Increased quantitative proteome coverage with (13)C/(12)C-based, acid-cleavable isotope-coded affinity tag reagent...

Area of Science:

• Proteomics
• Analytical Chemistry
• Biochemistry

Background:

• Quantitative protein profiling compares protein expression using isotope-coded affinity tag (ICAT) and tandem mass spectrometry (MS).
• Previous ICAT methods used deuterium (2H) labeling, causing HPLC retention time shifts that limited protein identification.
• A modified ICAT reagent with a cleavable bond and carbon-13 (13C) labeling offers potential improvements.

Purpose of the Study:

• To investigate a new ICAT reagent utilizing 13C labeling and an acid-cleavable bond.
• To develop and validate an MS data acquisition strategy for enhanced protein identification and quantification.
• To assess the impact of the new reagent and strategy on proteomic analysis efficiency and accuracy.

Main Methods:

• Utilized a novel ICAT reagent with an acid-cleavable biotin moiety and 13C substitution for heavy labeling.
• Developed a mass spectrometry (MS) data acquisition strategy involving a single survey scan followed by serial collision-induced dissociations (CIDs) of precursor ions.
• Applied the new ICAT reagent and MS strategy to analyze a six-protein mixture and a total yeast lysate digest.

Main Results:

• The new ICAT reagent eliminated HPLC retention time shifts associated with 2H labeling.
• The developed MS strategy increased protein identifications by approximately two-fold per experiment.
• Accurate protein quantification was maintained with the new method, comparable to previous approaches.

Conclusions:

• The combination of the new 13C-based ICAT reagent and the serial MS/MS acquisition strategy significantly enhances proteomic analysis.
• This approach allows for a greater number of protein identifications in a single experiment without sacrificing quantification accuracy.
• The method represents a substantial advance for quantitative proteomics, enabling deeper insights into biological systems.