title: Efficient Microscale Basic Reverse Phase Peptide Fractionation for Global and Targeted Proteomics.
abstract: 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.
authors.affiliation: Department of Biochemistry, Vanderbilt University School of Medicine , 607 Light Hall, 2215 Garland Avenue, Nashville, Tennessee 37232-0146, United States.
authors.forename: Hyoung-Joo
authors.initials: HJ
authors.lastname: Lee
authors.affiliation: Department of Biochemistry, Vanderbilt University School of Medicine , 607 Light Hall, 2215 Garland Avenue, Nashville, Tennessee 37232-0146, United States.
authors.forename: Hye-Jung
authors.initials: HJ
authors.lastname: Kim
authors.affiliation: Department of Biochemistry, Vanderbilt University School of Medicine , 607 Light Hall, 2215 Garland Avenue, Nashville, Tennessee 37232-0146, United States.
authors.forename: Daniel C
authors.initials: DC
authors.lastname: Liebler
mesh_terms: D045744:Cell Line, Tumor; D005591:Chemical Fractionation; D056148:Chromatography, Reverse-Phase / Q000379:methods / Q000592:standards*; D004351:Drug Resistance; D000069347:Erlotinib Hydrochloride / Q000494:pharmacology; D015870:Gene Expression / Q000187:drug effects; D006801:Humans; D008175:Lung Neoplasms / Q000188:drug therapy / Q000473:pathology; D008904:Miniaturization; D009363:Neoplasm Proteins / Q000032:analysis; D020543:Proteome / Q000187:drug effects*; D040901:Proteomics / Q000379:methods*
chemical_list: D009363:Neoplasm Proteins; D020543:Proteome; D000069347:Erlotinib Hydrochloride
keywords: basic reverse-phase liquid chromatography; drug resistance; iTRAQ; label-free quantitation; parallel reaction monitoring; protein tyrosine kinase