Quantitative Proteomic Analysis by iTRAQ

Isobaric tags for relative and absolute quantification (iTRAQ), a chemical labeling mass spectrometry method, has been performed following the manufacturer’s protocol (AB SCIEX, Framingham, MA, USA) [19 (link), 20 (link)]. Protein identification and relative quantification were carried out using ProteinPilot Software Version 4.5 (AB SCIEX) [21 (link)]. Function definitions of the variable protein contents were searched against the Swissport database (Release, 10/16/2013). Protein ratios were normalized using the overall median ratio for all the peptides in the sample for each separate ratio in every individual experiment. A confidence cutoff for protein identification > 95% was applied. The specific pathway alteration was identified by Metacore (GeneGo, St. Joseph, MI) or KEGG ontology analysis (Kyoto University, Japan) [22 (link)].