Both isobaric tags for relative and absolute quantitation (iTRAQ) and label-free methods are widely used for quantitative proteomics. Here, we provide a detailed evaluation of these proteomics approaches based on large datasets from biological samples. iTRAQ-label-based and label-free quantitations were compared using protein lysate samples from noninfected human lung epithelial A549 cells and from cells infected for 24？h with human adenovirus type 3 or type 5. Either iTRAQ-label-based or label-free methods were used, and the resulting samples were analyzed by liquid chromatography (LC) and tandem mass spectrometry (MS/MS). To reduce a possible bias from quantitation software, we applied several software packages for each procedure. ProteinPilot and Scaffold Q+ software were used for iTRAQ-labeled samples, while Progenesis LC-MS and ProgenesisF-T2PQ/T3PQ were employed for label-free analyses. R2 correlation coefficients correlated well between two software packages applied to the same datasets with values between 0.48 and 0.78 for iTRAQ-label-based quantitations and 0.5 and 0.86 for label-free quantitations. Analyses of label-free samples showed higher levels of protein up- or downregulation in comparison to iTRAQ-labeled samples. The concentration differences were further evaluated by Western blotting for four downregulated proteins. These data suggested that the label-free method was more accurate than the iTRAQ method. 1. Introduction Quantitative proteomics based on mass spectrometry (MS) is an important methodology for biological and clinical research allowing, for example, the identification of functional modules and pathways, or the monitoring of disease biomarkers [1, 2]. Relative quantitation of two or more samples for studies of differential protein expression is of particular importance. Quantitative results can be gained using stable isotope labels or label-free methods [3–5]. In general, isotope labels offer higher reproducibility in quantitation, and label-free methods require highly reproducible LC-MS/MS platforms [3]. Several labeling methods based on heavy isotopes such as 2H, 13C, 15N, and 18O have been developed and allow relative quantitation using MS. In vivo metabolic labeling methods such as stable isotope labeling by amino acids in cell culture (SILAC) were introduced for arginine [6], lysine [7], tyrosine [8], or leucine [9]. For direct labeling of proteins or peptides, two strategies are being generally used. Isotope coded affinity tag (ICAT) labeling allows enrichment and MS analysis of cysteine-containing peptides [10]. iTRAQ
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