PNNL

Abstract

The NCI Clinical Proteomic Tumor Analysis Consortium (CPTAC) employed a pair of reference xenograft proteomes for initial platform validation and ongoing quality control of its data collection for The Cancer Genome Atlas (TCGA) tumors. These two xenografts, representing basal and luminal-B human breast cancer, were fractionated and analyzed on six mass spectrometers in a total of 46 replicates divided between iTRAQ and label-free technologies, spanning a total of 1095 LC-MS/MS experiments. These data represent a unique opportunity to evaluate the stability of proteomic differentiation by mass spectrometry over many months of time for individual instruments or across instruments running dissimilar workflows. We evaluated iTRAQ reporter ions, label-free spectral counts, and label-free extracted ion chromatograms as strategies for data interpretation. From these assessments we found that differential genes from a single replicate were confirmed by other replicates on the same instrument from 61-93% of the time. When comparing across different instruments and quantitative technologies, differential genes were reproduced by other data sets from 67-99% of the time. Projecting gene differences to biological pathways and networks increased the similarities. These overlaps send an encouraging message about the maturity of technologies for proteomic differentiation.