Screening And Analysis Of Differential Expression Proteins From HIV/AIDS Damp-Heat Syndrome Based On ITRAQ-MS Technique

ObjectiveScreen differential expression proteins between HIV/AIDS damp-heat syndrome and healthy control group in serum using iTRAQ-MS quantitative technology.Then validate these proteins by ELISA experimental technique and analyse by bioinformatics to explore the function and interaction of these differentially expressed proteins and find the protein material basis of HIV/AIDS damp-heat syndrome.Methods1 According to the HIV/AIDS damp-heat syndrome diagnostic scale,select patients and healthy subjects of the same area as the control group.Their blood sample is extracted in the morning and centrifuged.Then choose the upper serum as samples.2 Evaluate the removal efficiency of high abundant proteins using the SDS-PAGE experiment.3 Mark low abundance proteins by the iTRAQ technique.4 Identify and quantitative analyse low abundance proteins in serum using the MS technique.Query database to identify and analyse MS original date using the Mascot 2.2 and Proteome Discoverer 1.4 software.Then Screen out differential expression proteins.5 Validate these proteins by ELISA experimental technique,analyse differential multiple and P value.6 Describe the properties of genes and gene products in living organisms from three aspects: biological process?cellcular component and molecular function using gene ontology analysis which is a standardized gene function classification system.7 Compare target protein sequence and Human protein sequence in the KEGG GENES database by KEGG Automatic Annotation Server.Annotate KO number of homology or similarity proteins to the related KEGG pathway.Results 1 A total of 30 cases are included in this study,including 24 cases with damp-heat syndrome and 16 healthy cases in the control group.There is no significant difference in age and sex composition level between the two groups.But CD4 and CD4/CD8 level in the damp-heat syndrome group is obviously lower compared with the healthy control group.2 Protein electrophoresis band is significantly increased and clear after high abundance proteins removal.The sheltering effect of high abundance proteins is obviously reduced.3 This study identify a total of 279 Protein Group and 2612 unique peptide segment,including 195 proteins with quantitative information in each of the labeled channels.4 Compare differential multiple and calculate the P value to screen differential expression proteins.Screening criteria: differential multiple ?1.2 or <0.83,P<0.05.This study identify a total of 24 differential expression proteins,of which 12 proteins is up-regulation and 12 proteins is down-regulation.5 There are only two proteins of platelet factor 4 and Gelsolin from the results of ELISA validation experiment which is accordant with mass spectrometry in upward or downward(P<0.05).6 A total of 48 protein sequences are annotated with 492 GO Term during the Annotation process.The ellcular component are involved 7 aspects of extracellular region,cell,membrane,extracellular matrix,and so on.The molecular function are involved 6 aspects of catalytic activity,receptor activity,transporter activity,binding,enzyme regulator activity,molecular transducer activity.The biological process are involved 16 aspects of cellular component organization or biogenesis,multicellular organismal process,single-organism process,immune system process,metabolic process,multi-organism process,and so on.7 A total of 7 KEGG signaling/metabolic pathways are extracted during KEGG pathway annotation,which is associated with the 24 target proteins sequence.The pathways are involved PPAR signaling pathway,cytokine-cytokine receptor interaction,vascular smooth muscle contraction,complement and coagulation cascades,Fc gamma R-mediated phagocytosis,staphylococcus aureus infection and systemic lupus erythematosus.Conclusion 1 This research method of screening differential expression proteins between HIV/AIDS damp-heat syndrome and healthy control group in serum using iTRAQ-MS quantitative technology is feasible and effective.2 The removal efficiency of high abundant proteins with Agilent multiple centrifugal affinity column Human-14 is significant.Human-14 can reduce sheltering effect of high abundance proteins to low abundance protein,which is the advantage of the study of differential expression proteins.3 Gelsolin has a greater correlation in the central nervous system injury and AIDS induced tumors.4 Staphylococcus aureus infection may be related to AIDS patients endoretention of diarrhea andloose stool associated symptoms.5 Alpha-1 antitrypsin deficiency may be the risk factors of HIV infection,but also is expected to become AIDS Damp-Heat Syndrome potential protein material basis.