Screening Of Differential Expression Saliva Proteins From Oral Lichen Planus Patients By Two-dimensional Fluorescence Difference In Gel Electrophoresis And Mass Spectrometry

Objective: Oral lichen planus (OLP) is a common oral mucosal disease ofunknown etiology and recurrent attacks easy. The World Health Organization(WHO) defined OLP as a precancerous condition because of its tendency tocancer. In our previous study, we had selected142differentially expressedgenes by the application of gene chip technology, and the gene was expressedby directing protein synthesis. With the completion of the human genomeproject, we gradually focus on the functional genomics. However, it is still notenough to solve the time and quantities of gene expression and even proteinpost-translational processing and modification depending on the sequence ofDNA and its expression only. Therefore, we can find the answer which thegenomics can not solve in proteomics. At present, with the development ofproteomics, the research is increasingly applied to every field of life scienceand medicine, especially in the study of the etiology and pathogenesis ofdisease. The traditional method of two-dimensional gel electrophoresis is notaccurate in protein quantification and proteomics studies in tissues and bloodmore,in saliva less. Saliva sample is simple, safe, low cost and noninvasive,and contains many helpful proteins to diagnosis of diseases earlier. So wetend to draw attention to the salivary proteomics development. Because of thehigher saliva protein types and larger protein concentration, there is nouniform standard and specification for extraction of protein and need furtherexploration. This study is to identify differential express saliva proteins fromoral lichen planus patients by two-dimensional fluorescence difference gelelectrophoresis and mass spectrometry, so as to explore the possiblemechanism of oral lichen planus associated with the change of proteinexpression in saliva.__________________________________________________________________________________________________ Methods：1Preliminary test about extraction of salivary proteinWe extracted fresh salivay protein by three methods ofmethanol/NH4HCO3precipitation method, phenol extraction method and2-DClean-Up Kit, and phenol extraction protein needed the ultrasonic treatmentof50s. It is necessary to ensure the optimum extraction method of salivaryproteins by observing SDS-polyacrylamide gel electrophoresis of proteinbands.2SDS-polyacrylamide gel electrophoresis of salivary protein9pairs of patients and matched healthy adults were selected. In order toreduce the influence of individual differences on the study,3cases of each partmixed as a test sample respectively. Saliva proteins were cleaned by the2-DClean-Up Kit and protein concentration was determined by Bradford method.We observed the salivary proteins bands by SDS-polyacrylamide gelelectrophoresis.3Saliva proteins were separated by two-dimensional fluorescence differencegel electrophoresis, and saliva proteins were identified by mass spectrometrytechnologySaliva proteins were separated by two-dimensional fluorescencedifference gel electrophoresis. The electrophoresis pattern can be obtained byTyphoon9410, and the different proteins were finded by DecyderTM2D6.5software. After deep purple staining, the differential protein spots can bepicked by Ettan Spot Picker in gel. Then the different protein spots weredigested by trypsin and were analyzed by liquid chromatography-massspectrometry (LC-MS).The mass spectrometry data were calculated bySEQUEST algorithm of Bio Works3.3.1data analysis in the software.Theproteins were identified by searching the database in NCBI.Results:1the results of pre-testsThere were thick transparent jelly in the proteins by methanol/NH4HCO3precipitation method and phenol extraction. And methanol/NH4HCO3 precipitation method of protein have caused significant protein loss. AfterSDS-polyacrylamide gel electrophoresis, the jelly bands showed that thesaliva protein were not completely dissolved into the DIGE buffer. In order toincrease the solubility of salivary proteins, the phenol extraction proteinsconcentration increased after ultrasonic treatment of50s. However, there wasstill a jelly, and salivary protein was not completely dissolved. The proteinextraction was no jelly found by2-D Clean-Up Kit, and SDS-polyacrylamidegel electrophoresis showed that all samples bands were clear, no significantprotein loss and degradation. Protein extraction with2-D Clean-Up Kit was fitfor saliva.2the results of SDS-PAGEThe saliva proteins of patients with oral lichen planus group and thenormal group were extracted by2-D Clean-Up Kit. SDS-polyacrylamide gelelectrophoresis showed that the protein bands were clear with no significantprotein loss and degradation.3the results of2-D DIGEThe images were good repeatability, high resolution and a wide range ofisoelectric point and molecular weight distribution by two-dimensionalfluorescence difference gel electrophoresis. The average of lichen planus andnormal human different salivary proteins were317±71by analysis ofDecyderTM2D6.5software, and identified4different protein spots of goodreproducibility by using liquid chromatography-mass spectrometry technology.The expression of secretory IgA1, zincα-2-glycoprotein, salivary amylase,serum albumin were rised in the saliva of patients with OLP.Conclusions:12-D Clean-Up Kit was suitable for the protein extraction without losingand degrading obvious protein.2There were some differential proteins in saliva from oral lichen planuspatients. Secretory IgA1, zinc α-2-glycoprotein, salivary amylase and serumalbumin in patients with oral lichen planus were up-regulated, indicating thatthey may be participate in the occurrence, development of oral lichen planus.