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.