| Extracellular vesicles(EVs)are double-layer phospholipid membrane structures secreted by cells.Through direct interaction with receptor membrane proteins or internalization of receptor cells,EVs can transfer their cargoes into recipient cells,thereby affecting the progression status of various diseases.In addition,EVs are widely distributed in various interstitial fluids and body fluids,and their interor proteins are stable due to the protection of the double-layer membrane structure.The specific isolation of EVs can effectively eliminate the interference of highly abundant free proteins in body fluids.In recent years,EVs based proteomic/phosphoproteomic studies have provided a novel approach for liquid biopsies.Currently reported EVs isolation protocols are less efficient and specific,so we developed 1,2-distearoyl-sn-glycero-3-phosphorylethanolamine(DSPE)and Ti(Ⅳ)bifunctionalized magnetic beads(BiMBs).Compared with ultracentrifugation,the superiority of BiMBs in terms of selectivity and enrichment efficiency was proved.Further,we used BiMBs for EVs isolation from urine samples to perform downstream phosphoproteomic studies.We analyzed EVs phosphoproteins in urine samples from prostate cancer patients and healthy controls to screen out differential expression phosphoproteins,and the relative quantitative analysis of phosphopeptide isomers was also conducted,which provided an effective and non-invasive approach for the study of diseases caused by post-translational modifications of proteins.Saliva is the most suitable biofluid for oral diseases diagnosis and prognosis,but due to the co-existance of enzymes and highly abundant mucin and other proteins,the evaluation based on salivary proteomics have been greatly hindered.The superiority of BiMBs in EVs isolation provides a basis for the postoperative evaluation of oral squamous cell carcinoma(OSCC)based on salivary EVs proteomics and phosphoproteomics.Differential expression analysis and parallel reaction monitoring coupling with parallel accumulation-serial fragmentation(prm-PASEF)method were performed for salivary samples from OSCC patients and controls,and a total of 7 global proteins and 10 phosphoproteins were screened out with significantly reduced intensities after chemotherapy.The biological pathways of these 17 proteins provide an in-depth analysis of OSCC pathogenesis and postoperative evaluation.Combined with machine learning technology,the proteins HEP2,MMP25,ACLY and KPCD were revealed as the biomarkers for the assessment of OSCC surgical outcomes.In order to address the challenge of limited sample volumn and the complicated operation process of phosphoproteomics,we have developed a novel bifunctional magnetic beads(Extracellular Vesicles TO Proteomics,EVTOP)that realized the separation of EVs from biofluids and in-tandem enrichment of the inner phosphopeptides.EVTOP comprises an energy-independent penetrating peptide(octaarginine)riched in guanidine groups which can effectively anchor into the membrane structure and co-functionality of Ti(Ⅳ)can effectively attach the phosphate groups of phospholipid of EV membrane.After tryptic digestion,the octa-arginine peptide is removed and the retained Ti(Ⅳ)can now enrich downstream phosphopeptides efficiently and selectively.The EVTOP method not only enables efficiently and selectively isolation of EVs from small volume sample but also enrich the phosphopeptides in situ after EV sample digestion,avoiding sample loss caused by desalting,different materials introducing and the tubes tranfering,etc.Cerebrospinal fluid(CSF)is obtained by spinal puncture,and the obtained volume is limited,in which80% of the proteins are derived from plasma,and the total protein concentration is about1% of plasma,which greatly limits the development of disease diagnosis and prognosis based on CSF.Using EVTOP,we performed differential expression analysis of phosphoproteins in CSF samples from patients with primary central nervous system lymphoma(PCNSL)and non-PCNSL controls.Combined with the prm-PASEF assay and machine learning,six phosphoproteins were screened out.These phosphoproteins were significantly reduced in intensity after chemotherapy and could separate prechemotherapeutic samples from post-chemotherapeutic samples,including osteopontin(SPP1)which has been reported as a biomarker for PCNSL diagnosis.Plasma EVs are derived from cells throughout the body,however low concentration of disease-related EVs and high content of plasma proteins greatly limit the development of plasma phosphoproteomics.In view of the performance of EVTOP,we applied this method to the study of plasma EVs phosphoproteomics.About 500 phosphopeptides were identified from only 10 μL of plasma sample,corresponding to 150 phosphoproteins.By analyzing the samples of colorectal cancer patients before and after surgery and healthy ones,KNG1,HPRG and PA2G4 were screened out as candidate biomarkers with good response for colorectal cancer treatment.Using EVTOP,the volume of body fluids used was significantly reduced,the identification of phosphoproteins was improved,the time of experimental operation was shortened,and the conventional barrier of EVs phosphoproteomics was broken.Collectively,this thesis reported BiMBs based protocol for the proteomic and phosphoproteomic studies of urinary EVs of prostate cancer,and the postoperative evaluation of OSCC by salivary EVs proteomics and phosphoproteomics.In order to overcome the challenges of limited volume of samples and the complicated operation of phosphoproteomics,we established a new method based on EVTOP,and realized the evaluation of the chemotherapeutic outcomes of PCNSL through the analysis of clinical samples.Finally,in view of the good performance of EVTOP,trace plasma phosphoproteomic studies were also conducted for colorectal cancer surgical outcomes assessment.Through these clinical applications,we have realized disease biomarker screening and treatment outcomes evaluation based on urine,saliva,CSF and plasma EVs,etc.,which may allow EVs proteomics and phosphoproteomics of various body fluids from same patient with certain disease,thereby realizing personalized medicine. |
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