Background Gastroesophageal junction adenocarcinoma (GEJA) is a malignant tumor located at the junction of the esophagus and stomach, the incidence of which is increasing year by year, while screening for early biomarkers is limited. Tandem mass tagging (TMT) coupled with liquid chromatography–tandem mass spectrometry (LC/MS/MS) has been used to screen for differential proteins in various cancers. Methods Differential proteins in GEJA and precancerous lesions were screened using TMT–LC/MS/MS, and then proteins that met expectations were selected for trend clustering analysis, combined with GO and KEGG analysis for functional annotation of differential proteins in GEJA. Then, parallel reaction monitoring and immunohistochemistry techniques were used to validate the accuracy of the proteomics data. Results Our group screened the differential proteins during GEJA progression using proteomics technology, analyzed the expression trends and functional regions involved in the differential proteins during carcinogenesis, and validated the accuracy of the experimental results. Conclusions The screening of differential proteins in GEJA carcinogenesis based on TMT–LC/MS/MS technology provides detailed information for the elucidation of GEJA progression process, pathogenesis, early screening and screening of candidate markers.

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