A Personalized Mass Spectrometric-Map of the Disease-Associated Urinary Proteins for Biomarker Discovery and Verification of Urological Diseases

Protein biomarker discovery in urine has gained prominence. One of the major challenges in urine biomarker discovery is the high biological variation between individuals. Therefore, after the discovery phase in biomarker discovery pipeline, potential urinary biomarkers must be verified by quantitation of multiple urinary proteins in a large number of individual samples. However, commercially-available immunoassays are usually very costly, and multiplexing of Enzyme-linked immunosorbent assays may be limited due to cross-reactivity of the antibodies. The availability of specific antibodies against novel candidate proteins and time required for development of new ELISA assays create other bottlenecks in the biomarker pipeline. Mass spectrometry-based (MS) approaches in quantitative proteomics have become a powerful tool for the analysis of complex body fluid proteomes. Multiple-reaction-monitoring (MRM) or selected-reaction-monitoring (SRM) is an MS scanning mode involving two stages of mass analysis, that is commonly performed using triple quadrupole MS instruments. The protein is digested as peptides using a suitable enzyme, commonly as trypsin, to achieve a reasonable molecular range for sensitive detection by MS. The triple quadrupole instrument selects a narrow Q1 window around each target mass of precursor ion instead of scanning the entire mass range. This results in improved sensitivity over full-scan instruments. Instead of a full scan for Q3 to detect all of the fragments from the targeted precursor ion, Q3 only scans a narrow m/z window centered on each targeted fragment ion. Although urinary proteins seem to be promising biomarkers for diagnosis of several kinds of urological diseases including bladder cancer, kidney diseases, and other benign urological diseases. Some of the reported protein biomarker candidates are identified in multiple diseases which may result in poor specificity for disease diagnosis purpose. The detailed absolute concentration distributions and redundancies as biomarkers of these proteins among different disease conditions are not clear. Additionally, most of the published works focused on using blood-based clinical specimens for the development of MRM-MS platforms. The non-optimized MRM-MS platform may result inaccurate quantitation results of urinary proteins. In this project, we plan to optimize the pre-analytical sample preparation, and development of a multiplexed-protein-quantitation assay (number of disease-associated-proteins ＞100) using a MRM-mass spectrometric method. The analytical effects of protein digestion workflows, analytical column size, peptide loading capacity, LC flow rate, MS scan cycle time, selection using differential ion mobility, Q1/Q3 transitions, for quantification of disease-associated-urine proteins will be evaluated. Then, the optimized workflow will be used to quantify multiple disease-associated-protein targets in health volunteers and patients of eight types common urological diseases including healthy control, hernia, renal cell carcinoma, transitional cell carcinoma of kidney cancer, bladder cancer, prostate cancer, urinary tract infection, hematuria, and benign prostatic hyperplasia. Special attention will be also given to standardization methods to enable widespread use of LC-MRM-MS. We will evaluate this targeted protein quantitation technology for urinary biomarker verification. The data contains concentration, biological variation, sensitivity, and specificity of these 106 targeted urine proteins in multiple diseases. The protein concentrations will be used to construct a personalized mass spectrometric-map of the 106 disease-associated urinary proteins and disease marker panels. The result will provide benchmarks for individual laboratories to evaluate the performance of non-invasive biomarkers in body fluids.

Project ID:PA10301-0331
External Project ID:NSC102-2113-M182-001-MY2