Phosphoproteomic subtyping and characterization of colorectal cancer by comprehensive phosphoproteomics of fresh frozen endoscopic biopsy specimens

Background and Aims: Phosphorylation is a key post-translational modification that regulates protein activity, localization, and interactions. By mapping phosphorylation patterns, phosphoproteomics provides real-time insights into how cancer cells reprogram their signaling networks to promote growth, survival, metastasis, and therapy resistance. While conventional deep phosphoproteomics requires a relatively large amount of clinical samples, we have developed a highly sensitive method using endoscopic biopsy samples which can offer excellent quality for hosphoproteomic analysis. In this study we evaluated the phosphoproteomic landscape in fresh-frozen endoscopic biopsies from patients with colorectal cancer (CRC). Methods: Endoscopic biopsy specimens were obtained from 102 treatment-naïve CRC patients and snap-frozen in liquid nitrogen within 20 seconds of collection. Ultra-deep proteome and phosphoproteome analyses were performed using tandem mass tag (TMT)-based multiplexing. Genomic profiling was conducted with a targeted, high-multiplex PCR-based next-generation sequencing (NGS) panel. Results: Ultrasensitive mass spectrometry-based proteomics using 204 naïve colorectal cancer specimens and 204 normal tissue adjacent to the tumor (NAT) quantified an average of 23985 phosphorylation sites. Consensus clustering clearly divided colorectal cancer into subtype 1 (proliferative type, 33%), subtype 2 (EMT type, 25%), Subtype 3 (Metabolic type, 20%), and subtype 4 (Abnormal RNA splicing type, 21%). Kinase activity profiles were obtained from phosphoproteome data and kinases that are specifically activated or inactivated in each subtype were identified. While concordance between these phosphoproteomic subtypes and the consensus molecular subtypes (CMS) was low, sidedness was associated with different kinome activity of tumor and NAT. Conclusions: This proof-of-concept study demonstrates that phosphoproteomic analysis can delineate CRC subtypes independent of CMS classification and provide detailed kinase activity landscapes. Our pioneering approach enables robust clinical phosphoproteomics from small endoscopic biopsies, offering a promising tool for monitoring therapeutic kinase activities and advancing precision oncology.