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09:24
Automated Sample Multiplexing by using Combined Precursor Isotopic Labeling and Isobaric Tagging (cPILOT)
Published on: December 18, 2020
Dimethyl multiplexed labeling combined with microcolumn separation and MS analysis for time course study in
Jue-Liang Hsu1, Sheng-Yu Huang, Shu-Hui Chen
1Department of Chemistry, National Cheng Kung University, Tainan, Taiwan.
Electrophoresis
|August 24, 2006
Summary
This study extends dimethyl labeling for multiplexed quantitative proteomics, enabling simultaneous analysis of four samples. The method efficiently monitors protein digestion kinetics using mass spectrometry.
Area of Science:
- Proteomics
- Analytical Chemistry
- Biochemistry
Background:
- Stable-isotope labeling is crucial for comparative proteomics.
- Dimethyl labeling offers a robust method for quantitative analysis.
- Existing methods are limited to binary sample labeling.
Purpose of the Study:
- To extend dimethyl labeling for simultaneous analysis of four samples.
- To develop a multiplexed labeling strategy for enhanced quantitative proteomics.
- To validate the method for monitoring enzymatic digestion.
Main Methods:
- Multiplex dimethyl labeling using formaldehyde (d0, d2) and sodium cyanoborohydride (d0, d3).
- Enzymatic digestion with endoproteinase Lys-C for increased labeling sites.
- Coupling with nano-liquid chromatography (LC) and electrospray ionization tandem mass spectrometry (ESI-MS/MS).
Main Results:
- Successful simultaneous labeling and analysis of four sample sets.
- Demonstrated no significant isotopic effect during RP LC elution.
- Monitored Lys-C digestion of hemoglobin, completing within 10-15 hours.
Conclusions:
- The multiplex dimethyl labeling method enables simultaneous detection of four sample sets.
- This approach retains the advantages of binary labeling while increasing sample capacity.
- The validated method is effective for quantitative proteomic studies and kinetic analyses.
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