FastCAT Accelerates Absolute Quantification of Proteins Using Multiple Short Nonpurified Chimeric Standards.

Area of Science:

• Biochemistry
• Proteomics
• Analytical Chemistry

Background:

• Accurate protein quantification is crucial for establishing clinical reference values in biopsies.
• Existing methods for absolute protein quantification can be complex and labor-intensive.

Purpose of the Study:

• To introduce and validate the FastCAT (Fast-track QconCAT) method for absolute molar protein quantification.
• To improve the accuracy, precision, and dynamic range of protein quantification in biological samples.

Main Methods:

• Development of stable-isotope labeled chimeric proteins (CPs) composed of quantotypic (Q)-peptides and reference (R)-peptides.
• Grouping of Q-peptides based on target protein abundance to optimize quantification.
• Benchmarking FastCAT against MS Western and application to neurological markers in human cerebrospinal fluid.

Main Results:

• FastCAT eliminates the need for CP purification and SDS-PAGE.
• Demonstrated improved accuracy, precision, and dynamic range in absolute protein quantification.
• Successfully quantified low ng/mL levels of neurological markers in cerebrospinal fluid.

Conclusions:

• FastCAT offers a robust and efficient method for absolute molar protein quantification.
• The method enhances the reliability of protein reference values for clinical applications.
• FastCAT shows significant potential for biomarker discovery and validation in complex biological matrices.