TMT Labeling under Acidic pH Overcomes Detrimental Overlabeling and Improves Peptide Identification Rates

Area of Science:

• Proteomics
• Analytical Chemistry
• Biochemistry

Background:

• Tandem Mass Tags (TMT) enable high-throughput proteomics quantification by multiplexing samples.
• Standard TMT labeling targets primary amines but can also label hydroxyl groups, reducing sensitivity and peptide identification.
• Overlabeling, particularly on serine, threonine, and tyrosine residues, compromises TMT-based proteomic analyses.

Purpose of the Study:

• To investigate the chemical mechanism behind TMT overlabeling.
• To develop an improved TMT labeling strategy to mitigate overlabeling.
• To enhance peptide and protein identification rates in quantitative proteomics.

Main Methods:

• Investigated the chemical nature of TMT overlabeling, identifying intramolecular catalysis by histidine residues.
• Developed a modified TMT labeling protocol utilizing acidic pH conditions.
• Compared the novel method against the standard TMT labeling procedure using proteomic analysis.

Main Results:

• Identified histidine-mediated intramolecular catalysis as the cause of TMT overlabeling on hydroxyl groups.
• The novel acidic pH TMT labeling method effectively prevented overlabeling.
• Achieved comparable labeling efficiency to the standard method while significantly reducing overlabeled peptides.
• Led to a 33.9% increase in unique peptide and a 20.9% increase in protein identifications.

Conclusions:

• Understanding the chemical mechanism of TMT overlabeling is crucial for method development.
• The developed acidic pH TMT labeling method offers a significant improvement for quantitative proteomics.
• This optimized TMT approach enhances analytical sensitivity and data quality, enabling deeper proteome coverage.