| Purpose:MALDI-TOF-MS(matrix-assisted laser desorption ionization time-of-flight mass spectrometry)was used to study the differential serum polypeptide profiles between bladder cancer patients and healthy people,to search for potential serum polypeptide biomarkers for bladder cancer and to establish a highly sensitive and specific serological diagnostic model for early bladder cancer,so as to provide important reference for clinical diagnosis of early bladder cancer.Methods:(1)Serum samples of 67 patients with bladder cancer,64 healthy persons and 30 patients with hematuria were collected.All bladder cancer patients and healthy persons were randomly divided into training set(32 patients with bladder cancer and 31volunteers)and validation set 1(35 patients with bladder cancer and 33 volunteers).Serum peptides were enriched and extracted by weak cation exchange magnetic beads(WCX-MB).Serum polypeptide profiles of each sample were detected by MALDI-TOF-MS technique.BioExplorer software was used to analyze and compare the profiles of bladder cancer group and healthy volunteers group in the training set,and to find the different polypeptide peaks.(2)ROC curve analysis(Receiver operating characteristics curve analysis)wasused to evaluate the diagnostic value of different polypeptides,and the internal algorithm of BioExplorer software was used to construct the serum polypeptide diagnostic model for bladder cancer.The sensitivity and specificity of the model for different stages of bladder cancer were calculated.(3)The sensitivity and specificity of the model were verified by the serum samples of 35 bladder cancer patients and 33 healthy controls in validation set 1.Serum samples from 30 hematuria patients were used as another validation set(validation set 2)to verify the specificity of the model.(4)39 patients with early bladder cancer were further divided into early low-grade bladder cancer group(20 cases)and early high-grade bladder cancer group(19 cases).They were applied to the diagnosis model for classification and compared with healthy volunteers.The sensitivity and specificity of the model for the diagnosis of early bladder cancer with different grades were obtained.(5)The expression of carcinoembryonic antigen(CEA)in serum of early bladder cancer,healthy volunteers and hematuria patients was detected by automatic chemiluminescence immunoassay.The diagnostic ability of CEA in early bladder cancer was analyzed by ROC curve.(6)Using nano-LC-ESI-MS/MS to identify the differential serum polypeptide sequences in the training set,and further identified the differential peptides according to the sequence database.(7)Enzyme-linked immunosorbent assay(ELISA)was used to detect the expression of proteins in serum of patients with bladder cancer and healthy volunteers,and to compare and analyze the differences between the two groups.Results:(1)By comparing and analyzing the serum polypeptide profiles of bladder cancer patients and healthy volunteers in the training set,50 polypeptide peaks were obtained,of which 17 were statistically different(P<0.05).The intensity of 13 polypeptide peaks were up-regulated in bladder cancer group,and 4 were down-regulated in bladder cancer group.(2)According to the intensity of the peak and the difference between the two groups,we screened five polypeptides(m/z 1954.9Da,m/z 2081.0Da,m/z 3938.3Da,m/z 3946.5Da and m/z 4268.8Da).Compared with the healthy volunteers group and hematuria group,the peak intensity of the five polypeptide peaks in the bladder cancer group was higher than that in the control group,and the expression in the early bladder cancer patients was also higher than that in the control group.There was significant difference(P<0.05).The ROC curves showed that the AUC values of each polypeptide peak were 0.778(95% CI: 0.659-0.898,P<0.001),0.862(95% CI: 0.768-0.955,P<0.001),0.859(95% CI: 0.769-0.950,P<0.001),0.837(95% CI: 0.741-0.933,P<0.001)and 0.787(95% CI: 0.675-0.900,P<0.001),respectively.The AUC value of five polypeptide peaks was 0.923(95% CI: 0.857-0.990,P<0.001).Finally,the diagnostic model of bladder cancer was constructed with these five polypeptides.The diagnostic sensitivity and specificity were 93.75% and 96.77%,respectively.The sensitivity and specificity of the model for early bladder cancer(Ta stage)were 92.31% and 93.75%.The sensitivity and specificity for the diagnosis of invasive T1-T4 stage of bladder cancer were 85.71% and 93.75%.(3)The sensitivity and specificity of validation set 1 were 91.43% and 90.91%.The diagnostic specificity of validation set 2 was 73.33%.(4)The sensitivity of five-peptide diagnostic model for early low-grade bladder cancer was 90%,and for early high-grade bladder cancer was 94.74%.ROC curve analysis showed that the AUC value of this model was 0.944(95% CI: 0.871-1.000,P<0.001)and 0.963(95%CI: 0.916-1.000,P<0.001)for the diagnosis of early and high-grade bladder cancer,respectively.(5)The average serum CEA concentration in patients with early bladder cancer was2.53±1.44ng/ml,2.21±1.50ng/ml in healthy volunteers and 2.32±0.99ng/ml in patients with hematuria.The CEA level of patients with early bladder cancer and healthy persons were analyzed.The AUC value was 0.593(95% CI: 0.458-0.727),P =0.174,there was no statistical difference between the two groups.The CEA level of early bladder cancer patients and hematuria patients were analyzed.The AUC value was0.529(95% CI: 0.391-0.666),P = 0.685,there was no significant difference between the two groups.(6)In the model,2 peptides from 5 serum peptides were identified as fetoprotein A(alpha-2-HS-glycoprotein,AHSG)and serum transferrin by nano-LC-ESI-MS/ MS.(7)The serum levels of AHSG and transferrin were detected by ELISA in all patients with bladder cancer.The serum levels of AHSG and transferrin were significantly higher than those of healthy volunteers(P<0.05).Conclusions:(1)The MALDI-TOF-MS technique based on WCX-MB was able to analyze the small polypeptide in complex serum samples,and screen the different polypeptide peaks between bladder cancer patients and healthy people by comparing the profiles,a serological diagnosis model of bladder cancer containing five polypeptides was established by MALDI-TOF-MS technology.This model had high sensitivity and specificity in diagnosis of bladder cancer.(2)The model had obvious advantages in the diagnosis of early bladder cancer,with a sensitivity of 92.31%,even early low-grade bladder cancer had a high sensitivity,and could be differentiated from healthy people and patients with hematuria symptoms.Therefore,this model could be used for early screening of bladder cancer,and had important significance for early diagnosis of bladder cancer.(3)Two peptides from 5 serum peptides were identified as fetoprotein A(alpha-2-HS-glycoprotein,AHSG)and serum transferrin by nano-LC-ESI-MS/MS.And they were both highly expressed in the serum of bladder cancer patients,these two proteins may be involved in the proliferation,invasion and metastasis of bladder cancer,which provided a new way to further study the pathogenesis of bladder cancer. |
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