Cost-effective isobaric tagging for quantitative phosphoproteomics using DiART reagents

Area of Science:

• Proteomics
• Biochemistry
• Analytical Chemistry

Background:

• Quantitative phosphoproteomics is crucial for understanding cellular signaling pathways.
• Existing methods for phosphopeptide enrichment and quantification can be costly and complex.

Purpose of the Study:

• To introduce and validate a novel isobaric tagging reagent, Deuterium isobaric Amine Reactive Tag (DiART), for quantitative phosphoproteomics.
• To assess the compatibility of DiART with titanium dioxide (TiO2) affinity purification.
• To demonstrate the accuracy and cost-effectiveness of DiART for large-scale phosphopeptide analysis.

Main Methods:

• Utilized DiART for isobaric tagging of phosphorylated and non-phosphorylated peptides.
• Employed TiO2 affinity purification for phosphopeptide enrichment.
• Analyzed peptide quantification using mass spectrometry and reporter ion ratios.
• Applied the DiART-TiO2 method to protein lysates from Aspergillus nidulans.

Main Results:

• Demonstrated compatibility of DiART with TiO2 enrichment for phosphorylated peptides.
• Achieved accurate quantification of phosphorylated peptides over a 15-fold dynamic range.
• Successfully quantified 744 unique phosphopeptides from 8 mg of Aspergillus nidulans cell lysate.
• Validated accurate quantification trends through comparison with theoretical values.

Conclusions:

• DiART is a feasible and accurate reagent for quantitative phosphoproteomic experiments.
• The DiART-TiO2 workflow offers a cost-effective approach for large-scale phosphopeptide identification and quantification.
• This method advances the study of phosphoproteomes in various biological systems.