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Simon Mysling , Thomas J. D. Jørgensen University of Southern Denmark Protein Research Group

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

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Simon Mysling , Thomas J. D. Jørgensen University of Southern Denmark Protein Research Group

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

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

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

  4. On-column accumulation of TCEP Threeconsecutiveinjectionswith 400 mM TCEP Injection 1 Injection 2 Injection 3

  5. On-column accumulation of TCEP Threeconsecutiveinjectionswith 400 mM TCEP Injection 1 Injection 2 Injection 3

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

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

  8. Reduction of Insulin using TCEP Quenchconditions - 0°C and pH 2.5 Insulin MH5+ 400 mM TCEP 2 min. incubation Insulin MH6+ Insulin MH4+ Insulin MH5+ 400 mM TCEP 10 min. incubation Insulin MH6+ Insulin MH4+ Insulin MH5+ Insulin MH6+ Chain B MH4+ Insulin MH4+ Chain B MH5+

  9. Reduction of Insulin using TCEP Quenchconditions - 0°C and pH 2.5 Insulin MH5+ 400 mM TCEP 2 min. incubation Insulin MH6+ Insulin MH4+ Insulin MH5+ 400 mM TCEP 10 min. incubation Insulin MH6+ Chain B MH4+ Insulin MH4+ Insulin MH5+ Insulin MH6+ Chain B MH4+ Insulin MH4+ Chain B MH5+ 10 min. incubationlessthan 5% reduction 50 min. Incubation ~15-20% reduction

  10. Reduction of Insulin using TCEP Quenchconditions - 0°C and pH 2.5 Insulin MH5+ 400 mM TCEP 2 min. incubation Insulin MH6+ Insulin MH4+ Insulin MH5+ 400 mM TCEP 10 min. incubation Insulin MH6+ Chain B MH4+ Insulin MH4+ Insulin MH5+ 400 mM TCEP 50 min. incubation Insulin MH6+ Chain B MH4+ Insulin MH4+ Chain B MH5+ 10 min. incubationlessthan 5% reduction 50 min. Incubation less than 20% reduction

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

  12. Electrochemicalreductioncell Reference electrode Able to reduce insulin efficiently, usingdirect infusion 12 uL internalvolume Solvent flow Runningconditions 50 bar (725 PSI) pressure limit 1% FA in solvent Working Electrode

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

  14. Electrochemicalreduction of insulin Insulin MH5+ Cell off 100 μL/min. Insulin MH6+ Chain B MH5+ Cell on 100 μL/min. Residence time: 7.2 s. Chain B MH4+ Relative intensity [AU] Insulin MH6+ Insulin MH5+ Chain B MH5+ Chain B MH4+ Chain A MH3+ m/z [Th]

  15. Electrochemicalreduction of insulin Insulin MH5+ Cell off 100 μL/min. Insulin MH6+ Chain B MH5+ Cell on 100 μL/min. Residence time: 7.2 s. Chain B MH4+ Relative intensity [AU] Insulin MH6+ Insulin MH5+ Chain B MH5+ Chain B MH4+ Chain A MH3+ m/z [Th]

  16. Electrochemicalreduction of insulin Insulin MH5+ Cell off 100 μL/min. Insulin MH6+ Reductionefficiency is dependent on residence time (Flow rate) Tweak reductionusing the desalting flow rate Chain B MH5+ Cell on 100 μL/min. Residence time: 7.2 s. Chain B MH4+ Relative intensity [AU] Insulin MH6+ Insulin MH5+ Chain B MH5+ Cell on 50 μL/min. Residence time: 14.4 s. Chain B MH4+ Chain A MH3+ m/z [Th]

  17. Impact on deuterium back-exchange Labeled insulin B-chain Deuterons Theoretical maximum Observed

  18. Impact on deuterium back-exchange Labeled insulin B-chain Deuterons Theoretical maximum Observed

  19. Impact on deuterium back-exchange Labeled insulin B-chain Deuterons Theoretical maximum Observed Main contributors to back-exchange Increasedesalting time Non-cooledcell in flowpath

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

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

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

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

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

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

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

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

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