Fluorescent isotope-coded affinity tag (FCAT). I: Design and synthesis

Area of Science:

• Proteomics
• Analytical Chemistry
• Biochemistry

Background:

• Quantitative proteomics requires precise methods for protein and peptide analysis.
• Existing isotope-coded affinity tags (ICAT) can be enhanced for improved quantification and isolation.
• Cysteine-containing proteins and peptides are crucial targets in proteomic studies.

Purpose of the Study:

• To introduce a novel fluorescent isotope-coded affinity tag (FCAT) for quantitative proteomics.
• To develop a new tool for absolute quantification and isolation of cysteine-containing peptides.
• To synthesize and characterize the light isotope-containing FCAT molecule.

Main Methods:

• Solid-phase organic synthesis of the FCAT reagent.
• Chemical labeling of cysteine-containing model peptides.
• Evaluation of the reactivity and cleavage efficiency of the FCAT tag.
• Assessment of the fluorescent tag for absolute quantification and antibody-based pull-down.

Main Results:

• Successful solid-phase synthesis of the light isotope FCAT molecule.
• Demonstrated good reactivity of FCAT with model cysteine-containing peptides.
• Effective cleavage of the fluorophore group from labeled peptides for mass spectrometry.
• Potential for antibody-based pull-down of FCAT-labeled peptides.

Conclusions:

• FCAT represents a promising new tool for quantitative proteomics.
• The fluorescent and isotopic features enable absolute quantification and peptide isolation.
• FCAT facilitates improved mass spectrometry-based analysis of cysteine-containing peptides.