iProXProteomicsChris Soon Heng TanHomo Sapienshttp://www.iprox.org/page/project.html?id=IPX0013133000christan@sustech.edu.cnSouthern University of Science and Technology, ShenzheniProXMicroproteins, encoded by small open reading frames (sORFs), are polypeptides with fewer than 100 amino acids with unique structural and functional characteristics. Protein mass spectrometry is currently the de facto approach to verify the existence of microproteins, but the short length and the low abundance of microproteins pose significant challenges to their detection. Covalent organic frameworks (COFs) with adjustable pore sizes and hydrophobicities have shown excellent performance in the enrichment of short bioactive peptides. Here, we created COF-coated magnetic nanoparticles with an average pore size of 2.72 nm and verified their utility for the enrichment of microproteins from cell lysate. The material identified about 5× more microproteins than uncoated particles, where an average of 109 microproteins per MS run were unveiled with 45 min MS analysis time, and a total of 142 unique microproteins were identified across three replicates with a stringent FDR of 0.01%. The material unveils the greatest number of microproteins and the best reproducibility compared to other methods. We observed that COFs and acid precipitation unveil a unique set of microproteins, which were combined to identify 195 unique microproteins using a total MS instrument time of 4.5 h. Application of COFs with quantitative proteomics identified seven microproteins differentially upregulated during ferroptosis, including three novel microproteins that are robustly confirmed by high-quality MS/MS spectra. These results indicate that the COF offers a robust tool for the identification of microproteins.Covalent Organic Framework with Acid Precipitation Enhances Microprotein Enrichment and Discovery of Ferroptosis-Associated Microproteins.Li Fengming F, Sui Jingchen J, Wang Xiaoqian X, Liao Bin B, Zhang Ying Y, Zhang Shuang S, Hu Hailiang H, Low Teck Yew TY, Du Changzheng C, Tan Chris Soon Heng CSHfalseCovalent Organic Framework with Acid Precipitation Enhances Microprotein Enrichment and Discovery of Ferroptosis-Associated MicroproteinsMicroproteins, 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.Thu Feb 26 00:00:00 GMT 2026PXD074935960641775710