Efficient Microscale Basic Reverse Phase Peptide Fractionation for Global and Targeted Proteomics

Analysis
of small biological samples would benefit from an efficient
microscale fractionation strategy that minimizes sample handling,
transfer steps, and accompanying losses. Here we describe a microscale
basic reverse phase liquid chromatographic (bRPLC) fractionation method
that offers high reproducibility and efficiency for peptide mixtures
from small (5–20 μg) samples. We applied our platform
to detect differentially expressed proteins from lung tumor cell lines
that are sensitive (11–18) and resistant (11–18R) to
the tyrosine kinase inhibitor erlotinib. Label-free analyses of 5–20
μg samples yielded identifications
of approximately 3,200 to 4,000 proteins with coefficients of variation
of 1.9–8.9% in replicate analyses. iTRAQ analyses produced
similar protein inventories. Label-free and iTRAQ analyses displayed
high concordance in identifications of proteins differentially expressed
in 11–18 and 11–18R cells. Micro-bRPLC fractionation
of cell proteomes increased sensitivity by an average of 4.5-fold
in targeted quantitation using parallel reaction monitoring for three
representative receptor tyrosine kinases (EGFR, PDGFRA, and BMX),
which are present at low abundance in 11–18 and 11–18R
cells. These data illustrate the broad utility of micro-bRPLC fractionation
for global and targeted proteomic analyses. Data are available through
Proteome eXchange Accession PXD003604.