qualitative analysis,spiking quality control peptides,and creating in-depth spectral libraries for each proteome used in the experiment.Robust and reproducible chromatographic separation using a 60-minute capillary flow gradient enables high-throughput analysis.Besides setting FDR at 1%,a roll-up statistic strategy was applied to improve the quantitation precision.Robust and straightforward SOPs are created for the HRMS1-DIA workflow,to define the breadth of instrumental aspects such as chromatographic retention time stability,ionization spray stability,and product ion distribution overlap with a common spectral library.The study was benchmarked across multiple Cancer Moonshot sites worldwide utilizing identical instrument platforms,procedures,and software,and demonstrated to be stable in a 24/7 operation mode for 7 consecutive days.To ensure the reliability of the results,a QC sample is defined and routinely applied in the study.The resulting data were processed individually and combined to evaluate proteome coverage and quantitative capabilities.Reported metrics used to evaluate workflow performance include sub-proteome coverage and differential expression analysis,inter-days data reproducibility,as well as comparative data overlap among all different laboratories.In our initial data,at 1%FDR,> 5,000 proteins from > 40,000 peptides of the QC sample,as well as > 7,000 proteins from > 50,000 peptides of the mixed proteome sample,are consistently detected and reliably quantified across all site.The ratios of the mixed three proteomes accurately reflect the expected values.