REVIEW: Methodological Toolbox for Identifying and Studying Micropeptides: From Genome to Function

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Aleksandr I. Lavrov^1,2, Nikita M. Shepelev^1,3, Olga A. Dontsova^1,3, and Maria P. Rubtsova^1,3,a*

¹Faculty of Chemistry, Lomonosov Moscow State University, 119991 Moscow, Russia

²Belozersky Institute of Physico-Chemical Biology, Lomonosov Moscow State University, 119992 Moscow, Russia

³Shemyakin–Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997 Moscow, Russia

^* To whom correspondence should be addressed.

Received: July 22, 2025; Revised: October 2, 2025; Accepted: October 6, 2025

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Micropeptides encoded by small open reading frames (sORFs) represent a novel, actively studied class of functional molecules regulating key cellular processes. Studying micropeptides is complicated by methodological challenges, in particular, their small size, low cellular abundance, and difficulty in generating specific antibodies. The review systematizes modern approaches to the identification and functional characterization of micropeptides. The main strategies for their discovery include the use of bioinformatic algorithms, global translation analysis via ribosome profiling, direct detection using mass spectrometry-based proteomics, and phenotypic screenings. The methods for confirming the functions of micropeptides and elucidating molecular mechanisms of their action genetic knockouts, affinity tagging for visualization, and investigation of protein-protein interactions. The review discusses key challenges and future prospects in the field, emphasizing the importance of an integrated multi-omics approach for the comprehensive micropeptidome mapping.

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KEY WORDS: micropeptides, small open reading frames, long non-coding RNAs, mass spectrometry, Ribo-Seq, affinity labeling, co-immunoprecipitation

DOI: 10.1134/S0006297925602242

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