In this chapter, we describe some of the approaches we employ in the analysis of iTRAQ data in our group, with an emphasis on practical issues that can occur in larger multi-run projects. Our pipeline starts with a well-established iTRAQ workflow, makes use of protein level quantitation using ProteinPilot, and continues either via a global analysis in the presence of a common reference, or by identifying pairwise comparisons of interest and applying a method taking the protein ratios and protein ratio confidence measures into consideration. Additionally we describe what issues can occur in the more subtle scenarios involving composite databases in multi-run situations, and an approach applicable in that setting.
Keywords: Data processing; Mass spectrometry; Quantitative proteomics; Replication; iTRAQ.