ESTIMATION OF RELATIVE PROTEIN ABUNDANCE AND STATISTICAL INFERENCE FROM MULTIPLE iTRAQ EXPERIMENTS WITHOUT USING MASTERPOOLS
Abstract
Isobaric tags for relative and absolute quantitation (iTRAQ) is a prominent mass spectrometry technology for protein identification and quantification, capable of analyzing multiple samples in a single experiment. Frequently, iTRAQ experiments are carried out using a ``masterpool'' in one of the channels, a reference sample used to estimate protein relative abundances in the biological samples, and to combine abundance estimates from multiple experiments. In this manuscript we show that using such a masterpool is counterproductive. Besides occupying a channel that can be used for an additional biological sample, we demonstrate that more precise estimates of protein relative abundance can be derived by using the available biological data instead of the masterpool. In addition, we introduce a simple statistical method to associate proteomic data from multiple iTRAQ experiments with a numeric response, and show that this approach is more powerful than the conventionally employed masterpool based approach. We illustrate our methods using data from replicate iTRAQ experiments on samples of pooled plasma, and from a large project carried out to detect proteins in circulation that covary with nutrient concentrations measured in a young, chronically undernourished child population in South Asia.