Reproducibility of Differential Proteomic Technologies in CPTAC Fractionated Xenografts

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 (source code is available from http://homepages.uc.edu/~wang2x7/Research.htm). From these assessments, we found that differential genes from
a single replicate were confirmed by other replicates on the same
instrument from 61 to 93% of the time. When comparing across different
instruments and quantitative technologies, using multiple replicates,
differential genes were reproduced by other data sets from 67 to 99%
of the time. Projecting gene differences to biological pathways and
networks increased the degree of similarity. These overlaps send an
encouraging message about the maturity of technologies for proteomic
differentiation.