The Identification Of Proteins Related To The Pathogenesis Of Immune Thrombocytopenia And Its Role In The Pathogenesis

Objective:Immune thrombocytopenia(ITP)is a complex autoimmune disease characterized by decreased platelet count with or without skin and mucosal bleeding.In the current study,the pathogenesis of ITP involves many aspects.Pathological antiplatelet antibodies,impaired megakaryocyte maturation,and platelet destruction mediated by cytotoxic T cell could lead to thrombocytopenia.In addition,impaired T cell function,imbalanced cytokine and bone marrow niche have also been considered the important reasons.However,its pathogenesis is not yet fully understood.As a powerful technology for discovering biomarkers,proteomics technology has been introduced into many hematological malignancies to understand complex biological systems and the function of proteins and find the relationship of protein-protein interaction.Quantitative liquid chromatography tandem mass spectrometry(LC-MS/MS)was performed to detect the differentially expressed proteins in bone marrow samples from active ITP patients and normal controls,then the pathogenesis related proteins of patients with immune thrombocytopenia were analyzed by bioinformatics methods to explore the pathogenesis of immune thrombocytopenia.Methods:In this experiment,the quantitative liquid chromatography tandem mass spectrometry(LC-MS/MS)was used to detect the differential expressed proteins in bone marrow samples of active immune thrombocytopenia and normal control group.The bone marrow samples of 20 patients with newly diagnosed immune thrombocytopenia and 20 normal donors were collected.After mononuclear extraction with lymphocyte separation solution,the PierceTM Top 12 Abundant Protein Depletion Spin Columns kit was used to remove the high abundant protein.Then the protein extraction,trypsin digestion,HPLC fractionation and LC-MS/MS analysis was conducted sequentially.All identified proteins were systematically analyzed by bioinformatics including protein function annotation,functional classification of differentially expressed proteins,functional enrichment of differentially expressed proteins,enriched-based clustering of differentially expressed proteins and protein-protein interaction network.After analyzing the molecular function,biological process,protein domain and KEGG pathway of the identified differential proteins,we determined the target proteins.Then the quantitative identification of these proteins was processed using targeted proteome quantification technique based on mass spectrometry-parallel reaction monitoring(PRM).Results:1.A total of 829 proteins were identified in this study,of which 613 proteins were quantifiable.When the fold-change cut off was set to 1.5(patients vs.controls)and a t-test P<0.05 was used as the significance threshold,26 upregulated proteins and 69 downregulated proteins were identified.2.Bioinformatics analysis found that two up-regulated proteins(ORM1 and vWF)and two down-regulated proteins(PPBP and SPARC)were all related to the TNF-αsignaling pathway involved in the pathogenesis of ITP.3.Targeted proteome quantification technique based on mass spectrometry-parallel reaction monitoring(PRM)were used to quantify the four proteins(ORM1,vWF,PPBP,and SPARC)involved in immune system processes.The protein levels of ORM1 and vWF were clearly higher in ITP patients than in controls,while the protein levels of SPARC and PPBP were obviously lower in ITP patients than in controls.Conclusions:The bioinformatics analysis identified differentially expressed proteins in bone marrow that are involved in the TNF-α signalling pathway and are related to the activation of immune function in ITP patients,which could provide a new thought for the study in the loss of immunity tolerance of ITP patients.