This paper describes and compares two methods for estimating the variance function associated with iTRAQ (isobaric tag for relative and absolute quantitation) isotopic labeling in quantitative mass spectrometry based proteomics. Measurements generated by the mass spectrometer are proportional to the concentration of peptides present in the biological sample. However, the iTRAQ reporter signals are subject to errors that depend on the peptide amounts. The variance function of the errors is therefore an essential parameter for evaluating the results, but estimating it is complicated, as the number of nuisance parameters increases with sample size while the number of replicates for each peptide remains small. Two experiments that were conducted with the sole goal of estimating the variance function and its stability over time are analyzed, and the resulting estimated variance function is used to analyze an experiment targeting aberrant signaling cascades in cells harboring distinct oncogenic mutations. Methods for constructing conservative p-values and confidence intervals are discussed.
- Mixture model
- Nuisance parameter