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Fluorescent isotope-coded affinity tag 2: peptide labeling and affinity capture.

Zuly Rivera-Monroy¹, Guenther K Bonn, András Guttman

¹Horváth Laboratory of Bioseparation Sciences, Institute of Analytical Chemistry and Radiochemistry, University of Innsbruck, Innsbruck, Austria.

Electrophoresis

|March 17, 2009

Summary

This summary is machine-generated.

A new heavy isotope-coded fluorescent affinity tag (FCAT) enables absolute quantification and selective capture of cysteine-containing peptides. This method effectively labels and isolates tryptic peptides for mass spectrometry analysis.

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Area of Science:

Proteomics
Analytical Chemistry
Biochemistry

Background:

Cysteine-containing peptides are crucial in various biological processes.
Accurate quantification and selective isolation of peptides are essential for proteomic studies.
Fluorescent isotope-coded affinity tag (FCAT) offers a method for peptide labeling and quantification.

Purpose of the Study:

To synthesize and characterize the heavy isotopic form of the FCAT reagent.
To evaluate the utility of both light and heavy FCAT tags for labeling tryptic peptides.
To demonstrate the selective capture and analysis of FCAT-labeled peptides.

Main Methods:

Synthesis of the heavy isotopic form of FCAT.
Labeling of tryptic peptides from various proteins (alpha-lactalbumin, fetuin, BSA, phosphorylase b) using light and heavy FCAT.
Affinity capture of labeled peptides using anti-FITC antibody or iminodiacetic-acid-coated beads.
Separation by RP HPLC and analysis by MALDI-TOF MS.

Main Results:

The heavy FCAT reagent demonstrated comparable reactivity and chromatographic behavior to the light version.
Effective labeling of tryptic peptides was achieved with both FCAT forms.
Selective peptide capture was successfully performed using antibody-coated pipette tips or IDA beads.
RP HPLC coupled with MALDI-TOF MS enabled separation and analysis of differentially labeled peptides.

Conclusions:

The heavy FCAT reagent is a viable tool for quantitative proteomics.
FCAT technology facilitates absolute quantification and selective enrichment of cysteine-containing peptides.
This method provides a robust approach for analyzing complex peptide mixtures.