In order to explore the proteomic characteristics of colorectal cancer, we collected cancer tissues and adjacent normal tissues of postoperative colorectal cancer patients for proteomic detection, based on ultra-high performance liquid chromatography-mass spectrometry (UHPLC-MS) and data-independent acquisition mode (DIA). All samples were processed by DIA individually to assess the proteome differences. MS1 and MS2 data were all acquired, and samples acquisition by random order. The iRT kit (Ki3002, Biognosys AG, Switzerland) was added to all of the samples to calibrate the retention time of extracted peptide peaks. The statistical analysis of the DIA dataset was performed by Spectronaut 15(Biognosys AG, Switzerland) including data normalization and relative protein quantification. After t-Test and corrected by Benjaminiand Hochberg algorithm, different expressed proteins were filtered.