Beyond the known cuts: trypsin specificity in native proteins

Marcelo Gaspar; Bohdana Sokolova; Amir Ata Saei; José C. Marques; Roman A. Zubarev

doi:10.1039/D5CC02378E

Beyond the known cuts: trypsin specificity in native proteins†

Marcelo Gaspar,

^abe Bohdana Sokolova,^c Amir Ata Saei,

^cd José C. Marques

^be and Roman A. Zubarev

*^bcfg

Author affiliations

* Corresponding authors

^a Faculty of Exact Sciences and Engineering, University of Madeira, Campus Universitário da Penteada, 9020-105 Funchal, Portugal

^b ISOPlexis Centre for Sustainable Agriculture and Food Technology, University of Madeira, 9020-105 Funchal, Portugal

^c Division of Chemistry I, Department of Medical Biochemistry and Biophysics, Karolinska Institutet, SE-17 177 Stockholm, Sweden
E-mail: roman.zubarev@ki.se

^d Department of Microbiology, Tumor and Cell Biology, Karolinska Institutet, SE-17 177 Stockholm, Sweden

^e i3N – Institute for Nanostructures, Nanomodelling and Nanofabrication, Department of Physics, University of Aveiro, 3810-193 Aveiro, Portugal

^f Department of Pharmacological & Technological Chemistry, I.M. Sechenov First Moscow State Medical University, Moscow, Russia

^g Department of Pharmaceutical and Toxicological Chemistry, Medical Institute, Peoples’ Friendship University of Russia named after Patrice Lumumba (RUDN University), 6 Miklukho-Maklaya St, Moscow, Russia

Abstract

Above-filter digestion proteomics (AFDIP) was applied to quantify trypsin cleavage preferences in native HeLa cell lysates. Lysine sites were cleaved faster than arginine ones, with cleavage rates modulated by the peptide's size and isoelectric point. These trends, absent in denatured proteomes, highlight trypsin's context-dependent behavior and inform protein engineering for optimal digestibility.

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Article information

DOI: https://doi.org/10.1039/D5CC02378E
Article type: Communication
Submitted: 29 Apr 2025
Accepted: 16 Jul 2025
First published: 23 Jul 2025
This article is Open Access

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Chem. Commun., 2025, Advance Article

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Beyond the known cuts: trypsin specificity in native proteins

M. Gaspar, B. Sokolova, A. A. Saei, J. C. Marques and R. A. Zubarev, Chem. Commun., 2025, Advance Article , DOI: 10.1039/D5CC02378E

This article is licensed under a Creative Commons Attribution 3.0 Unported Licence. You can use material from this article in other publications without requesting further permissions from the RSC, provided that the correct acknowledgement is given.

Chemical Communications

Beyond the known cuts: trypsin specificity in native proteins†

Abstract

Supplementary files

Article information

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Beyond the known cuts: trypsin specificity in native proteins

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