| Summary: | 碩士 === 長庚大學 === 生物醫學研究所 === 99 === Bladder cancer mortality increases annually in Taiwan. The main detection tool for bladder cancer is cystoscopy. However, this method is an invasive and costly procedure. Urine is stored in bladder for hours and can be collected non-invasively. Therefore, urine can be a good material for discovery of bladder cancer biomarkers. Multivesicular body derived exosomes present in saliva, plasma, and urine. Previous studies pointed out that exosomes correlate with diseases, and can be a material for biomarker searching. In this work, we compared the proteomic profiles of urinary exosomes between controls and bladder cancer patients using isotopic dimethyl labelling and two-dimensional LC/MS/MS. The differentially-expressed exosomal proteins will be potential non-invasive biomarkers for detection of bladder cancer.
Exosomes extracted from 9 hernia and 9 bladder cancer patients were pooled as a control and a bladder cancer samples, respectively. After digestion of exosomal proteins, peptides of hernia and bladder cancer samples were labelled with light and heavy dimethylation reagents, respectively. The labelled peptides were analyzed by on-line SCX/RP LC/MS/MS. The quantification of proteins were performed by MaxQuant software. We further used the dimethylation platform to compare the exosome proteins expressions of 9 hernia and 9 bladder cancer individuals without pooling samples.
We have indentified total 3873 exosomal proteins in two comparisons, and 107 proteins show higher different concentration levels between bladder cancer and hernia patients. 29 of 107 proteins in urinary exosomes could directly quantified by multiple-reaction-monitoring (MRM)-MS. MRM-MS validated 29 proteins expression levels between 5 hernia and 5 bladder cancer patients’ exosomes, and most results were consistent with that we have seen in discovery phase. Then we validated more clinical sample. We quantified 29 proteins expression levels in 12 hernia, 28 bladder cancer, 5 hematuria and 3 urinary tract infection patients’ exosomes. 22 of 29 proteins had significant differentially expressed level between hernia and bladder cancer patient, and higher expressed level of 3 proteins in bladder cancer patients were not due to hematuria or urinary tract infection.
In this work, we implemented two comparisons to searching differentially-expressed exosomal proteins, and we validated these proteins by MRM-MS. The validation results are consistent with prediction. In the future, we will increase the sample number to raise the reliability of these potential bladder cancer biomarkers.
|
|---|
