Chemical labelling of active serum thioester proteins for quantification

Area of Science:

• Biochemistry
• Immunology
• Proteomics

Background:

• Serum proteins C3, C4, and α-2 macroglobulin belong to the ancient C3/α-2M thioester protein family.
• These proteins feature an intrachain thioester bond crucial for their function, enabling covalent linkage to targets via acyl transfer.
• Current methods for quantifying active C3 in clinical settings face limitations, including inability to distinguish active from inactive forms or reliance on unstable reagents.

Purpose of the Study:

• To develop and optimize a site-specific chemical labeling method for thioester-containing proteins in serum.
• To demonstrate the selective pull-down and identification of intact C3, α-2M, and C4 using a bioreporter-linked nucleophile.
• To explore the potential of this chemical approach for a robust, antibody- and enzyme-free quantitative assay for active C3 in clinical samples.

Main Methods:

• Utilized a biotinylated nucleophile to target the thioester bond of C3, C4, and α-2M.
• Optimized conditions for selective labeling and pull-down of intact thioester proteins from serum under high salt concentrations.
• Employed streptavidin-coated resin for capture and MALDI-TOF MS for identification of the pulled-down proteins.

Main Results:

• Successfully demonstrated the site-specific modification and pull-down of full-length C3, α-2M, and C4 from human sera.
• Confirmed the identity of the captured proteins using MALDI-TOF MS.
• Investigated the feasibility of quantifying active C3 in patient sera, highlighting the method's potential for clinical applications.

Conclusions:

• Developed a robust, enzyme- and antibody-free chemical method for detecting active thioester proteins in biological fluids.
• This novel approach offers a promising alternative for quantifying active C3, overcoming limitations of existing antibody-based and haemolytic assays.
• The technique provides a foundation for developing accurate and reliable diagnostic tools for assessing complement system activity.