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Related Experiment Video

Updated: May 24, 2026

Computational Prediction of Amino Acid Preferences of Potentially Multispecific Peptide-Binding Domains Involved in Protein-Protein Interactions

Computational Prediction of Amino Acid Preferences of Potentially Multispecific Peptide-Binding Domains Involved in Protein-Protein Interactions

Published on: January 26, 2024

Model studies on iTRAQ modification of peptides: sequence-dependent reaction specificity.

John E Wiktorowicz¹, Robert D English, Zheng Wu

¹Department of Biochemistry and Molecular Biology, The University of Texas Medical Branch , Galveston, Texas 77555, United States.

Journal of Proteome Research

|February 17, 2012

Summary

The iTRAQ reagent can cause unintended peptide modifications, specifically O-acylation, which impacts mass spectrometry (MS) quantification accuracy. Reversing these modifications with hydroxylamine is recommended for reliable iTRAQ-based peptide measurements.

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Last Updated: May 24, 2026

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Optimization of Synthetic Proteins: Identification of Interpositional Dependencies Indicating Structurally and/or Functionally Linked Residues

Optimization of Synthetic Proteins: Identification of Interpositional Dependencies Indicating Structurally and/or Functionally Linked Residues

Published on: July 14, 2015

Area of Science:

Proteomics
Analytical Chemistry
Biochemistry

Background:

The iTRAQ (isobaric Tandem Mass Tag) reagent enables multiplexed peptide quantification for relative protein measurements.
Validating the chemical specificity of iTRAQ reactions is crucial for accurate proteomic analyses.
Previous studies have focused on the general reactivity of iTRAQ, but sequence-specific modifications require further investigation.

Purpose of the Study:

To investigate the chemical specificity of the iTRAQ reagent, particularly its reactivity with synthetic peptides.
To identify sequence-dependent O-acylation modifications of hydroxyl-containing residues (serine, threonine, tyrosine) by iTRAQ.
To assess the impact of these modifications on mass spectrometry (MS) quantification and explore methods for their reversal.

Main Methods:

Synthesized two sets of peptides to study iTRAQ reactivity with varying amino acid sequences.
Investigated O-acylation of hydroxyl-containing residues, focusing on proximity to histidyl residues.
Utilized liquid chromatography-tandem mass spectrometry (LC-MS/MS) with retention time targeting in complex protein extracts.
Performed experiments using hydroxylamine to reverse iTRAQ-induced O-acylation.

Main Results:

iTRAQ reagent exhibited variable O-acylation of non-sequence-specific hydroxyl residues.
Significant sequence-specific O-acylation occurred at seryl, threonyl, and tyrosyl hydroxyls adjacent to histidyl residues.
These sequence-dependent modifications were confirmed in complex protein mixtures using LC-MS/MS.
Hydroxylamine effectively reversed iTRAQ-induced O-acylation with minimal N-acylation cleavage.

Conclusions:

Sequence-dependent O-acylation by the iTRAQ reagent can interfere with accurate MS quantification.
Understanding and mitigating these specific chemical modifications is essential for reliable proteomic data.
Reversal of O-acylation using hydroxylamine is a valuable modification to the iTRAQ protocol for improved quantification accuracy.