An Artificial Antibody‐Based Toolbox Accelerates Validation of Hidden Microproteins Encoded by the Dark Genome

Microproteins encoded by small open reading frames are a pivotal blind spot redefining the conventional protein-coding assumptions. However, the annotation of the "dark proteome" remains time- and labor-consuming due to the limited efficiency, sensitivity, and comprehensiveness of existing validation methods. To address these issues, we developed a comprehensive toolbox called CLAIMID to achieve accelerated and ultrasensitive validation at multiple biological scales. As the core of CLAIMID, molecularly imprinted polymers (MIPs), which are synthesized artificial antibodies toward putative microproteins, provide ultrasensitive and precise annotation in combination with surface-enhanced Raman scattering (SERS) detection. The excellent specificity, comparable to antibodies, of MIPs enables high anti-interference against biological matrix. The adaptability of MIPs engineering confers the rigorous validations by CLAIMID at multiple scales (single living cells, cell populations, and tissues) and diverse detection formats (SERS-based immunoassay and imaging, and mass spectrometric identification). Through CLAIMID, we rapidly confirmed the protein-level translation of four predicted microproteins-previously supported only by computational or ribosome profiling data-across various cell lines, and further identified three as potential tumor biomarkers, thereby demonstrating its university to putative microproteins. Together, we present an annotation toolbox with unparalleled efficiency, sensitivity, and scalability, moving forward for the advent of intriguing microprotein biology era.