An Efficient, Amine-Specific, and Cost-Effective Method for TMT 6/11-plex Labeling Improves the Proteome Coverage,...

Area of Science:

• Proteomics
• Analytical Chemistry
• Biochemistry

Background:

• Tandem Mass Tags (TMT) are essential for multiplexed quantitative proteomics.
• TMT reagents can react with hydroxyl groups (serine, threonine, tyrosine) under alkaline conditions, causing overlabeling.
• Overlabeling compromises analytical sensitivity, precision, and protein identification rates.

Purpose of the Study:

• To develop a novel method to suppress TMT overlabeling.
• To improve peptide/protein identification and quantitative accuracy in proteomics.
• To enhance statistical power for detecting significant protein changes.

Main Methods:

• A new TMT labeling method was developed using acidic conditions.
• This method utilizes significantly reduced TMT reagent amounts (one-fifth of manufacturer's recommendation).
• The new method was systematically evaluated against standard and TMT-reduced methods using yeast/human proteome samples.

Main Results:

• The acidic method reduced overlabeled peptides by 9-fold compared to the standard method and 6-fold compared to the TMT-reduced method.
• Significant enhancement in peptide and protein identification rates was observed.
• Quantitative accuracy and precision were improved, leading to a 42% and 12% increase in statistically significant yeast protein detection, respectively.

Conclusions:

• The developed acidic TMT labeling method effectively suppresses overlabeling.
• This approach substantially improves quantitative proteomics, enhancing data reliability and statistical power.
• The method offers a more sensitive and accurate alternative for TMT-based proteomic analyses.