To investigate the early changes in protein function that induce micro-metastasis in early-stage non-small cell lung cancer, we conducted proteomic analysis of tissue that had been completely resected. We selected sixteen patients whose tumors were pathological stage I adenocarcinoma with hobnail cell morphology. We compared their proteomic profiles between patients whose cancers recurred and did not recur after 5 years of follow-up. Proteins extracted from frozen tumor tissue were saturated by CyDye labeling and subjected to two-dimensional difference gel electrophoresis (2D-DIGE). We found approximately 2500 protein spots by DeCyder-DIA software analysis. A random forest classifier to the spot volume data sets revealed 30 extracted spots that were potentially effective marker proteins for distinguishing the recurrence and non-recurrence groups. Among them, Mann-Whitney U-test analysis showed that 15 spots were candidate marker proteins. Finally, 11 unique proteins corresponding to the 15 candidate spots were found using an improved in-gel digestion method and MALDI-TOF MS and nanoLC-ESI MS analysis. Among them, aldehyde dehydrogenase and tropomyosin were considered to have undergone protein modifications such as phosphorylation, acetylation, and glycosylation. We have thus identified some biomarkers that can predict tumor recurrence in patients with early-stage non-small cell lung cancer including hobnail-type adenocarcinoma. A further study is required to confirm the utility of these biomarkers.

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