Quantitative Proteomics And Kinaseomics Studies Of Liver Cancer Based On SWATH-MS Technology

Hepatocellular carcinoma(HCC)is one of the most common malignant tumor worldwide,and has been ranked as the second most frequent fatal cancer in China,with short-term survival rate and long-term mortality rate.The discovery of diagnosis and drug targeting markers of HCC was still urgent problems to be solved.Multiple signaling pathways are closely related to the development and progression of HCC,and the cross-talks among the signal pathways form a complex network.The study of dynamic protein and kinase changes in the whole signaling pathways helps to reveal the complex pathogenesis of HCC,and is beneficial to find sensitive and specific diagnosis and drug targeting marker molecules of HCC.The majority of HCCs in China are caused by Hepatitis B virus(HBV)infection.Based on the important status and function of key proteins and kinases in the development of HCC,we focused on the quantitative analysis of proteins and kinases in HBV related HCC.The Sequential window acquisition of all theoretical mass spectra(SWATH-MS),a label-free quantitative technology,and the chemical proteome technology,a strategy of kinase enrichment by variety of small molecule inhibitors combined with mass spectrometry analysis,were used to achieve panoramic quantitative identification of proteins and kinases of the clinical specimens(HCC tissue and adjacent normal tissue).Further,western blotting was carried out with clinical samples to validate the proteomic results.The study provides some data for revealing the molecular mechanism of the development and progression of HCC,and for the discovery of sensitive and specific diagnosis markers and drug targeting markers of HCC.SWATH-MS is a lable-free quantitative proteome technology,which can avoid the quantitative deviation of traditional mass spectra for low abundance protein,with less sample,simple sample preparation,high protein coverage and accurate,suitable for clinical sample analysis.A quantitative proteomic study of tumour and adjacent nontumour liver tissue was performed using a SWATH-MS strategy.A total of 4,216 proteins were reliably quantified and 338 were differentially expressed,with 191 proteins highly-expressed and 147 lowly-expressed in HCC tissues compared with adjacent non-tumourous(non-HCC)tissues.KEGG pathway analysis showed that differentially expressed proteins involved in the regulation of different functions.Notably,our study suggested sophisticated metabolic pathway changing in HCC,including the highly-expressed of the glycolysis,pentose phosphate pathway and fatty acid biosynthesis,and the lowly-expressed of gluconeogenesis;serine,glycine and sarcosine biosynthesis /metabolism;and fatty acid-oxidation.Twenty-seven metabolic enzymes,including PCK2,PDH and G6 PD,were significantly changed in this study.To our knowledge,this study identified hundreds of differentially expressed proteins,which together form a rich resource for novel drug targets or diagnostic biomarker discovery.Chemical proteomic technique was used to quantity the kinome of HCC tissues and non-HCC tissues.The serine/threonine kinase inhibitor Purvalanol B and the receptor tyrosine kinase inhibitor SU6668 were incubated with EAH Sepharose 4B,or the highly potent broad-spectrum kinase inhibitor VI16832 was incubated with ECH Sepharose 4B to enrich the kinases in HCC tissues and non-HCC tissues,then the differential kinases were quantitatively analyzed using the label-free quantitative SWATH-MS strategy.A total of 383 kinases were reliably quantified,and 93 were significantly differentially expressed,including 80 up-regulated kinases and 13 downregulated kinases in HCC.Among the 93 differentially expressed kinases,there were 73 protein kinases,which were distributed in the whole 9 human kinase groups.In addition,the differential kinases are involved in complex signal pathwaysof HCC,including the MAPK signaling pathway,PI3K/Akt/m TOR signaling pathway,VEGF signaling pathway,Wnt signaling pathway and Hedgehog signaling pathway.Key kinases,including VEGFR-2,CDK6 and m TOR,were significantly changed in these pathways.Our results indicate that the chemical proteomic strategy can effectively identify key kinases involved in the development of HCC.Besides,the key kinases involved in the complex signal pathways of HCC,and the small molecule kinase inhibitors(SMKIs)were discussed in this study.In addition to the multi-target SMKI sorafenib,which has been approved by the Food and Drug Administration(FDA)for the treatment of hepatocellular carcinoma,the SMKI lenvatinib(targeting VEGFR-2)for the treatment of renal cell carcinoma and thyroid cancer,and other multi-target(including VEGFR-2)SMKIs for other cancer treatment,such as pazopanib,vandetanib,axitinib,regorafenib,cabozantinib,ponatinib and nintedanib,have potential for the treatment of HCC.At the same time,the SMKIs(targeting CDK4 and CDK6)palbociclib and ribociclib(just approved by FDA)for breast cancer treatment,and the small molecule drugs(targeting m TOR)sirolimus,temsirolimus and everolimus for the treatment of renal cell carcinoma,might be a potential treatment for HCC too.Over all,the quantitative kinomic study of HCC tissues and non-HCC tissues using chemical proteomic strategy not only provide some data for the discovery of biomarkers and drug targets,but also provide a new strategy for exploring the kinome change in other cancers.In conclusion,SWATH-MS strategy was used to achieve non-directional,panoramic quantitative identification of proteins and kinases of the clinical specimens(HCC tissue and adjacent normal tissue).A number of differential proteins and kinases,which play an important role in the development of hepatocellular carcinoma were quantified,together form a rich resource for novel drug targets or diagnostic biomarker discovery,and provide a new perspective to explore the treatment of HCC.