iTRAQ-Based and Label-Free Proteomics Approaches for Studies of Human Adenovirus Infections

الملخص EN

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.