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Efficient Microscale Basic Reverse Phase Peptide Fractionation for Global and Targeted Proteomics
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DOI：10.1021/acs.jproteome.6b00102.png)
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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 mu g) 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 mu g 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.
Keyword:
basic reverse-phase liquid chromatography
drug resistance
label-free quantitation
iTRAQ
protein tyrosine kinase
parallel reaction monitoring
期刊
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3.6
论文数:
9.4K
被引数:
2.3W
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