The Study Of Total Protein Sample Preparation Methods In Proteomics Of Pancreatic Tissue Glycoproteins And Its Applications In Pancreatic Carcinoma Tumor Markers Screening

Proteomics is the most actively interesting field in life science after humangene project. The separation and identification of proteins in cells or tissues arekey problems in proteomics research. Two-dimensional electrophoresis (2-DE)suffers from several unavoidable technical limitations, although it has beenwidely used as the separation tool in proteomics. Recently because of highresolving power, reproducibility and automation, multidimensional highperformanceliquid chromatography represents an attractive alternative to twodimensionalployacryl- amide gel electrophoresis (2-DE) for the separation ofmacromolecules. It is recognized as a powerful tool in proteomics research.However, proteomic research is newly founded, there are still many problemsneed improved, especially the preparation of tissue protein sample, a all purposemethod which is refer to all tissue has not been founded yet, and the commonlyused method for extraction of tissue total protein is not appropriate for extractionof pancreatic tissue total protein.We compared different method to extract total protein of pancreatic tissue, then the protein level was measured and the proteins was abruption using SECRPLC,detected MALDI-TOF MS respectively. Establishment of the bestextraction method of pancreatic tissue at last and applied it in pancreaticcarcinoma tumor markers screening.Exp.1.Establishment of homogenization method of pancreatic tissueObjectives Contrast different homogenization method to extract total protein ofpancreatic tissue to find the best method to homogenize of pancreatic tissue.MethodsThe pancreatic tissue of health SD rat were homogenized by differentmethod and extract the total protein of pancreatic tissue by using total proteinextract kit then the protein level was measured and the protein was abruptiousing SEC-RPLC, detected MALDI-TOF-MS respectively. ResultsTheextraction amount of total protein with liquid nitrogen grind combined withfreeze-thaw homogenization and liquid nitrogen grind combined with ultrasonichomogenization methods was(6.7±0.5)mg ,(6.9±0.2)mg and the number ofMALDI-TOF MS spectra peak was (304.2±22.3) and (331.6±30.5) respectively.The extraction amount of total protein with mechanical homogenizationcombined with freeze-thaw homogenization and mechanical homogenizationcombined with ultrasonic homogenization methods was(5.4±0.2)mg,(5.6±0.2)mg and the number of MALDI-TOF MS spectra peak was(263.6±19.7) and (289.8±12.2) respectively. ConclusionThe liquid nitrogengrind combination ultrasonic homogenization method is the best homogenizemethod of pancreatic tissue and possess tissue chemical property compared withothers. Exp.2.Study of extraction method of total protein in pancreatic tissueObjectives Contrast different method to extract total protein of pancreatic tissueto establish the best method to extract total protein of pancreatic tissue.Methods One-step, three-step sequential protein extraction method and threedifferent pH lysate methods to extract the total protein of health SD ratpancreatic tissue respectively. Then the protein level was measured and theprotein was abruptio using SEC-RPLC, taken into MALDI-TOF-MSrespectively. ResultsThe extraction amount of total protein with one-step, threestepand three different pH lysate methods was(18.7±2.8)mg,(23.2±1.0)mg, (18.3±0.3) mg and the number of MALDI-TOF MS spectra peak was(404±37.4), (447.8±23.3) and (399.8±43.6) respectively. The results betweeneither two of three kinds of methods were compared. ConclusionThe three-stepsequential protein extraction method has the higher protein extraction rate andthe better protein dissolubility as compared with one-step protein extractionmethod.The three different pH lysate methods is the best for acidic proteincompared with other two extract methods.Exp.3. Specificity glycoproteins/peptide of pancreatic carcinoma tissue preliminary screeningObjectivesDetect the total protein in pancreatic cancer tissues using MALDITOFMS. Found to the significant deviation glycoproteins/peptides then try tovalue the relationship between the significant deviation glycoproteins/peptidesand the tissue type of pancreatic cancer. MethodsExtract total protein in 6 pancreatic cancer tissues using Exp.2. method,the protein was abruptioed usingAFC-RPLC, taken into MALDI-TOF MS. ResulResults The positive expression rates of 5733D glycoprotein/peptide in 6 pancreatic cancer tissues are 50%. ConclusionThe high level expression of 5733D protein/peptide in thepancreatic cancinoma tissue of pancreatic cancer, maybe 5733D protein/peptideshould become new biomarker candidates in diagnosis of pancreatic cancer andan important index to reflex the prognosis of pancreatic cancer patients.