In-Line Sample Processing System with an Immobilized Trypsin-Packed Fused-Silica Capillary Tube for the Proteomic Analysis of a Small Number of Mammalian Cells

Omics analysis at
single-cell resolution has helped to demonstrate
the shaping of cellular heterogeneity on the basis of the expression
of various molecules. However, in-depth proteomic analysis of low-quantity
samples has remained challenging because of difficulties associated
with the measurement of large numbers of proteins by shotgun proteomics
using nanoflow liquid chromatography tandem mass spectrometry (nano-LC/MS/MS).
To meet such a demand, we developed a method called in-line sample
preparation for efficient cellular proteomics (ISPEC) in which cells
were captured, directly lysed, and digested with immobilized trypsin
within fused-silica capillaries. ISPEC minimized sample loss during
the sample preparation processes with a relatively small number of
mammalian cells (<1000 cells) and improved the stability and efficiency
of digestion by immobilized trypsin, compared to a conventional preparation
method. Using our optimized ISPEC method with nano-LC/MS/MS analysis,
we identified 1351, 351, and 60 proteins from 100 cells, 10 cells,
and single cells, respectively. The linear response of the signal
intensity of each peptide to the introduced cell number indicates
the quantitative recovery of the proteome from a very small number
of cells. Thus, our ISPEC strategy facilitates quantitative proteomic
analysis of small cell populations.