Microscale Reversed-Phase Liquid Chromatography/Capillary Zone Electrophoresis-Tandem Mass Spectrometry for Deep and Highly Sensitive Bottom-Up Proteomics: Identification of 7500 Proteins with Five Micrograms of an MCF7 Proteome Digest.

Capillary zone electrophoresis-tandem mass spectrometry (CZE-MS/MS) has been well recognized for bottom-up proteomics. It has approached 4000-8000 protein identifications (IDs) from a human cell line, mouse brains, or Xenopus embryos via coupling with liquid chromatography (LC) prefractionation. However, at least 500 μg of complex proteome digests were required for the LC/CZE-MS/MS studies. This requirement of a large amount of initial peptide material impedes the application of CZE-MS/MS for deep bottom-up proteomics of mass-limited samples. In this work, we coupled microscale reversed-phase LC (μRPLC)-based peptide prefractionation to dynamic pH-junction-based CZE-MS/MS for deep bottom-up proteomics of the MCF7 breast cancer cell proteome starting with only 5 μg of peptides. The dynamic pH-junction-based CZE enabled a 500 nL sample injection from as low as a 1.5 μL peptide sample, using up to 33% of the available peptide material for an analysis. Two kinds of μRPLC prefractionation were investigated, C18 ZipTip and nanoflow RPLC. C18 ZipTip/CZE-MS/MS identified 4453 proteins from 5 μg of the MCF7 proteome digest and showed good qualitative and quantitative reproducibility. Nanoflow RPLC/CZE-MS/MS produced over 7500 protein IDs and nearly 60 000 peptide IDs from the 5 μg of MCF7 proteome digest. The nanoflow RPLC/CZE-MS/MS platform reduced the required amount of complex proteome digests for LC/CZE-MS/MS-based deep bottom-up proteomics by 2 orders of magnitude. Our work provides the proteomics community with a powerful tool for deep and highly sensitive proteomics.