Updated project metadata. We report methods development for quantitative proteomic analysis of samples with limited number of cells. The improvements include suspension trapping (S-Trap) for sample processing, longer analytical column length (75cm), and a segmented data dependent acquisition approach. The modified approach was applied to the proteomic analysis of adult-derived mouse microglia and optimized to the cell count expected from mouse brain (~300K microglial cells). With the improved approach, over 4500 quantifiable proteins can be obtained (in biological triplicate) with an average CV of 10%.