Development of Chemical Labeling Metabolomics and Siscapa for Measurement of Minor Bladder Cancer-Associated Biomarkers

Base on the high morbidity and mortality, diagnosis and treatment of bladder cancer is an important issue for the better clinical practice. Taking the advantage of non-invasive sampling, urine is a good source for studies of urological diseases. In this project, we will focus on the development of mass spectrometry (MS)-based platforms to detect preciously-discovered biomarker candidates of bladder cancer, particularly focus on the minor bladder cancer-associated protein biomarkers. We will also develop the chemical labeling reaction for multiple functional groups for comparative metabolomics of bladder cancer urine. Overall, the project will separate into targeted proteomic and metabolomic profiling parts. Targeted proteomic part  To discover specific biomarkers from tissue proteome, recently we employed a strategy combining laser capture microdissection, iTRAQ labeling, and LC-MS/MS analysis to profile proteomic changes in fresh-frozen bladder tumor tissue specimens. 130 proteins were up-expressed in bladder tumor tissue cells of four patients. Only seven differentially expressed proteins were selected as potential bladder cancer biomarkers for further verification. To systematically verify the remaining discovered tissue-original biomarker candidates in urine as non-invasive biomarkers, a targeted and sensitive MS-based multiplexed detection platform, multiple-reaction-monitoring (MRM), will be developed in this project. For those low abundant proteins with poor sensitivity due to limited loading amount by LC-MRM-MS, Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) assay will be developed to quantify low abundant urinary proteins which have important roles in cancer biology or clinical utility. To the best of our knowledge, urinary proteins have not been detected by SISCAPA assay before. In the metabolomics part  we have previously published the amine- and phenol-containing sub-metabolome of bladder cancer urine. 75.2% of urinary metabolites which downloaded from the HMDB website have hydroxyl group. Therefore, hydroxyl group (-OH) is the next functional group that I plan to further analyze using modified dansylated labeling protocols to get a hydroxyl submetabolome of urine and for integration with amine/phenol urine sunmetabolome. Amine and phenol are the most active functional groups for dansylation reaction. With the modifications in reaction conditions, dansylation can be used to label on unactive-alcohol containing metabolites, enriched hydroxyl-containing metabolites, and even carboxylic acid. In this project, I plan to test several dansylation protocols and evaluate the spectra complexity, quantification accuracy, metabolite identification rate after database search, consumption volume of samples, reaction efficiency and, interference from untargeted functional groups, bioinformatics analysis to decide the most suitable workflow to obtain the combination of amine, phenol, and hydroxyl containing metabolome for a more comprehensive bladder cancer urine metabolome. In summary, this project will focus on the development of analytical technology for important low-abundant proteins and metabolites detection. According to quantification results of the higher abundant urinary proteins by MRM-MS, lower abundant urinary proteins by SISCAPA, and novel hydroxyl-associated metabolites by chemical labeling metabolomics, we will use the bioinformatic and statistic tools to generate a biomarker panel for differentiation of bladder cancer from control individuals non-invasively.

Project ID:PA10701-0465
External Project ID:MOST106-2113-M182-001-MY2