Updated publication reference for PubMed record(s): 31610113. Novel mass spectrometry (MS)-based proteomic tools with extremely high sensitivity and high peak capacity are required for comprehensive characterization of protein molecules in mass-limited proteome samples. We previously reported a nanoflow RPLC-CZE-MS/MS (nanoRPLC-CZE-MS/MS) system for deep bottom-up proteomics of low micrograms of human cell samples (Yang et al. Anal. Chem. 2018, 90, 10479-10486). In this work, we further improved the sensitivity of the nanoRPLC-CZE-MS/MS system drastically via employing bovine serum albumin (BSA) treated sample vials, improving the nanoRPLC fraction collection procedure, and using a shorter capillary for fast CZE separation. The improved nanoRPLC-CZE produced a high peak capacity of 8500 for peptide separation. The improved system identified 6500 proteins from a MCF7 proteome digest starting with only 500-ng peptides using a Q-Exactive HF mass spectrometer. The system actually consumed roughly 100-ng peptides. The improved system produced a comparable number of protein identifications (IDs) to our previous system with 10-fold less sample consumption. The improved system is also comparable to the two-dimensional (2D) nanoRPLC-MS/MS system developed by the Mann’s group regarding the number of protein IDs starting with 500-ng human cell proteome digests but with 4-fold lower sample consumption. We further coupled single spot solid phase sample preparation (SP3) method to the improved nanoRPLC-CZE-MS/MS for bottom-up proteomics of 5000 HEK293T cells, resulting in 3689 protein IDs with the consumption of a peptide amount that corresponded to only roughly 1000 cells. This work represents the first study of a small number of human cells using CZE-MS/MS.