| Bladder cancer (BCa) is the most common cancer of the urinary system, and ranks at the first in the cancers related to urinary system in China. It is commonly accepted that early diagnosis and treatment of BCa are beneficial to the patients. Although the study related to BCa biomarkers has been extensively carried out, the biomarker application in clinical diagnosis is still limited. Exploration of new BCa biomarkers with high sensitivity and specificity is badly required in BCa study. Herein, we proposed a comprehensive approach that consisted of three key technique considerations for expedition the discovery and verification efficiency of BCa biomarkers.As bladder is an organ to collect urine, urine is thought of as an ideal material to study and examine the bladder cancer biomarkers. On the other hand, urine is a body fluid whose content is often controlled by diet and other physiological conditions, and contains several high abundance proteins. The efficiency of screening BCa biomarkers directly from urine was found unsatisfactory. In this study, we proposed to collect the secreted proteins from BCa cell lines and to define the BCa related proteins using proteomics analysis, and to further verify the candidates in the patient urine. The comprehensive strategy based on the secretion proteins from cells to the BCa biomarker from urine is anticipated to enhance the research efficiency and reduce the experimental difficulty. Proteomics techniques are currently in progress, and none of the techniques could fully address all the issues of proteomic investigation. Hence, we designed a strategy for searching the indicative proteins of BCa, in which 2DE was undertaken to isolate the intact proteins and RP-LC was used to separate the tryptic digestion peptides. How to accurately quantitate and evaluate the differential proteins is a key question of proteomics technique in exploration of the disease biomarker. To reach the goal, quantification derived from different techniques should be better applied on similar scale of physical parameters, and be processed quantitative and verification at large scale. In this study, we attempted to integrate quantitative profiling (2DE-MS/MS and iTRAQ) and targeted MRM technique for efficient screening and validation, respectively.We selected two BCa-related cell lines (5637 and T24) and a relative normal cell line (SV-HUC-1), which are well characterized and accepted in BCa study. The secreted proteins from the three cell lines were collected and undergone with two quantitative proteomics in 2DE which defined 72 and 79 differential proteins in 5637 and T24, repectively. It merits to noting that the overlapping rate of these differential proteins in two groups was low and many proteins displayed multiple spots on 2DE image, indicating that the two BCa cell lines secreted diverse proteins with severe modifications. According to the quantitative analysis by iTRAQ,218 and 535 differential proteins were defined in 5637 and T24. The abundance distribution of the secretomes in SV-HUC-1 was basically comparable with that in 5637, but was very different from that in T24. Importantly, the literature searching and bioinformatics analysis revealed that most of the differential secreted proteins could exist in blood or urine. Taking the complementary information together, we finally defined a total of 724 BCa-related secretion proteins in the two cell lines, which were considered as the candidates of BCa urine biomarkers.Next, we surveyed the MS/MS characteristics of the urine proteins using SWATH profiling technique, specifically focusing on these BCa candidates. A total of 94 such proteins were identified in urine by SWATH. With evaluation of theoretical prediction towards the peptides derived from the 94 candidates and the MS/MS spectra gained from SWATH,17 proteins were selected as the urine targets for further verification. MRM approach was employed to specifically detect the candidates in 47 urine samples (23 BCa patients and 24 health people). The MRM data were acquired with strict quality control with CV less than 20%. Upon statistical appraisement, a total of the 10 proteins whose abundances in the urines of BCa patients were found significantly different from those in the health ones. Of the potential indicators for bladder cancer, analysis of receiver operating characteristics (ROC) curve analysis further revealed the combination of complement component 3 (CO3)and lactose dehydrogenase B (LDHB) was more sensitive and efficient in distinguishing healthy and disease urine. |
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