Multiplexed absolute quantification for proteomics using concatenated signature peptides encoded by QconCAT genes

Area of Science:

• Proteomics
• Analytical Chemistry

Background:

• Absolute protein quantification is crucial for biomarker discovery and systems biology.
• Current methods often rely on stable isotope-labeled peptides, but their availability is limited.
• Relative quantification methods are insufficient for absolute measurements.

Purpose of the Study:

• To develop a novel approach for absolute protein quantification.
• To create artificial QconCAT proteins as internal standards for mass spectrometry.
• To provide a detailed protocol for QconCAT synthesis and application.

Main Methods:

• Design and synthesis of artificial QconCAT proteins, which are concatamers of tryptic peptides.
• Expression, labeling, purification, and characterization of QconCAT proteins.
• Application of QconCATs for absolute quantification of target proteins in complex samples using mass spectrometry.

Main Results:

• Successful design, synthesis, and characterization of QconCAT proteins.
• Demonstrated utility of QconCATs for absolute quantification of multiple proteins simultaneously.
• Established a comprehensive protocol for QconCAT-based absolute quantification.

Conclusions:

• QconCAT proteins offer a robust solution for absolute protein quantification.
• This method addresses the limitations of standard labeled peptides, facilitating broader adoption.
• The protocol enables accurate protein quantification within approximately 29 days.