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Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification
Published on: November 15, 2017
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Protocol for high-throughput semi-automated label-free- or TMT-based phosphoproteome profiling
Claire Koenig1, Ana Martinez-Val1, Previn Naicker2
1Novo Nordisk Foundation Center for Protein Research, Copenhagen, Denmark.
STAR Protocols
|September 2, 2023
Summary
We present a modular pipeline for Tandem Mass Tags data-dependent acquisition (TMT-DDA) and label-free quantification data-independent acquisition (LFQ-DIA) to improve phosphoproteome profiling. This guide helps researchers choose the best workflow for their needs.
Area of Science:
- Proteomics
- Mass Spectrometry
- Biochemistry
Background:
- Tandem Mass Tags data-dependent acquisition (TMT-DDA) and label-free quantification data-independent acquisition (LFQ-DIA) are leading workflows for deep proteome and phosphoproteome profiling.
- Scalable and efficient methods are crucial for comprehensive phosphoproteome analysis.
Purpose of the Study:
- To present a modular pipeline for TMT-DDA and LFQ-DIA workflows.
- To provide guidance on selecting the optimal workflow based on sample characteristics and experimental goals.
Main Methods:
- The pipeline integrates protein lysate extraction, clean-up, digestion, and phosphopeptide enrichment.
- It includes TMT-labeling, peptide/phosphopeptide fractionation, and pre-mass spectrometry desalting steps.
Main Results:
- The presented pipeline enables scalable phosphoproteome profiling using both TMT-DDA and LFQ-DIA.
- Detailed protocols and workflow selection guidance are provided.
Conclusions:
- This modular pipeline offers a flexible and scalable approach to phosphoproteome profiling.
- Researchers can optimize their experimental design by choosing the most suitable workflow (TMT-DDA or LFQ-DIA) based on their specific needs.
