1. Simon Mysling, Thomas J. D. Jørgensen University of Southern Denmark Protein Research Group June 12 th 2013 H/D exchange: New Developments in Technology The 61 st annual ASMS conference Electrochemical Reduction of TCEP-resistant Disulphide Bonds For use in Hydrogen/Deuterium exchange monitored by Mass Spectrometry

2. Disulphide bond reduction in HDX experiments Chemical reductant Tris(2-carboxyethyl)phosphine (TCEP) Important step when analyzing disulphide bond-containing proteins R–S–S–R to R–SH HS–R Improve proteolytic digestion Improve sequence coverage (previously disulphide-linked peptides observable) Reduction should be rapid, and run a quench conditions - pH 2.5, 0°C Spike sample with reductant and incubate prior to injection

3. Reduction at quench conditions using TCEP Cline, D. J.; Thorpe, C. Biochemistry 2004, 43, 15195 pH 2.5 TCEP efficiency is severely reduced at pH 2.5 High concentrations Accumulation in RP columns Extensive washing

4. Three consecutive injections with 400 mM TCEP On-column accumulation of TCEP Injection 2 Injection 3 Injection 1

5. Injection 2 Injection 3 Injection 1 On-column accumulation of TCEP Three consecutive injections with 400 mM TCEP

6. Reduction at quench conditions using TCEP TCEP efficiency is severely reduced at pH 2.5 High concentrations Accumulation in RP columns Extensive washing Some disulphide bonds are less vulnerable to TCEP reduction Difficult to analyze using HDX-MS Cline, D. J.; Thorpe, C. Biochemistry 2004, 43, 15195 pH 2.5

7. Insulin H S H S H S H S H S H S Reduction Chain B Chain A +

8. Reduction of Insulin using TCEP Quench conditions - 0°C and pH 2.5 Insulin MH 5 + Insulin MH 4 + Insulin MH 6 + Chain B MH 4 + Chain B MH 5 + 400 mM TCEP 10 min. incubation 400 mM TCEP 2 min. incubation Insulin MH 5 + Insulin MH 4 + Insulin MH 6 + Insulin MH 5 + Insulin MH 4 + Insulin MH 6 +

9. Reduction of Insulin using TCEP Quench conditions - 0°C and pH 2.5 Insulin MH 5 + Insulin MH 4 + Insulin MH 6 + Chain B MH 4 + Chain B MH 5 + 400 mM TCEP 10 min. incubation 400 mM TCEP 2 min. incubation Insulin MH 5 + Insulin MH 4 + Insulin MH 6 + Insulin MH 5 + Insulin MH 4 + Insulin MH 6 + 10 min. incubation less than 5% reduction 50 min. Incubation ~15-20% reduction Chain B MH 4 +

10. Reduction of Insulin using TCEP Quench conditions - 0°C and pH 2.5 Insulin MH 5 + Insulin MH 4 + Insulin MH 6 + Chain B MH 4 + Chain B MH 5 + 400 mM TCEP 50 min. incubation 400 mM TCEP 10 min. incubation 400 mM TCEP 2 min. incubation Insulin MH 5 + Insulin MH 4 + Insulin MH 6 + Insulin MH 5 + Insulin MH 4 + Insulin MH 6 + 10 min. incubation less than 5% reduction 50 min. Incubation less than 20% reduction Chain B MH 4 +

11. Reduction at quench conditions using TCEP Alternative reduction methods could be valuable in many situations TCEP efficiency is severely reduced at pH 2.5 High concentrations Accumulation in RP columns Extensive washing Some disulphide bonds are less vulnerable to TCEP reduction Difficult to analyze using HDX-MS Cline, D. J.; Thorpe, C. Biochemistry 2004, 43, 15195 pH 2.5

12. Electrochemical reduction cell Working Electrode Solvent flow Reference electrode 1% FA in solvent Able to reduce insulin efficiently, using direct infusion 12 uL internal volume Running conditions 50 bar (725 PSI) pressure limit

13. Is electrochemical reduction, at quench conditions: - Stable and reproducible? - Going to increase back- exchange markedly? Reduction cell Pepsin column Digestion chamber – 10°C Trap and analytical column – 0.2°C From loop To desalting trap Injection - Still efficient?

