectrum, mobility spectrum, and 2D-IMS-MS with reduced accumulation β-lactoglobulin mass spectra, mobility resolved spectra, and 2D-IMS-MS when accumulation is 20 ms and Δ6=150 V with ladder of resulting fragment ions. Fragments in purple are identified only from the mobility resolved spectra.

Figure S17. Mass spectra of intact Ubiquitin at varied accumulation times
Intact ubiquitin at 100 ms (Top) and 20 ms accumulation (Bottom).

Figure S18. Ubiquitin CIDtims mass spectrum, mobility spectrum, and 2D-IMS-MS with reduced accumulation
Ubiquitin mass spectra, mobility resolved spectra, and 2D-IMS-MS when accumulation is 20 ms and Δ6=150 V with ladder of resulting fragment ions. Fragments in purple are identified only from the mobility resolved spectra. Figure S19. BSA CIDtims spectrum, mobility spectrum, and 2D-IMS-MS at 0.5 M BSA mass spectra, mobility resolved spectra, and 2D-IMS-MS when concentration is 0.5 M, tunnel-in 1.5 mbar, 20 ms accumulation, and when Δ6=150 V with ladder of resulting fragment ions. Fragments in purple are identified only from the mobility resolved spectra. Figure S20. BSA CIDtims spectrum, mobility spectrum, and 2D-IMS-MS with ICC BSA mass spectra, mobility resolved spectra, and 2D-IMS-MS when concentration is 2 M, tunnel-in 1.5 mbar, ICC is 3.5 mio, and when Δ6=150 V with ladder of resulting fragment ions. Fragments in purple are identified only from the mobility resolved spectra. Figure S21. Ubiquitin isCID mass spectrum and ladder Spectrum and resulting fragment ladder of ubiquitin after activation by isCID.

Figure S22. Cytochrome C isCID mass spectrum and ladder
Spectrum and resulting fragment ladder of cytochrome C after activation by isCID. Figure S23. β-lactoglobulin isCID mass spectrum and ladder Spectrum and resulting fragment ladder of β-lactoglobulin after activation by isCID.

Figure S24. Carbonic Anhydrase isCID mass spectrum and ladder
Spectrum and resulting fragment ladder of carbonic anhydrase after activation by isCID. Figure S25. BSA isCID mass spectrum and ladder Spectrum and resulting fragment ladder of BSA after activation by isCID.

Figure S26. Ubiquitin CID spectrum and ladder
A. CID tandem mass spectrum of 12+ charge state of Ubiquitin. B. Ladder indicates sequence information provided by pooling the product ions generated by dissociating the 10+, 11+, and 12+ charge states of ubiquitin.

Figure S27. Venn diagram of CID-generated product ions for indicated charge states.
Venn diagrams indicate the number of unique and shared sequence informative product ions generated from dissociation of each indicated charge state for the proteins A. ubiquitin B. cytochrome C C. βlactoglobulin D. carbonic anhydrase and E. BSA Figure S28. Tandem mass spectrum of cytochrome C following CID A. CID tandem mass spectrum of 15+ charge state of Cytochrome C. B. Ladder indicates sequence information provided by pooling the product ions generated by dissociating the 8+, 14+, and 15+ charge states of cytochrome C.

Figure S29. Tandem mass spectrum of β-lactoglobulin following CID
A. CID tandem mass spectrum of 15+ charge state of β-lactoglobulin. B. Ladder indicates sequence information provided by pooling the product ions generated by dissociating the 13+, 14+, and 15+ charge states of β-lactoglobulin.

Figure S30. Tandem mass spectrum of Carbonic anhydrase following CID
A. CID tandem mass spectrum of 33+ charge state of carbonic anhydrase. B. Ladder indicates sequence information provided by pooling the product ions generated by dissociating the 31+, 32+, and 33+ charge states of carbonic anhydrase. Figure S31. Tandem mass spectrum of BSA following CID A. CID tandem mass spectrum of 50+ charge state of BSA. B. Ladder indicates sequence information provided by pooling the product ions generated by dissociating the 48+, 49+, and 50+ charge states of BSA.