Statistical inference from multiple iTRAQ experiments without using common reference standards

J Proteome Res. 2013 Feb 1;12(2):594-604. doi: 10.1021/pr300624g. Epub 2013 Jan 16.

Abstract

Isobaric tags for relative and absolute quantitation (iTRAQ) is a prominent mass spectrometry technology for protein identification and quantification that is capable of analyzing multiple samples in a single experiment. Frequently, iTRAQ experiments are carried out using an aliquot from a pool of all samples, or "masterpool", in one of the channels as a reference sample standard to estimate protein relative abundances in the biological samples and to combine abundance estimates from multiple experiments. In this manuscript, we show that using a masterpool is counterproductive. We obtain more precise estimates of protein relative abundance by using the available biological data instead of the masterpool and do not need to occupy a channel that could otherwise be used for another biological sample. 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 four replicate iTRAQ experiments on aliquots of the same pool of plasma samples and from a 406-sample project designed to identify plasma proteins that covary with nutrient concentrations in chronically undernourished children from South Asia.

Publication types

  • Research Support, Non-U.S. Gov't

MeSH terms

  • Blood Proteins / chemistry*
  • Calibration
  • Child
  • Child Nutrition Disorders / blood*
  • Chromatography, Liquid
  • Humans
  • Nepal
  • Peptide Fragments / analysis*
  • Proteomics
  • Reference Standards
  • Tandem Mass Spectrometry / methods
  • Tandem Mass Spectrometry / standards*
  • Tandem Mass Spectrometry / statistics & numerical data*
  • Trypsin / chemistry

Substances

  • Blood Proteins
  • Peptide Fragments
  • Trypsin