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

Microproteins, 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.