1. New Dual Electrode µ-PrepCell™ for Efficient Reduction of Disulfide Bonds in Proteins/Peptides
ASMS 2018
San Diego, CA, USA

2. Outline Cell configuration and workwise Cell evaluation
- Influence of flow rate and %FA
- Long-term stability
- Cell to cell repro
Reduction of Fab fragment
Conclusion
The lecture will explore on-line EC/MS as a powerful technique to investigate various oxidation and reduction processes in life sciences.

3. Cell Configuration Inversed applied potential Ti inlet block
Pt counter electrode

4. Instrumental Setup FIA-EC-MS1 3 2 5 4
1) Sample infusion pump, 2) injection valve, 3) HPLC loading pump,
4) ROXY Potentiostat equipped with dual electrode µ-PrepCell, 5) MS

5. Pulse Settings
Pulse settings: E1 = 1.5V, t1 = 1s, E2 = 0V, t2 = 0.1s, ts = 40 ms

6. Structure of Intact and Reduced Insulin
SH HS SH
Chain A
Chain B
Intact insulin:
2 inter disulfide bonds between chain A and B
1 intra disulfide bond in chain A between Cys 6 - Cys 11
Reduced insulin:
Formation of chain A and B
and reduced disulfide bond between Cys 6 - Cys 11

7. MS Spectra of Intact and Reduced Insulin
Cell OFF: intact
Cell ON: reduced
[M + 6H+]6+
[M + 5H+]5+
Insulin
Chain B
[M + 4H+]4+
[M + 5H+]5+
Chain A
[M + 3H+]3+
Pulse settings: E1=1.5V, E2=0V, t1=1.0s, t2=0.10s

8. MS Voltammogram
Insulin Reduction vs. Voltage

9. Reduction Efficiency - Insulin
99%
Insulin 10 µg/mL, diluted in 1%FA 50% Acetonitrile/H2O, 50 µL/min,
for clarity only Chain B recorded

10. Flow Rate vs. Reduction Efficiency
optimal flow rate range
20 – 150 µL/min
Insulin 10 µg/mL, diluted in 1%FA 50% Acetonitrile/H2O

11. Acidity vs. Reduction Efficiency
98%
92%
67%
56%

12. Reproducibility (short-term)
5 consecutive injections of insulin 

13. Reproducibility (long-term)
100 injections of insulin 

14. Cell to Cell Reproducibility
Cell # Cell # Cell #3
3 Different cells, randomly assembled

15. Cell Contamination/Fouling
Pt counter electrode
Ti inlet block
No visible contamination or fouling of cell (inlet block & counter electrode),
after several days of operation

16. Reduction of Avastin® Fab Fragment
50 kDa
Cell off
Cell on
E= 1.5V Lc
75 kDa
25 kDa
100 kDa
Left: Spectrum of Avastin Fab digest consisting of intact 100 KDa fragment and different digest forms of 75, 50 and 25 kDa. Separated by on-line Capillary LC-EC-MS. Cell off
Right Spectrum after reduction, E = 1.5 V, clearly visible light chain (Lc) with mass of Da.

17. Spectrum of Light Chain (Lc)
Annotation of some charge states, A16 - A25

18. TP237, 10:30 am - 2:30 pm

19. Conclusions EC Reduction
The new dual electrode µ-PrepCell for disulfide bond reduction in proteins provides the following features:
High reduction efficiency
Robust and stable reduction w/o cell fouling
Good cell to cell reproducibility
Ideal for on-line reduction of proteins, mAbs, etc., in top-down, bottom-up and HDX-MS Proteomics 19

20. Acknowledgement Dr. Theo M. Luider et al., Erasmus MC, Rotterdam.
The Netherlands
For the data on Avastin Fab reduction 20