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An improved nanoflow RPLC-CZE-MS/MS system with high peak capacity and sensitivity for nanogram bottom-up proteomics.

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 samples. We reported a nanoRPLC-CZE-MS/MS system for deep bottom-up proteomics of low micrograms of human cell samples in previous work (Yang et al. Anal. Chem. 2018, 90, 10479-10486). In this work, we 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 short capillary for fast CZE separation. The improved nanoRPLC-CZE produced a 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 produced a comparable number of protein identifications (IDs) to our previous system and the two-dimensional (2D) nanoRPLC-MS/MS system developed by the Mann's group with 10-fold and 4-fold less sample consumption, respectively. We 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.