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Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification
Published on: November 15, 2017
iTRAQ-labeling of in-gel digested proteins for relative quantification
Carla Schmidt1, Henning Urlaub
1Bioanalytical Mass Spectrometry Group, Max Planck Institute for Biophysical Chemistry, Am Fassberg 11, 37077 Göttingen, Germany.
Methods in Molecular Biology (Clifton, N.J.)
|June 23, 2009
Summary
This study details a chemical labeling quantification strategy using Isobaric Tag for Relative and Absolute Quantitation (iTRAQ) reagents for in-gel digested proteins. This method is effective for analyzing moderately complex protein samples, such as tri-snRNPs.
Area of Science:
- Proteomics
- Analytical Chemistry
- Biochemistry
Background:
- Mass spectrometry (MS)-based protein quantification is increasingly used in proteomics.
- Stable isotope labeling (metabolic or chemical) is common, with chemical labeling gaining attention.
- Label-free quantification is emerging but not yet fully established.
Purpose of the Study:
- To present a comprehensive quantification strategy for in-gel digested proteins using Isobaric Tag for Relative and Absolute Quantitation (iTRAQ) reagents.
- To establish a robust workflow for chemical labeling-based protein quantification.
Main Methods:
- Protein separation via gel electrophoresis.
- In-gel digestion and peptide extraction.
- Peptide labeling with iTRAQ reagents.
- Sample pooling and LC-MS/MS analysis.
- Database searching for protein identification and quantification.
Main Results:
- The workflow successfully quantifies proteins in moderately complex samples (300-400 components).
- Demonstrated efficacy using varying amounts of 25S [U4/U6.U5] tri-snRNPs.
Conclusions:
- The presented iTRAQ-based workflow provides a reliable method for protein quantification.
- This strategy is suitable for comparative proteomic studies of moderate complexity.
