High-Resolution Enabled 5-plex Mass Defect-Based N, N-Dimethyl Leucine Tags for Quantitative Proteomics.

Area of Science:

• Proteomics
• Analytical Chemistry
• Biochemistry

Background:

• Mass spectrometry (MS)-based proteomics is crucial for biological research.
• Isobaric labeling methods can suffer from ratio compression, limiting quantification accuracy.
• Existing labeling strategies may lack sufficient multiplexing capabilities.

Purpose of the Study:

• To develop a novel, cost-effective 5-plex mass defect labeling strategy for enhanced quantitative proteomics.
• To overcome limitations of existing reporter ion quantification methods, such as ratio compression.
• To increase multiplexing capacity in mass spectrometry-based analyses.

Main Methods:

• Development of 5-plex mass defect N, N-dimethyl leucine (mdDiLeu) tags.
• Utilizing a combination of mass difference and mass defect for labeling.
• MS1-centric quantification strategy.
• High-resolution mass spectrometry (Orbitrap Fusion Lumos) for analysis.
• Proof-of-principle experiments using Saccharomyces cerevisiae lysate.

Main Results:

• The mdDiLeu tags enable accurate MS1-centric quantification, avoiding ratio compression.
• The 5-plex strategy increases multiplexing by leveraging mass difference isotopologues.
• Labeled peak clusters are separated by 5 Da in MS1 spectra, with distinct isotopic peaks.
• The synthesis of mdDiLeu tags is a single, straightforward reaction step.

Conclusions:

• The developed 5-plex mdDiLeu labeling strategy offers a cost-effective and accurate approach for quantitative proteomics.
• This method enhances multiplexing and improves quantification accuracy compared to traditional isobaric labeling.
• The accessibility of the synthesis makes this technique broadly applicable in various laboratories.