96DRA-Urine: A high throughput sample preparation method for urinary proteome analysis.

Mass spectrometry (MS)-based urinary proteomics is increasingly used for clinical research. A critical step in urinary proteomic analysis comprises the implementation of a reliable sample preparation method with high yields of peptides and proteins. In this study, we developed a urinary sample preparation method, DRA-Urine (Direct reduction/alkylation in urine), which urinary proteins were directly reduced/alkylated in urine, and then precipitated by acetone, washed and digestion on an ultrafilter unit. The qualitative and quantitative comparison of different urinary sample preparation methods (in-solution methods and ultrafilter-assisted methods) showed that DRA-Urine could achieve better results. Adapting DRA-Urine protocol to a 96-well format, namely 96DRA-Urine, shortened the time for buffer change and improved sample preparation throughput. The results showed that 96DRA-Urine displayed similar proteomic performance to DRA-Urine. Finally, the 96DRA-Urine method was used in a label-free, small pilot biomarker discovery analysis for differential urinary proteome analysisof bladder cancer urine. The results showed that urinary proteins could differentiate bladder cancer (BCa) patients from healthy controls and distinguish high-grade BCa from low-grade BCa with area under the curve (AUC) values of 0.972 and 0.847, respectively. Consequently, the 96DRA-Urine method might be a high-throughput method for preparing body fluid samples used in clinical research but needs to be further verified.