Covalent Organic Framework with Acid Precipitation Enhances Microprotein Enrichment and Discovery of Ferroptosis-Associated Microproteins

ABSTRACT: Microproteins, defined as polypeptides fewer than 100 amino acids, are encoded by small open reading frames (sORFs) and exhibit unique structural and functional characteristics. Despite their short length and recent discoveries, microproteins have already been found to play critical roles in diverse cellular functions. Nevertheless, the identification and verification of novel microproteins by mass spectrometry face significant challenges due to their short length and low abundance. Covalent organic frameworks (COFs) with adjustable pore size and hydrophobicity have shown excellent performance in the enrichment of short bioactive peptides. Here, we created COF-coated magnetic nanoparticles with a pore size of 2.7 nm (termed Fe3O4@COF2.7) and verified its utility for the enrichment of microproteins. The material shows outstanding performance in enriching microproteins, identifying about 6X more microproteins than uncoated particles where an average of 251 microproteins were unveiled with a 45-minute MS analysis time. Furthermore, the material unveils the most significant number of microproteins with the best reproducibility compared to other methods. In addition, we observed Fe3O4@COF2.7 and acid precipitation unveil a unique set of microproteins which were combined to achieved a comprehensive identification of 713 unique microproteins with a total MS run of 4.5 hours. This integrated approach significantly enhanced the efficiency and coverage of protein detection, demonstrating the synergistic benefits of combining different techniques for robust microprotein identification for advancing microprotein research and opening up new avenues in proteomics.

ORGANISM(S): Homo Sapiens

SUBMITTER: Chris Soon Heng Tan  

PROVIDER: PXD074935 | iProX | Thu Feb 26 00:00:00 GMT 2026

REPOSITORIES: iProX