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Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification
Published on: November 15, 2017
Comparison of protein expression ratios observed by sixplex and duplex TMT labeling method
Navin Rauniyar1, Benbo Gao, Daniel B McClatchy
1Department of Chemical Physiology, The Scripps Research Institute, 10550 North Torrey Pines Road, La Jolla, California 92037, USA.
Journal of Proteome Research
|December 11, 2012
Summary
Isobaric tandem mass tag (TMT) labeling enables protein quantification. Sixplex TMT analysis identified more significant protein expression changes in rat brain development than duplex TMT, validated by metabolic labeling.
Area of Science:
- Proteomics
- Biochemistry
- Mass Spectrometry
Background:
- Stable isotope labeling via isobaric derivatization of peptides is a key proteomics technique.
- Amine-reactive isobaric tandem mass tag (TMT) reagents facilitate concurrent protein identification and quantification across samples.
- TMT offers duplex and sixplex formats for comparative proteomic analyses.
Purpose of the Study:
- To evaluate the performance of duplex versus sixplex TMT reagents for quantifying protein expression changes.
- To compare the number of significantly altered proteins identified by each TMT format.
- To validate TMT-based findings using an independent metabolic labeling strategy.
Main Methods:
- Proteomic analysis of rat brain tissue at postnatal day 1 (p1) and day 45 (p45).
- Comparison of protein expression ratios (p45/p1) using triplicate duplex TMT analysis versus single sixplex TMT analysis.
- LC-MS/MS analysis for protein identification and quantification.
- Validation using (15)N metabolic labeling for relative protein quantification.
Main Results:
- A high correlation (0.79) was observed in relative protein abundance between duplex and sixplex TMT analyses.
- Sixplex TMT analysis identified a greater number of proteins with significant fold changes (p < 0.05, |log2(ratio)| ≥ 1) compared to duplex TMT.
- Most proteins with significant fold changes were quantified with high precision, supported by multiple spectra.
- TMT-derived fold changes were corroborated by (15)N metabolic labeling results.
Conclusions:
- Sixplex TMT reagents provide enhanced sensitivity for detecting protein expression changes compared to duplex TMT.
- Both duplex and sixplex TMT methods offer reliable protein quantification, especially for proteins identified by multiple spectra.
- TMT-based proteomic analysis, validated by metabolic labeling, is effective for studying developmental changes in complex biological systems like the rat brain.
