Food shortages are one of the most serious problems caused by global warming and population growth in this century; consequently, it is important to increase food production. Especially, climate changes influence the magnitude and frequency of hydrological fluctuations, which create an unfavorable environment for the growth and development of crops. Because the accurate analysis of a large number of proteins promotes the knowledge of biological systems, gel-free/label-free proteomics has been performed on agricultural materials. Based on the results from proteomic analyses, the roles of key proteins in crops related to stress tolerance were carefully confirmed with molecular biological techniques. These approaches contribute to not only understanding stress-tolerant mechanisms in crops, but also producing crops with environmental-stress tolerance.

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.

Ovarian clear cell carcinoma (OCCCa) shows chemoresistance and clinical outcomes at advanced stages are generally unfavorable. To identify proteins involved in OCCCa, shotgun proteomics analysis was applied using formalin-fixed and paraffin-embedded samples. Analysis of 1521 proteins revealed that 52 were differentially expressed between four OCCCa and 12 non-OCCCa samples. Of the highly expressed proteins in OCCCa, we focused on left-right determination factor (LEFTY). In 143 cases of ovarian epithelial carcinoma, LEFTY expression at both mRNA and protein levels was significantly higher in OCCCas compared with non-OCCCas.

Since there is a close relationship between epithelial-mesencymal transition (EMT)/cancer stem cell (CSC) features and chemoresistance in OCCCas, we next examined a link between LEFTY expression and EMT/CSC properties. OCCCa cell lines that were treated with TGF-β, underwent morphological changes toward an EMT appearance, along with increased expression of LEFTY and Snail. The cells also showed CSC properties, as demonstrated by increases in the aldehyde dehydrogenase (ALDH)1^high activity population, number of spheroid formation, and expression of several CSC markers. Inhibition of LEFTY expression induced decreases in the number of spindle-shaped cells and CSC features, while cells stably overexpressing LEFTY exhibited enhancement of such EMT/CSC properties. Finally, treatment of cells with TGF-β led to increased LEFTY expression and activation of Akt, which subsequently induced inactivation of GSK-3β, while inhibition of GSK-3β resulted in increased expression of both LEFTY and Snail. In conclusion, we reported the first time that LEFTY may be an excellent OCCCa-specific molecular marker and the functional rule may be the establishment and maintenance of phenotypic characteristics of OCCCas through modulation of EMT/CSC properties mediated LEFTY/Akt/GSK-3β/Snail axis.

Current proteome analysis method is an effective method to comprehensively analyze the proteins, but it is still difficult to identify and quantify the proteins of a specific cell in a single analysis due to the complexity of the proteome samples. We have been working on the development of basic technologies to solve this problem and improve the identification and quantification efficiency. We have focused on the use of monolithic columns to improve separation efficiency and have developed the RiMS (removal of interference mixture spectra) method, which improves the accuracy of isobaric labelling quantification. We applied this method to the analysis of pluripotent stem cells, resulting improved quantification accuracy of cell type-specific proteins. In this paper, we will introduce the RiMS method with an overview of the methods used for quantitative proteome analysis.