Differential Protein Analysis Of Fluorescence 2-dimensional Difference In Gel Electrophoresis Profiles From Malignant And Inflammatory Pleural Effusion

Recently,lung cancer has been one of the most common malignant tumors with the highest rate of morbidity and mortality in many countries.Because of lacking sensitive and precise methods for early detection,the five-year survival rate of lung cancer is only arround 15%.Thus it is important to find some more specific and sensitive tumor biomarkers for this disease especially from the human body fluid such as plasma or pleural effusion.Among them,pleural effusion is usually induced directly by lung disease and has a more simple protein composition than plasma;therefore,it is considered as a more ideal sample than other body fluids for lung cancer biomarker screening.Although malignant pleural effusion usually develops in the later stages of lung caner,it always contains the specific tumor associated secreted proteins and turns out to be suitable for primary biomarker screening.Furthermore,pleural effusion protein composition analysis is also helpful for understanding the molecular biology mechanism of lung diseases.In the past decade,high-throughput proteomic techniques offer a new and exciting way to discover tumor biomarkers.To study the proteome map of pleural effusion,it is necessary to remove high-abundant proteins to enhance the detection of low-abundant ones.The depletion approach can effectively improve the sensitivity of this protein separation technology.In addition,non-malignant pleural effusion(such as inflammations) should also be used as a control to establish a differential proteomic analysis for tumor biomarker screening.In this study,we successfully construct a high-abundant protein depletion technique in pleural effusion proteomic analysis;then,differential proteome patterns were compared between lung adenocarcinoma and benign inflammatory pleural effusions(para-pneumonic and tuberculous effusion) with fluorescence two-dimensional differential gel electrophoresis (2D-DIGE) method to highlight the clues for tumor biomarker searching.METHODS AND RESULTSA preliminary proteomic analysis was first performed to evaluate the efficiency of high-abundant protein depletion by using traditional two-dimensional gel electrophoresis and silver staining.3 lung adenocarcinoma and 2 tuberculous pleural effusion samples were analyzed.Our results showed that additional 778 protein spots (spot numbers increased 80%) were detected after high-abundant protein depletion. Meanwhile,some protein SPOTS with differential expression were excised and underwent matrix-assisted laser desorption/ time-of-flight mass spectrometry analysis.One of differentially expressed proteins was identified as 14-3-3ζ,a member of 14-3-3 protein family.Its appearance in pleural effusion was further confirmed by Westem blot.This is the first time 14-3-3ζwas reported as a secreted protein appeared in human pleural effusion.Based on our primary results,10 lung adenocarcinoma,6 tuberculous and 4 parapneumonic patients' pleural effusions were further studied.2D-DIGE,a recent advance in 2DE technology,was employed to analyze the differetial expressions of proteome profiles among 3 lung disorders.It showed that 10 differetially expressed protein spots were found between cancer and TB,34 spots between cancer and pneumonia,and 16 spots between TB and pneumonia,respectively.As 19 of the altered spots were common to different groups,there were actually 41 differentially expressed protein spots among these three diseases.Among them,6 altered spots were found between malignant and non-malignant(pneumonia plus TB) pleural effusions.4 of them were up-regulated,and the other 2 spots were down-regulated in malignant pleural effusions with a similar pattern of change. CONCLUSIONS1.80%more protein spots were detected by using the new high-abundant protein depletion method than traditional protein separation technology.It clearly showed that the high-abundant protein removal technology can efficiently improve the detection rate of low-abundant proteins.2.14-3-3ζ,a member of 14-3-3 protein family,was first reported as a secreted protein appeared in human pleural effusion which was further confirmed by Western blot.It directly provide the evidence that the high-abundant protein depletion method can help us to find some important low-abundant proteins cannot be detected by routine methods.3.With 2D-DIGE analysis,41 differentially expressed protein spots were found between lung adenocarcinoma,TB and pneumonia.6 altered spots were common between malignant and non-malignant(pneumonia plus TB) pleural effusions which may associate with the origin and development of lung adenocarcinoma.