NHS-Ester Tandem Labeling in One Pot Enables 48-Plex Quantitative Proteomics.

Area of Science:

• Proteomics
• Mass Spectrometry
• Biochemistry

Background:

• Stable-isotope labeling is crucial for quantitative proteomics.
• Hybrid strategies combining isotopic and isobaric tags increase multiplexity.
• Existing methods can involve sample loss and quantification inaccuracies.

Purpose of the Study:

• To introduce a novel hybrid-isotope labeling method, NETLOP (NHS-ester tandem labeling in one pot).
• To demonstrate NETLOP's utility for high-plex quantitative proteomic analysis.
• To improve accuracy and reduce sample loss in multiplexed proteomics.

Main Methods:

• Optimized 16-plex TMTpro labeling of lysine residues.
• Performed 2- or 3-plex mTRAQ labeling of peptide N-termini using NHS-ester reagents.
• Applied NETLOP to HeLa cell samples for LC-MS/MS analysis.

Main Results:

• Successfully performed proof-of-principle 32-plex and 48-plex quantitative proteomic analyses in single experiments.
• NETLOP requires no intermediate sample cleanup, minimizing sample loss.
• NETLOP avoids retention time shifts, ensuring quantification accuracy.

Conclusions:

• NETLOP offers a robust and efficient hybrid-isotope labeling strategy for high-plex quantitative proteomics.
• The method is adaptable to other NHS-ester reagents for even greater multiplexity.
• NETLOP is suitable for diverse biological and clinical proteomics applications.