Comprehensive Characterization of the Phosphoproteome of Gastric Cancer from Endoscopic Biopsy Specimens

Abstract

Protein kinases are known to abnormally activate in cancer tissue and to occur oncogenesis. Thus, kinases have been studied as therapeutic targets in anti-cancer therapy. To optimize anti-cancer therapy by using kinase inhibitors, clinical phosphoproteomics are recently applied for monitoring phospho-signaling in cancer tissue from patients. In this study, we focused on phosphoproteomics of endoscopic biopsies, which can be rapidly frozen within 20 seconds after collection. Because surgical tissues are affected by ischemia during surgery, endoscopic biopsies have an advantage in monitoring physiological status in cancer tissues.

Since endoscopic biopsies are much smaller than surgical tissues, we developed a highly sensitive phosphoproteomics which are specialized for endoscopic biopsies. After trypsinization of protein lysates from endoscopic biopsies, phosphopeptides are enriched with Fe-IMAC/C¹⁸ Stage-Tip and labeled with Tandem Mass Tag reagent. Labeled phospho-peptides are further fractionated by SCX/C¹⁸ Stage-Tip for deep phosphoproteomics with mass spectrometer combined with nano-flow liquid chromatography.

We succeeded in identification of more than 10,000 class 1 phosphosites. Comparison between gastric cancer and normal mucosa revealed cancer-specific activation of phospho-signaling such as DNA damage signaling. Kinome profiling from the phosphoproteomics of endoscopic biopsies further demonstrated activation of regulatory kinases in DNA damage signaling. Our phosphoproteomics of endoscopic biopsies can be used to screen new therapeutic targets based on kinome profiling of cancer tissues. Furthermore, phosphoproteomics of endoscopic biopsies is expected to be applied to the monitoring of kinase activity in various diseases.