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.