Solid phase peptide synthesis. edit. The established method for the production of synthetic peptides is known as solid phase peptide synthesis (SPPS).Read more
Note: Content is periodically reviewed for consistency with published literature.ArticleApril 14, 2025
After 75 Years, an Alternative to Edman Degradation: A Mechanistic and Efficiency Study of a Base-Induced Method for N-Terminal Peptide Sequencing
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- Harnimarta Deol
Harnimarta Deol
Department of Chemistry, The University of Texas at Austin, Austin, Texas 78712, United States
- Ava Raeisbahrami
Ava Raeisbahrami
Department of Chemistry, The University of Texas at Austin, Austin, Texas 78712, United States
- Phuoc H.T. Ngo
Phuoc H.T. Ngo
Department of Chemistry, The University of Texas at Austin, Austin, Texas 78712, United States
- Jagannath Swaminathan
Jagannath Swaminathan
Erisyon Inc, Austin, Texas 78752, United States
- Ophelia Papoulas
Ophelia Papoulas
Department of Molecular Biosciences, The University of Texas at Austin, Austin, Texas 78712, United States
- Edward M. Marcotte*
Edward M. Marcotte
Department of Molecular Biosciences, The University of Texas at Austin, Austin, Texas 78712, United States
*Email: [email protected]
- Eric V. Anslyn*
Eric V. Anslyn
Department of Chemistry, The University of Texas at Austin, Austin, Texas 78712, United States
*Email: [email protected]
Journal of the American Chemical Society
Cite this: J. Am. Chem. Soc. 2025, 147, 16
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Published April 14, 2025
research-article
Copyright © 2025 American Chemical Society
Abstract
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The sequencing of peptides via N-terminal amino acid removal is a classic reaction termed Edman degradation. This method involves repeated treatment of the N-terminal amino group of a peptide with phenyl isothiocyanate (PITC), followed by treatment with trifluoroacetic acid. Spurred by the need for an alternative non-acid-based chemistry for next-generation protein sequencing technologies, we developed a base-induced N-terminal degradation method. Several N-terminal derivatization reagents carrying supernucleophiles were tested. After rounds of iterative designs, compound DR3, with a N-hydroxysuccinimide as a leaving group and hydrazinecarboxamide as the supernucleophile, demonstrated the highest yield for the peptide derivatization step and the most efficient elimination of the N-terminal amino acid in just 1% of a hydroxide salt. The method successfully removed all 20 amino acids at the N-terminus in high yield. The technique demonstrates compatibility with oligonucleic acids, which differs from Edman degradation due to their inherent sensitivity to acidic environments. To demonstrate the practical application of our approach, we sequenced amino acids sequentially from a peptide, effectively determining the sequence of an unknown peptide. Notably, our methodology was successfully applied to mixtures of peptides derived from protein samples, where a significant fraction of the peptides derivatized with DR3 underwent elimination of their N-terminal amino acid upon addition of base. Overall, although our method does not outperform Edman degradation in efficiency, it serves as a valuable alternative in cases where base-induced cleavage is advantageous, particularly for preserving acid-sensitive functionalities.
ACS Publications
Copyright © 2025 American Chemical Society
Supporting Information
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The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/jacs.5c03385.
Detailed information on the materials and methods, experimental procedures, LC-MS, HRMS, MS-MS, MALDI, and NMR spectra (PDF)
- ja5c03385_si_001.pdf (9.03 MB)
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Cited By
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This article is cited by 4 publications.
- Tomasz A. Leski, Sean M. Brown, Zachary T. Johnson, Scott N. Dean, Ellen R. Goldman, David A. Stenger. Optimization of the Quantum-Si Platinum Single-Molecule Protein Sequencing Platform Toward Improved Complex-Matrix Protein Identification. ACS Omega 2026, 11 (23) , 34047-34057. https://doi.org/10.1021/acsomega.6c01140
- Xiao-Juan Wang, Shuai-Hua Shi, Jie Zhang, Wei-Kang Zhai, Xing-Hua Wang, Yuan-Yuan Gao, Wen-Jun Han, Lan-Tao Liu, Xinxiang Lei, Gao-Wei Li. A 19F-Labeled Isothiocyanate Derivatizing Agent for the Chiral Discrimination of Cyclic Secondary Amines. Analytical Chemistry 2025, 97 (41) , 22768-22776. https://doi.org/10.1021/acs.analchem.5c04345
- Harnimarta Deol, Matthew H. Dixon, Hazel A. Fargher, Jacob Mayhugh, Bipin Pandey, Hyun Meen Park, Ava Raeisbahrami, Eric V. Anslyn. The Use of 5-Exo- and 6-Exo-Trig Cyclizations in the Manipulation and Degradation of Biotic and Abiotic Polymers. The Journal of Organic Chemistry 2025, 90 (41) , 14349-14362. https://doi.org/10.1021/acs.joc.5c01633
- Roanne Deanne Aves, Janwa El-Maiss, Divya Balakrishnan, Naveen Kumar, Mafalda Abrantes, Jérôme Borme, Vihar Georgiev, Pedro Alpuim, César Pascual García. A Graphene Field-Effect Transistor-Based Biosensor Platform for the Electrochemical Profiling of Amino Acids. Biosensors 2026, 16 (2) , 83. https://doi.org/10.3390/bios16020083
Journal of the American Chemical Society
Cite this: J. Am. Chem. Soc. 2025, 147, 16
Click to copy citationCitation copied!
Published April 14, 2025
Copyright © 2025 American Chemical Society
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