Capillary isoelectric focusing-tandem mass spectrometry and reversed-phase liquid chromatography-tandem mass spectrometry for quantitative proteomic analysis of differentiating PC12 cells by eight-plex isobaric tags for relative and absolute quantification.

ABSTRACT: We report the application of capillary isoelectric focusing for quantitative analysis of a complex proteome. Biological duplicates were generated from PC12 cells at days 0, 3, 7, and 12 following treatment with nerve growth factor. These biological duplicates were digested with trypsin, labeled using eight-plex isobaric tags for relative and absolute quantification (iTRAQ) chemistry, and pooled. The pooled peptides were separated into 25 fractions using reversed-phase liquid chromatography (RPLC). Technical duplicates of each fraction were separated by capillary isoelectric focusing (cIEF) using a set of amino acids as ampholytes. The cIEF column was interfaced to an Orbitrap Velos mass spectrometer with an electrokinetically pumped sheath-flow nanospray interface. This HPLC-cIEF-electrosp

SUBMITTER: Zhu G 

PROVIDER: S-EPMC3759156 | biostudies-literature | 2013 Aug

REPOSITORIES: biostudies-literature