Quantitative TMT-based proteomic profiling reveals stage-specific protein dysregulation in colorectal cancer

Background: Colorectal cancer (CRC) is a leading cause of cancer-related deaths in the U.S. and its territories, with incidence increasing particularly early-onset CRC, and is often diagnosed at advanced stages. Quantitative proteomics using Tandem Mass Tag (TMT) provides an opportunity to characterize stage-associated molecular alterations. This study aimed to profile the proteomic landscape of CRC in a cohort of Hispanics living in Puerto Rico (HPR) and identify molecular differences across disease stages. Methods: Tissue samples from healthy controls (n=21), early-stage CRC (n=11), and advanced-stage CRC (n=12) were obtained from the Puerto Rico Familial Colorectal Cancer Registry (PURIFICAR). Samples underwent TMT-based quantitative proteomics followed by liquid chromatography- tandem mass spectrometry (LC-MS/MS). Normalized protein abundance was processed in MetaboAnalyst 5.0. Compositional differences across study groups were evaluated using principal component analyses (PCAs) and PERMANOVA permutation testing for statistical evaluation. Differential protein abundance was assessed using a one-factor analysis with |fold change| ≥1.5 and p≤0.05. Dysregulated proteins were annotated using UniProt and Ingenuity Pathway Analysis (IPA). Results: PCA showed significant proteomic differentiation among all pairwise comparisons, confirmed by PERMANOVA testing: early-stage CRC versus healthy controls (p=0.001), advanced-stage CRC versus healthy controls (p=0.001), and advanced versus early-stage CRC (p=0.021). Early-stage CRC had 203 differentially abundant proteins relative to healthy controls (72 upregulated, 131 downregulated), with 55.2% unique to this stage. Advanced-stage CRC showed 131 (77 upregulated, 54 downregulated), of which 33.6% were unique. Only twenty-eight (28) proteins were differentially abundant between advanced and early-stages. Venn diagram revealed no proteins common to all three contrasts (0 in the three-way intersection). Early-stage CRC showed dysregulation of DEF1, AZU1, and FCGBP, while advanced-stage CRC demonstrated changes in MMP9, ANXA3, and DEF1. Pathway analysis of classified dysregulated proteins predominantly as enzymes, transporters, and membrane-associated proteins. Conclusion: Quantitative proteomic profiling identified distinct proteomic profiles associated with CRC stage within this HPR cohort. Early- and advanced-stage CRC demonstrated different dysregulation patterns, with early-stage disease showing greater overall proteomic remodeling and a unique protein composition compared with advanced-stage disease. These stage-associated differences warrant further validation to determine their relevance for early CRC detection and biological characterization.