14. Cell off 100 μL/min. Insulin MH 5 + Insulin MH 6 + Insulin MH 5 + Insulin MH 6 + Chain B MH 5 + Chain B MH 4 + Chain B MH 5 + Chain B MH 4 + Chain A MH 3 + Relative intensity [AU] m/z [Th] Cell on 100 μL/min. Residence time: 7.2 s. Electrochemical reduction of insulin

15. Cell off 100 μL/min. Insulin MH 5 + Insulin MH 6 + Insulin MH 5 + Insulin MH 6 + Chain B MH 5 + Chain B MH 4 + Chain B MH 5 + Chain B MH 4 + Chain A MH 3 + Relative intensity [AU] m/z [Th] Cell on 100 μL/min. Residence time: 7.2 s. Electrochemical reduction of insulin

16. Cell off 100 μL/min. Cell on 50 μL/min. Residence time: 14.4 s. Insulin MH 5 + Insulin MH 6 + Insulin MH 5 + Insulin MH 6 + Chain B MH 5 + Chain B MH 4 + Chain B MH 5 + Chain B MH 4 + Chain A MH 3 + Relative intensity [AU] m/z [Th] Cell on 100 μL/min. Residence time: 7.2 s. Tweak reduction using the desalting flow rate Electrochemical reduction of insulin Reduction efficiency is dependent on residence time (Flow rate)

17. Deuterons16.114.312.9 Back-exchange28.0%36.0%42.2% Cell active XXO Cell present XXO Buffer 0.23% FA1% FA Desalting 0.5 min. 300 ul/min 3 min. 50 ul/min 3 min. 50 ul/min Deuterons Labeled insulin B-chain Impact on deuterium back-exchange Observed Theoretical maximum

18. Deuterons16.114.312.9 Back-exchange28.0%36.0%42.2% Cell active XXO Cell present XXO Buffer 0.23% FA1% FA Desalting 0.5 min. 300 ul/min 3 min. 50 ul/min 3 min. 50 ul/min Labeled insulin B-chain Impact on deuterium back-exchange Observed Theoretical maximum Deuterons

19. 16.114.312.9 Back-exchange28.0%36.0%42.2% Cell active XXO Cell present XXO Buffer 0.23% FA1% FA Desalting 0.5 min. 300 ul/min 3 min. 50 ul/min 3 min. 50 ul/min Impact on deuterium back-exchange Observed Theoretical maximum Non-cooled cell in flowpath Increase desalting time Main contributors to back-exchange Labeled insulin B-chain Deuterons

20. - Only slightly improved deuterium back- exchange - Considerable decrease in reduction efficiency Electrochemical reduction was not found to alter deuteration patterns Placing the reduction cell within a cooled environment: -Alleviated by diluting samples 10x when quenching exchange -Other buffers could have less dramatic effects PBS and ammonium acetate had a negative impact on the reduction Other observations

21. Insulin hexamers T6 hexamers Stable assemblies R6 hexamers Very stable assemblies Insulin hexamers T6 hexamers Stable assemblies R6 hexamers Very stable assemblies

22. Full deut.T6 hexamer Full exchange Undeuterated 10s exchange-in 100s exchange-in 1000s exchange-in

23. Full deut.T6 hexamer Full exchange Undeuterated 10s exchange-in 100s exchange-in 1000s exchange-in

24. Full deut.T6 hexamer Full exchange Undeuterated 10s exchange-in 100s exchange-in 1000s exchange-in

25. Full deut.T6 hexamer Full exchange Undeuterated 10s exchange-in 100s exchange-in 1000s exchange-in

26. Full deut.T6 hexamer 90° EX-1 exchange kinetics reflecting the stability of insulin hexamers

27. Acknowledgements Finsenlaboratory, DK Michael Ploug Antec, NL Agnieszka Kraj Biolab, DK Kim Stjerne Britta Gribsholt Sabine Amon Protein Research Group University of Southern Denmark Novozymes, DK Rune Salbo Funding The Lundbeck Foundation Thomas J. D. Jørgensen Morten Beck Trelle