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.