Carrier-Assisted Single-Tube Processing Approach for Targeted Proteomics Analysis of Low Numbers of Mammalian Cells

Heterogeneity
in composition is inherent in all cell populations,
even those containing a single cell type. Single-cell proteomics characterization
of cell heterogeneity is currently achieved by antibody-based technologies,
which are limited by the availability of high-quality antibodies.
Herein we report a simple, easily implemented, mass spectrometry (MS)-based
targeted proteomics approach, termed cLC–SRM (carrier-assisted
liquid chromatography coupled to selected reaction monitoring), for
reliable multiplexed quantification of proteins in low numbers of
mammalian cells. We combine a new single-tube digestion protocol to
process low numbers of cells with minimal loss together with sensitive
LC–SRM for protein quantification. This single-tube protocol
builds upon trifluoroethanol digestion and further minimizes sample
losses by tube pretreatment and the addition of carrier proteins.
We also optimized the denaturing temperature and trypsin concentration
to significantly improve digestion efficiency. cLC–SRM was
demonstrated to have sufficient sensitivity for reproducible detection
of most epidermal growth factor receptor (EGFR) pathway proteins expressed
at levels ≥30 000 and ≥3000 copies per cell for
10 and 100 mammalian cells, respectively. Thus, cLC–SRM enables
reliable quantification of low to moderately abundant proteins in
less than 100 cells and could be broadly useful for multiplexed quantification
of important proteins in small subpopulations of cells or in size-limited
clinical samples. Further improvements of this method could eventually
enable targeted single-cell proteomics when combined with either SRM
or other emerging ultrasensitive MS detection.