Deuterium-Free, Three-Plexed Peptide Diethylation for Highly Accurate Quantitative Proteomics

Area of Science:

• Proteomics
• Analytical Chemistry
• Biochemistry

Background:

• Deuterium-based isotopic labeling in quantitative proteomics can cause retention time shifts, impacting accuracy.
• Conventional dimethylation using deuterium has limitations in precision and accuracy for complex proteome analysis.

Purpose of the Study:

• To introduce a novel three-plexed peptide diethylation method for enhanced proteome quantification.
• To compare the accuracy and precision of this new method against deuterium-based dimethylation and isobaric labeling.

Main Methods:

• Development of a three-plexed peptide diethylation using 13C isotopologues of acetaldehyde.
• Application of the method in single-shot and multidimensional LC-MS/MS analysis of the HeLa proteome.
• Time-resolved profiling of Xenopus laevis early embryogenesis using the diethylation method.

Main Results:

• The 3-plexed diethylation method demonstrated significantly superior accuracy and precision over deuterium-based dimethylation.
• In Xenopus laevis embryogenesis, diethylation outperformed isobaric labeling, yielding over two times more differentially expressed proteins.
• The method proved effective in both single-shot and multidimensional LC-MS/MS analyses.

Conclusions:

• The novel 3-plexed diethylation method offers a cost-effective and highly accurate alternative for quantitative proteomics.
• This technique improves proteome quantification accuracy and precision, outperforming existing labeling strategies.
• The method is suitable for applications requiring up to three-plex multiplexity in quantitative proteomics.