Toward Biomarker Development in Large Clinical Cohorts: An Integrated High-Throughput 96-Well-Plate-Based Sample...

We describe a cheap, robust, fast, high-throughput, and flexible proteomic sample processing method based on a regular 96-well plate by acetone precipitation under low centrifuge speed (96PACS), which enables predigestion processing of 96 samples within 2 h. Tested on a complex Huh-7 total lysate, 96PACS produced comparable proteome coverage and even showed better reproducibility than FASP. Quantitative performance of 96PACS was further tested using data-independent acquisition and parallel reaction monitoring quantitation in a set of 6 benchmark samples consisting of 6 serial dilutions of BSA spiked in complex E. coli proteome background. The protocol was also successfully modified for automation and was validated in a comparative label-free proteomic study to develop serum markers for early detection of liver fibrosis and necroinflammation in patients chronically infected with hepatitis B virus.