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TMT Sample Preparation for Proteomics Facility Submission and Subsequent Data Analysis
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Assessing Budding Yeast Phosphoproteome Dynamics in a Time-Resolved Manner using TMT10plex Mass Tag Labeling
Andrew W Jones1,2, Helen R Flynn1, Frank Uhlmann3
1Mass Spectrometry Proteomics Science Technology Platform, The Francis Crick Institute, London NW1 1AT, UK.
STAR Protocols
|July 21, 2020
Summary
This study details a protocol for analyzing yeast phosphoproteome dynamics using Amine-reactive Tandem Mass Tag 10plex (TMT10plex) labeling and mass spectrometry. The method enables multiplexed protein identification and quantitative analysis for researchers.
Area of Science:
- Proteomics
- Yeast molecular biology
- Biochemistry
Background:
- Phosphoproteome dynamics are crucial for understanding cellular signaling pathways.
- Tandem Mass Tag (TMT) labeling facilitates high-throughput protein quantification.
- Multiplexed analysis enhances efficiency in complex biological samples.
Purpose of the Study:
- To provide a detailed protocol for phosphoproteome analysis in Saccharomyces cerevisiae.
- To characterize phosphoproteome dynamics using TMT10plex labeling.
- To establish a robust method for time-resolved proteomic studies.
Main Methods:
- Utilized Amine-reactive Tandem Mass Tag 10plex (TMT10plex) labeling.
- Applied tandem mass spectrometry (MS/MS) for protein identification and quantification.
- Prepared and analyzed 20 Saccharomyces cerevisiae samples in a time-resolved manner.
Main Results:
- Successfully applied TMT10plex labeling for multiplexed protein identification.
- Enabled quantitative analysis of phosphoproteome dynamics in yeast.
- Developed a comprehensive protocol for biological and mass spectrometry sample preparation.
Conclusions:
- The TMT10plex labeling protocol is effective for studying yeast phosphoproteome dynamics.
- This method allows for detailed characterization of signaling pathways.
- The protocol serves as a valuable resource for future proteomic research.
