Exploration Of Molecular Biomarkers Of Benign And Malignant Pleural Effusions By Analysis Of Proteomics

Objective The differential diagnosis between benign and malignant pleural effusions is one of the common clinical problems.The aim of this study was then to explore novel molecular biomarkers of benign and malignant pleural effusions,and to primarily assess the clinical value of the potential markers for differential diagnosis.Methods The study used two proteomic techniques: two-dimensional gel electrophoresis(2-DE)and isobaric tags for relative and absolute quantitation(iTRAQ).The first experiments route used 2-DE method to separate and search proteins,matrix-assisted laser desorption/ionization time of flight mass spectrometry(MALDI-TOF-MS)to identify the differentially expressed proteins,and ELISA verifies the candidate protein biomarkers.The second experiments route used iTRAQ labeling coupled with two-dimensional liquid chromatography-tandem mass spectrometry(2D LC-MS/MS)proteomic method to separate,identify and relative quantificate proteins in pleural effusion samples.The differentially expressed proteins then processed GO analyzing(including biological process,cell component,molecular function)based on gene ontology(GO)database.KEGG Pathway analysis was used to search and identify the most closely related pathological pathways.Moreover,protein-protein interaction(PPI)network was built up.So as to further understand the differences in protein composition between benign and malignant pleural effusions and to select proteins with potential as biomarkers.Results Based on 2-DE experimental route,comparing malignant group with benign group,43 significantly different protein spots(up or down regulated?2 times)were found,including 9 up regulated spots and 34 down regulated spots.And 7 spotswere identified(up or down regulated?3 times)by mass spectrometry,the immunoglobulin ?(Ig?)and haptoglobin(Hp)were picked out and validated by ELISA.The results indicated that Ig? showed no statistical significance between two groups,while Hp showed the statistical significance(P<0.05).The diagnostic sensitivity and specificity of Hp in malignant pleural effusion were 75.00% and 52.38% at diagnostic cut-off point of 389.02 ?g/L.Based on iTRAQ-2D LC-MS / MS experimental route,151 differentially expressed proteins(the relative quantitative change of isotope labeling ?1.2 times)were identified in benign and malignant pleural effusion,of which 84 were raised and 67 were down.The GO analysis showed that in the part of biological process,the differentially expressed proteins exist in the multicellular organismal process,single-multicellular organism process and response to stress;in the part of molecular function,the differentially expressed proteins exist in the protein binding,in the part of cellular component,the differentially expressed proteins exist in the extracellular proteins.The KEGG Pathway analysis and PPI network showed that except enriched in processes of complement and coagulation cascades,the differentially expressed proteins enriched in PI3K-Akt signaling pathway,too.A signaling pathway has a close relationship with tumor development.Considering both fold change and protein-protein interaction,the data showed that ?-enolase(ENO1),phosphoglycerate kinase1(PGK1)and Tenascin(TNC)were the most promising candidates.Conclusions Based on two-dimensional gel electrophoresis,Hp is expected to be used for differential diagnosis of benign and malignant pleural effusion.Based on iTRAQ techniques,we had a better understanding on the different protein constitution of MPE and BPE.Moreover,ENO1,PGK1 and TNC were found to be differentially expressed in benign and malignant pleural effusions.In summary,the application of proteomics techniques is expected to provide greater help in the search of molecular biomarkers for benign and malignant pleural effusions,and iTRAQ may be moreadvantageous than two-dimensional gel electrophoresis.