Optimizing and integrating depletion and precipitation methods for plasma proteomics through data-independent acquisition-mass spectrometry

The application of plasma proteomics is a reliable approach for the discovery of biomarkers. However, the utilization of mass spectrometry-based proteomics in plasma encounters limitations due to the presence of highabundant proteins (HAPs) and the vast dynamic range. To address this issue, we conducted an optimization and integration of depletion and precipitation strategies eliminating interference from HAPs. The optimized procedure involved utilizing 40 mu L of beads for the removal of 1 mu L of plasma, and maintaining a ratio of 1:1:1 between plasma, urea, and trichloroacetic acid for the precipitation of 50 mu L of plasma. To facilitate highthroughput processing, experimental procedures were carried out utilizing 96 -well plates. The depletion method identified a total of 1510 proteins, whereas the precipitated method yielded a total of 802 proteins. The integration of these methods yielded a total of 1794 proteins, including a wide concentration range spanning over 8 orders of magnitude. Furthermore, these approaches exhibited a commendable level of reproducibility, as indicated by median coefficients of variation of 14.7 % and 21.1 % for protein intensities, respectively. The integrative method was found to be effective in precisely quantifying yeast proteins that were intentionally spiked in plasma at predetermined rations of 5, 2, 0.5, and 0.2 with a high genuine positive recovery with a range of 71 % to 91 % of all yeast proteins. The use of a complementary and finely tuned approach involving depletion and precipitation demonstrates tremendous potential in the field of discovering protein biomarkers from largescale cohort studies.