An Efficient Approach to Evaluate Reporter Ion Behavior from MALDI-MS/MS Data for Quantification Studies Using Isobaric Tags

Stephanie M. Cologna, Christopher A. Crutchfield, Brian C. Searle, Paul S. Blank, Cynthia L. Toth, Alexa M. Ely, Jaqueline A. Picache, Peter S. Backlund, Christopher A. Wassif, Forbes D. Porter, Alfred L. Yergey*

*Corresponding author for this work

Research output: Contribution to journalArticlepeer-review

2 Scopus citations


Protein quantification, identification, and abundance determination are important aspects of proteome characterization and are crucial in understanding biological mechanisms and human diseases. Different strategies are available to quantify proteins using mass spectrometric detection, and most are performed at the peptide level and include both targeted and untargeted methodologies. Discovery-based or untargeted approaches oftentimes use covalent tagging strategies (i.e., iTRAQ, TMT), where reporter ion signals collected in the tandem MS experiment are used for quantification. Herein we investigate the behavior of the iTRAQ 8-plex chemistry using MALDI-TOF/TOF instrumentation. The experimental design and data analysis approach described is simple and straightforward, which allows researchers to optimize data collection and proper analysis within a laboratory. iTRAQ reporter ion signals were normalized within each spectrum to remove peptide biases. An advantage of this approach is that missing reporter ion values can be accepted for purposes of protein identification and quantification without the need for ANOVA analysis. We investigate the distribution of reporter ion peak areas in an equimolar system and a mock biological system and provide recommendations for establishing fold-change cutoff values at the peptide level for iTRAQ data sets. These data provide a unique data set available to the community for informatics training and analysis.

Original languageEnglish (US)
Pages (from-to)4169-4178
Number of pages10
JournalJournal of Proteome Research
Issue number10
StatePublished - Oct 2 2015
Externally publishedYes


  • fold-change
  • iTRAQ
  • quality control
  • quantitative proteomics
  • time-of-flight

ASJC Scopus subject areas

  • Biochemistry
  • Chemistry(all)


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