Recent research has shown that genomic regions once considered noncoding may contain short open reading frames encoding functional microproteins. Microproteins such as PIGBOS1 and NoBody are associated with cancer hallmarks. PIGBOS1 increases resistance to ER stress and apoptosis, while NoBody is crucial for nonsense-mediated mRNA decay, both regulating essential biological processes in cancer cells.

In this study, microproteins were enriched from various stages of colorectal cancer (CRC) cell lines, including SW1116 (stage 1), SW480 (stage 2), SW48 (stage 3), COLO320DM (stage 3), COLO205 (stage 4), and CCD841CoN (noncancer control cells), using 0.25% acetic acid precipitation. These samples were analyzed using a nanoElute® system coupled with a Bruker tims-TOF Pro mass spectrometer. Additionally, publicly available TMT-10 plex MS raw data from CRC tissues (stages 1 to 4) were downloaded from CPTAC and reanalyzed.

From the CRC cell lines, 207 microproteins were identified, with 167 showing differential expression. These microproteins exhibited significant motifs associated with functions like DNA binding and morphogenesis. SwissProt-identified microproteins were linked to cellular biosynthesis, RNA splicing, and mitochondrial activities, participating in cancer pathways. Using BLASTp, InterPro domain, and BioPlex interactome analyses, OpenProt (unannotated) microproteins were characterized, revealing sequence similarities with domains linked to RNA recognition and TCP-1-like chaperonins. Interactome analysis uncovered connections to growth factor binding, the NOD-like receptor pathway, and SCF-dependent proteasomal processes.

Reanalysis of CPTAC data identified 205 microproteins, with 66 showing differential expression. In this dataset, SwissProt-identified microproteins were involved in muscle system processes and immune response suppression, suggesting roles in cancer extracellular matrix (ECM) remodeling and immune evasion. KEGG pathways highlighted their involvement in IL-17 and NOD-like receptor signaling. OpenProt microproteins resembled actin proteins and were part of actin family proteins and ribosomal subunits, with interactome data associating them with actin cytoskeleton pathways.

Notably, PIGBOS1 and NoBody were identified and quantified in both cell lines and CPTAC data. With a cutoff value of -1 < |Log2FC| < 1, and p=0.05, PIGBOS1 was downregulated in all stages of CRC cell lines relative to the control except stage 1 (SW1116). Conversely, NoBody was downregulated in early-stage CRC cell lines (SW1116, SW480, SW48). However, both PIGBOS1 and NoBody were not significantly differentially expressed in the CPTAC CRC tissue data given the same cutoff.