A New Method For Protein Tyrosine Phosphorylation Analysis Based On Centrifugal Enrichment Devices And Antibodies

Proteomics is a large-scale,high-throughput,systematic study of the composition and function of all proteins in a cell,tissue or organism.Post-translational modification plays an important role in the function and activity regulation of proteins.Therefore,proteomics studies should not only provide qualitative and quantitative data for proteins,but also include information on the modification types and sites of proteins.There are two commonly used strategies in proteomics research: "Top-down" and "Bottom-up"."Top-down" directly analyzes the whole protein to achieve protein sequence identification,based on determination of the protein molecular weight and interpretation of fragmentation spectra."Bottom-up" is currently the most widely used proteomics research strategy.Its workflow includes digestion of the complex protein samples into peptides,peptides separation by chromatography or other methods,and liquid chromatography-mass spectrometry(LC-MS)analysis.Peptides are identified by matching the fragmentation spectra to protein database to obtaine information of the corresponding proteins.This strategy is widely applied for large scale identification and quantification of complex proteomic samples.In recent decades,there are great advances in mass spectrometry technology.In particular,biological mass spectrometry has been widely used in the analysis and identification of proteins,providing highthroughput,rapid,accurate and sensitive detection methods for proteomics research.Through the study of the abnormal changes of protein expression,modification and function in the biological process,people can deeply understand the physiological processes and pathological changes of organism.Proteomics study provides a theoretical basis for revealing the mechanisms of human diseases in the cellular and molecular levels,targets for drug development,and molecular markers for early diagnosis and prognosis of human diseases.Protein tyrosine phosphorylation is an important post-translational modification and has become one of the most active areas in proteome research in recent years.Protein tyrosine phosphorylation plays key regulatory roles in numerous signal transduction process,including cell proliferation,cell cycle progression,apoptosis,angiogenesis,cell migration,gene transcription,and metabolic response.It is a key factor in cell signal transduction and plays a key role in the occurrence &development of tumors.The study of tyrosine phosphorylation and the activity of it corresponding tyrosine kinases is of great significance for the research of drug targets for cancer treatment.However,tyrosine phosphorylation only represents less than 0.1% of the total cellular protein phosphorylation.Therefore,there is a great challenge to identify tyrosine phosphorylation in real complex samples.Common used methods for tyrosine phosphopeptides enrichment include metal oxide affinity chromatography and antiphosphotyrosine antibody immunoprecipitation.However,both methods have drawbacks.Titanium dioxide enriches serine,threonine and tyrosine phosphopeptides without bias and the identified results contained only 1%~2% tyrosine phosphopeptides.A large number of non-tyrosine phosphopeptides would inhibit the tyrosine phosphopeptide signal in mass spectrometry analysis.Although the method of antibody immunoprecipitation is a specific enrichment method for tyrosine phosphopeptides,there are still a large amount of non-specificly adsorped non-phosphopeptides due to the high complexity of the sample and the limited affinity of antibody.In view of these problems,we established a strategy of phosphopeptides enrichment using combination of titanium dioxide and antibody immunoprecipitation.We developed a new centrifugal device by combined application of titanium dioxide(TiO2)and C18 reverse phase packing materials for phosphopeptides enrichment and separation,in which the enrichment,washing,elution and fractionation of phosphopeptides are integrated in the self-made centrifugal device.Next,the obtained phosphopeptides are antibody immunoprecipited by anti-phosphotyrosine antibody for tyrosine phosphopeptide enrichment.This enrichment device simplifys procedure,reduces sample loss,minimizes interference,and provides parallel sample processing,which significantly improves throughput of the analysis.We investigated and optimized the experimental conditions of this enrichment strategy to obtain optimized enrichment results.For the centrifugal enrichment device,we examined the mass ratio of peptides to TiO2 and the acetonitrile content in the elution buffer.The results showed that high enrichment effiency was obtained,when the mass ratio of peptides to TiO2 was 1: 4,and the content of acetonitrile was 2%,5%,8%,10% and 40%,respectively.Enrichment from 1mg mouse liver protein leads to the identification of 10793 phosphorylation sites,corresponding to 7333 phosphorylated peptides.Next,we studied the experimental conditions for antibody enrichment,including the manufacturer of antibody,the initial amount of proteins,and washing methods.Tandem enrichment by anti-phosphotyrosine antibody results in efficient enrichment and large scale identification of phosphotyrosine peptides by mass spectrometry.As a result,we successfully enriched and identified 849 phosphotyrosine peptides corresponding to 545 proteins from mouse liver,which have consistent functions with the reported tyrosine phosphorylated proteins.The above resultsprovide strong support for further study of tyrosine phosphorylation,and demonstrate the potential of this enrichment strategy in proteomic research.Liver cancer is one of the most common tumors in China,and its mortality rate is only lower than lung cancer.Most patients with hepatocellular carcinoma have lost their chance of surgical resection when they were diagnosed,so it is important to find effective liver cancer drug therapy target and early diagnosis marker.Using proteomic strategy to compare the difference in expression and modification of tyrosine phosphorylated protein in liver cells or tissues between normal and pathological conditions provides theoretical basis for clinical diagnosis,pathology study and drug screening of liver cancer.We used the tyrosine phosphopeptide enrichment strategy established by mouse liver sample for the enrichment and identification of tyrosine phosphopeptides in hepatocellular carcinoma tissues and paracancer tissues.A total of 970 non-redundant tyrosine phosphorylated proteins and 1492 tyrosine phosphorylation sites were identified in liver cancer tissues from 77 patients.962 non-redundant tyrosine phosphorylated protein and 1424 tyrosine phosphorylation sites were identified in paracancer tissues.The above results may facilitate the mechanism study of tyrosine phosphorylation in the pathophysiology of liver.This experiment further demonstrates the feasibility of our new tyrosine phosphopeptides enrichment strategy using the selfmade centrifugal enrichment device followed by antibody immunoprecipitation for clinical samples.Possibe application of this centrifugal device in the enrichment and identification of other proteins post-translation modification is also expected.