Enhanced Affinity Capture MALDI-TOF MS: Orientation of an Immunoglobulin G Using Recombinant Protein G

Abstract

Although immobilization of antigen-specific immunoglobulins onto matrix-assisted laser desorption/ionization (MALDI) targets allows the specific detection and enrichment of an antigen from complex biological fluids, the process of antibody immobilization is not optimal. The principal reason is that the antibody can bind to the template in various orientations, many of which block antigen recognition. An affinity capture MALDI mass spectrometry methodology was developed by covalently immobilizing an Fc receptor (recombinant protein G) onto MALDI gold targets for the purpose of orientating an immunoglobulin G, with the Fab domains pointing away from the target surface. The pregnancy and cancer marker, human chorionic gonadotropin beta core fragment (hCGbetacf), was our chosen test substance. To optimize the methodology, different surface densities of protein G and immunoglobulin were achieved by employing varying concentrations for immobilization. Captured amounts of hCGbetacf were compared using an external standard (cytochrome c). Orientation of immunoglobulin resulted in an approximately 3-fold increase in MALDI signal compared to using randomly immobilized antibody. Higher antibody concentrations resulted in diminished MALDI signals, which were explained by steric hindrance. Purification and enrichment of hCGbetacf was achieved from a test solution containing contaminant peptides and proteins using oriented immunoglobulins on-target.

Fabrication of protein chips based on 3-aminopropyltriethoxysilane as a monolayer

“…Protein A can bind with the Fc part of the antibody which reduces the non-specific binding of the antibody. Therefore, the use of protein A leads to highly efficient immunoreactions, and enhances detection system performance (Neubert et al 2002;Susmel et al 2000;Tanaka and Mastsunaga 2000). Protein adsorption experiments were performed with bovine serum albumin (BSA) as a model protein to analyze surface adsorption.…”

“…The fabrication of nanoscale structures with SAMs to combine protein A with solid substrates has attracted much attention because marked interest exists in two-dimensional molecular assemblies and their potential applications in molecular devices, sensors and surface engineering. Therefore, the use of protein A leads to highly efficient immunoreactions and improves the performance of detection systems (Neubert et al 2002;Susmel et al 2000;Tanaka and Mastsunaga 2000).…”

Development and Characterization of Novel Monoclonal Antibodies Against Tartrate-Resistant Acid Phosphatase 5

“…The development of techniques of proteomic analysis is facilitating these studies. The use of techniques such as two-dimensional polyacrylamide gel electrophoresis (2D-PAGE), (26,27) matrix-assisted laser desorption/ionization time-of-flight mass spectrometry MALDI-TOF-MS (28,29) and surface-enhanced laser desorption/ionization time-of-flight mass spectrometry (SELDI-TOF-MS) (30,31) to compare normal and diseased cells and tissues have led to the discovery of a large number of disease-related proteins. Many of these newly discovered proteins are present at very low levels in specimens.…”

Activated Surfaces for Laser Desorption Mass Spectrometry: Application for Peptide and Protein Analysis

“…In this study the affinity purification was performed using magnetic beads followed by MALDI-TOF analysis. Neubert et al [114] demonstrated the use of recombinant protein G with affinity capture MALDI-TOF. In this approach an Fc receptor was covalently immobilised on a MALDI gold target in order to orient an immunoglobulin G with Fab domains directed towards the target surface.…”