High-affinity capture of recombinant streptococcal protein G and Its application in serum proteomics with immunoglobulin G-coated magnetic nanoparticles

A new solid-phase extraction agent, IgG@ Fe 3 O 4 -PEI, was successfully developed by conjugating immunoglobulin G (IgG) with amino-functionalized magnetic nanoparticles (Fe 3 O 4 -PEI). The IgG@Fe 3 O 4 -PEI composite was characterized using various techniques including infrared spectroscopy, Raman spectroscopy, magnetic property testing, Zeta potential measurements, energy spectrum analysis, scanning electron microscopy (SEM), and transmission electron microscopy (TEM). Because of the presence of a specific binding domain in the Fc fragment of IgG, the IgG@Fe 3 O 4 -PEI composite demonstrated highly selective adsorption of recombinant streptococcal G protein (rSPG) under optimal experimental conditions. At pH 6.0, the IgG@Fe 3 O 4 -PEI composite (0.5 mg) achieved a maximum adsorption efficiency of 93.96 % for rSPG at a concentration of 100 μg mL -1 . Adsorption model simulations indicated that the adsorption behavior followed the Langmuir adsorption model, with a theoretical maximum adsorption capacity of 125.0 mg g -1 . The enriched rSPG was eluted and recovered using Britton-Robinson (BR) buffer at pH 10.0, achieving a recovery efficiency of 95.8 %. In practical applications, the IgG@Fe 3 O 4 -PEI magnetic nanoparticles effectively enriched rSPG from an Escherichia coli expression system, and SDS-PAGE analysis confirmed the successful isolation of highly pure rSPG. Furthermore, the IgG@Fe 3 O 4 -PEI magnetic nanoparticles were used to capture proteins from human serum samples. After enrichment and processing, 430 proteins were identified, including 34 that were undetectable in untreated serum samples. This validated the high sensitivity and specificity of this method in serum proteomics, demonstrating its potential for isolating and identifying low-abundance proteins in complex biological samples. The developed system offers a valuable tool for advancing human serum proteomics research and targeted protein analysis.