Dual Fragmentation Isobaric Tags for Metabolomics.

Area of Science:

• Proteomics
• Analytical Chemistry
• Biochemistry

Background:

• Isobaric tags are crucial for quantitative proteomics, relying on specific fragmentation patterns for reporter ion generation.
• Current isobaric tags have limited structural diversity, restricting isotopic labeling options and synthetic availability.
• A need exists for novel isobaric tagging strategies with enhanced flexibility and isotopic capacity.

Purpose of the Study:

• To develop and present novel dual fragmentation isobaric tagging strategies.
• To demonstrate the creation of diverse isobaric tags with variable reporter and balancer masses.
• To introduce a new method for generating constant mass reporters via sequential fragmentation.

Main Methods:

• Designed and synthesized two distinct dual fragmentation isobaric tagging systems.
• Utilized trimethylamine neutral loss and cyclization for the first tag type.
• Employed sequential fragmentation, including C-S bond cleavage, for thiol-reactive tags.

Main Results:

• Achieved high efficiency in reporter ion formation through dual fragmentation.
• Developed six-plex thiol-reactive isobaric tags with up to 13 isotopes in the balancer region.
• Demonstrated low variability (RSD 14%, R^2 0.98) in six-plex thiol analysis.
• Observed significant decreases in glutathione and cysteinyl-glycine in BSO-treated endothelial cells, confirming metabolic disruption.

Conclusions:

• A new dual fragmentation approach enables the generation of constant mass reporters.
• This method expands the possibilities for designing diverse isobaric tags with tunable isotopic compositions.
• The developed tags show promise for quantitative proteomics and thiol metabolism studies.