Optimization Of ¹⁸O Labeling Method And Development Of Analysis Tool And Its Application In Study Of Metastsis-related Membrane Proteome In Nasopharyngeal Carcinoma

Proteomics has become an important part of life science research and has been widely used in life science fields. Because there are transcriptional and translational regμlation of gene expression and post-translational modification of proteins, proteomics is much more complicated than genomics. Therefore, proteomics research is much more dependent on technology than genomic research. Therefore, the development of methods and techniques with high throughput, high sensitivity and high accuracy has been the challenge for proteomics research.With advances in the proteomics techniques and methods, proteomics research has new feature:a research focus has moved from the protein expression profile to precise quantitative and functional studies of proteins. Currently quantitative proteomics technology based on stable isotope labeling have become a new proteomics technology. However, quantitative proteomics technology is still in development stage and the research techniques need to be improved and analysis tools need to be developed.¹⁸O labeling technique is one of the earlier techniques for quantitative proteomics. The technique based on the principle of enzyme kinetics, where proteins are labeled with ¹⁸O by enzyme catalytic reaction.¹⁸O labeling technique is simple, mild technique with no by-products. However, there are some disadvantages of ¹⁸O labeling technique:（1） ¹⁸O-label is easily lost at the presence of the enzyme, thus affecting the accuracy of quantitation; （2） Data analysis is relatively complex; （3） Lack of computational tools for quantitative analysis.Membrane proteins play important roles in cell recognition, signal transduction material transport and etc. Abnormal expression of membrane proteins often lead to the changes of signal transduction, cell adhesion, motility and etc. which are related with tumor invasion and metastasis. Therefore, the study on tumors’ membrane proteome has become an important part of proteomic research.Based on the above considerations, we first optimized and improved ¹⁸O labeling method. The loss of ¹⁸O-label is the major weakness of this method. For this, We use solid-phase trypsin as a labeling enzyme and, after the reaction, the samples were filtered using Ziptip to remove solid-phase trypsin. The stability of ¹⁸O-label of the filtered samples stored under different pH conditions was analyzed. The resμlts showed that Ziptip filtration can significantly improve the stability of ¹⁸O-label, compared with the heat treatment method. There was no obvious loss of 180-label during continuous monitoring of three days. Further analysis of the impact of the Ziptip filtration on the results of quantitative analysis showed that the differences of quantitation are less than 5% during continuous monitoring of three days. There were no significant loss of ¹⁸O-label during the filter samples were separated by isoelectric focusing （IEF）. Further analysis also showed that the ¹⁸O labeled efficiency of peptides was no significant correlation with their molecμlar weight and theoretical isoelectric point. In addition, the volume of the samples handled by Ziptip was merely 10-20μl in the present study. Therefore, the optimized and improved ¹⁸O labeling method provided by us can be used for quantitative analysis of trace samples, which is of great significance for scarce clinical samples, such as laser capture microdissection （LCM） purified tissue samples and other limited samples.Considering the lack of computational tools for ¹⁸O labeling data anlaysis, We developed an computer tool named OxyQuantToolkit. The tool is developed using java language and therefore has cross-platform property. The tool has functions covering mμltiple aspects of ¹⁸O labeling data processing. A large number of computing tasks can be done automatically by the tool during ¹⁸O labeling data anslysis. The results of quantitative analysis can be output automatically. At the same time,the tool has functions for data arrangement and data dump, which has a positive effect on effective data management. In addition, the tool contains a data visualization programming package, which can be used for viewing mass spectrometry data in mzxml format. The development of OxyQuantToolkit provided the foundation for facilitating and broadening the application of ¹⁸O labeling quantitative proteomics technology.On the basis of the improvement of ¹⁸O labeling method and the development of analysis tool for ¹⁸O labeling data analysis, We use the improved ¹⁸O labeling method for the quantitative analysis of the membrane proteins from high metastatic nasopharyngeal carcinoma（NPC） 5-8F cells and non metastatic NPC 6-10B. A total of 503 proteins were identified, with 44% of membrane protein and 89.7% of the proteins’ expression ratio in 5-8F versus 6-10B were between 0.5-2 （mean 1.13）. Statistic analysis of the quantitative results of 395 proteins identified in three repeated experiments demonstrated that the average standard deviation was 0.06, showing high repeatability. The results showed that the improved ¹⁸O labeling has great applicable value. In addition, according to the standard provided in literatures （changing more than twice and identified in three repeated experiments）,31 differential expressed membrane proteins between 5-8F and 6-10B were found, of which 6 were downregμlated and 25 were upregμlated in 5-8F. Bioinformatics analysis showed that some differential expressed proteins, such as ITGB1 and EphA2, are related to cell adhesion, tumor invasion and metastasis. The discovery of these proteins provided the targets for the study of metastatic mechanisms and prevention and treatment of nasopharyngeal carcinoma. Further comparative analysis of the phosphoproteins in the membrane fraction of 5-8F and 6-10b cells found that EphA2 S897 phosphorylation site which is key to promoting metastasis are differentially expressed, and activated EGFR can phosphorylate the site, indicating that EGFR promotes NPC metastasis possibly by phosphorylating the site.An improved ¹⁸O labeling method based on removal of solid-phase trypsin using Ziptip has been established. The method has features such as high stability of label, high accuracy of quantitation and being suited to the analysis of trace samples; the platform-independent analysis tool, OxyQuantToolkit, for ¹⁸O labeling-based quantitative proteomics was developed, which provide the foundation for facilitating and broadening the application of 180 labeling technique. A java-based programming package for mass spectrometry data visualization was developed and a viewer for viewing mass spcetrometry data in mzxml format was also implemented; Comparative proteomic analysis of membrane proteins between NPC cell lines with different metastatic potential are performed using improved 180 labeling method, and 31 metastasis-related membrane proteins were identified, which provided the targets for the study of metastatic mechanism and the prevention of NPC. Comparative phsosphoproteomic analysis of membrane proteins between NPC cell lines with different metastatic potential are performed and the differential phsosphorylation level of EphA2 S897 site was identified, which is key to promoting metastasis. Further analysis confirms that activation of the EGFR can phosphorylate the site, which provide a important clue for studying the mechanism of EGFR that promoting NPC metastasis.