Updated project metadata. In this study, we systematically optimized the micro-flow LC-MS/MS system based ssDDA proteomics. Our results show that combination of 2 – 3 h gradient with prosit rescore method identified the highest number of proteins and peptides. There is no benefit to increase column length. A single 3 h run identified about 9,200 proteins from 50 µg of Arabidopsis protein digest. The micro-flow LC-MS/MS system based ssDDA proteomics can be widely used for routine proteomics analysis of large cohort samples.