Develop Novel Quantitation Methods for Peptides without Using Standards by Coulometric Mass Spectrometry

Scheme 1. Our LC/EC/MS apparatus and approach for quantitation of tyrosine-containing peptides.

Quantitation methods for peptides using mass spectrometry have advanced rapidly. These methods rely on using standard and/or isotope-labeled peptides, which might be difficult or expensive to synthesize.

To tackle this challenge, we present a new approach for absolute quantitation without the use of standards or calibration curve based on coulometry combined with mass spectrometry (MS)^1-3.

In this approach, which we call coulometric mass spectrometry (CMS), the mass spectrum of a target peptide containing one or more tyrosine residues is recorded before and after undergoing electrochemical oxidation. We record the total integrated oxidation current from the electrochemical measurement, which according to Faraday’s Law of coulometry, provides the number of moles of oxidized peptide. The ion intensity ratio of the target peptide before and after oxidation provides an excellent estimate of the fraction of the peptide that has been oxidized, from which the total amount of peptide is calculated.

The striking strength of CMS is that it needs no standard peptide, but CMS does require the peptide to contain a known number of oxidizable groups. To illustrate the power of this method, we analyzed various tyrosine-containing peptides such as GGYR, DRVY, oxytocin, [Arg8]-vasotocin and angiotensinogen 1-14 with a quantification error ranging from -7.5% to +2.4 %.

This approach is also applicable to quantifying phosphopeptides and could be useful in proteomics research.

References

1. P. Zhao, R. N. Zare*, H. Chen*, J. Am. Soc. Mass Spectrom. 2019, 30, 2398-2407.
2. P. Zhao, Y. Guo*, H. D. Dewald*, H. Chen*, Int. J. Mass Spectrom. 2019, 443, 41-45.
3. C. Xu, Q. Zheng, P. Zhao, J. Paterson, H. Chen*, J. Am. Soc. Mass Spectrom. 2019, 30, 685–693.
4. Takats, Z.; Wiseman, J. M.; Gologan, B.; Cooks, R. G*. Science, 2004, 306, 471.
5. Miao, Z.; Chen, H*. J. Am. Soc. Mass Spectrom. 2009, 20, 10.
6. T. A. Brown, H. Chen*, R. N. Zare*, Angew. Chem. Int. Ed., 2015, 54, 11183 –11185
7. T. A. Brown, H. Chen*, R. N. Zare, J. Am. Chem. Soc. 2015, 137, 7274.
8. T. A. Brown, N. Hosseinei-Nassab, H. Chen*, R. N. Zare*, Chemical Science, 2016, 7, 329.
9. Y. Cai, J. Wang, Y. Zhang, Z. Li, D. Hu, N. Zheng*, H. Chen*,  J. Am. Chem. Soc., 2017, 139, 12259.
10. Li, J.; Dewald, H. D.; Chen, H*. Anal. Chem., 2009, 81, 9716.
11. Y. Zhang, W. Cui, H. Zhang, H. D. Dewald, H. Chen*, Anal. Chem. 2012, 84, 3838.
12. Y. Zhang, H. D. Dewald, H. Chen*, J. Proteome Res. 2011, 10, 1293.
13. Q. Zheng, H. Zhang*, H. Chen*, Int. J. Mass Spectrom. 2013, 353, 84.
14. Q. Zheng, H. Zhang, S. Wu, H. Chen*, J. Am. Soc. Mass Spectrom. 2016, 27, 864-875.
15. Q. Zheng, H. Zhang*, L. Tong, S. Wu, H. Chen*, Anal. Chem. 2014, 86, 8983-8991
16. Q. Zheng, Y. Liu*, Q. Chen*, M. Hu, R. Helmy, E. C. Sherer, C. J. Welch, H. Chen*, J. Am. Chem. Soc., 2015, 137, 14035-14038
17. R. Cai, M. Lu, E. Aguilera, Y. Xi, N. Akhmedov, J. Petersen, H. Chen*, X. Shi*, Angew. Chem. Int. Ed., 2015, 54, 8772 –8776
18. H. Peng, R. Cai, C. Xu, H. Chen*, X. Shi*, Chemical Science, 2016, 7, 6190–6196.
19. J. Wang, S. Zhang, C. Xu, L. Wojtas, N. G. Akhmedov, H. Chen, X. Shi,* Angew. Chem. Int. Ed., 2018, 57, 6915 –6920.
20. M. Lu, Y. Su, P. Zhao, X. Ye, Y. Cai,  X. Shi,* E. Masson,* F. Li, J. L. Campbell, H. Chen*, Chem. – Eur. J., 2018, 24, 2144-2150
21. X. Ye,  P. Zhao,  S. Zhang,  Y. Zhang, Q. Wang, C. Shan, L. Wojtas, H. Guo*, H. Chen*, X. Shi,* Angew. Chem. Int. Ed., 2019, 58, 17226 –17230.
22. J. Wang, C. Wei, X. Li, P. Zhao, C. Shan, L. Wojtas, H. Chen, X. Shi*, Chem. Eur. J. 2020, 26, Accepted.
23. T. Yuan, P. Zhao, S. Teng, Y. Yi, J. Wang, C. Shan, L. Wojtas, J. Jean, H. Chen, X. Shi*, Chem, 2020, Accepted.
24. H. Chen, L. S. Eberlin, R. G. Cooks*, J. Am. Chem. Soc. 2007, 129, 5880.
25. H. Chen, L. S. Eberlin, M. Nefliu, R. Augusti, R. G. Cooks*, Angew. Chem. Int. Ed., 2008, 47, 3422.
26. P. Liu, P. Zhao, R. G. Cooks*, H. Chen*, Chemical Science, 2017, 8, 6499.
27. P. Zhao, T. White, R. G. Cooks,* Q. Chen,* Y. Liu, H. Chen*,  J. Am. Soc. Mass Spectrom. 2018, 29, 2317–2326.
28. Y. Cai, D. Adams, H. Chen*, J. Am. Soc. Mass Spectrom. 2014, 25, 286.
29. Y. Cai, Y. Liu, R. Hemly, H. Chen*, J. Am. Soc. Mass Spectrom. 2014, 25, 1820.
30. Y. Cai, Q. Zheng, Y. Liu, R. Helmy, J. A. Loo, H. Chen*, Eur. J. Mass Spectrom. 2015, 21, 341–351.