Enhanced affinity capture MALDI-TOF MS: orientation of an immunoglobulin G using recombinant protein G.
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.
Neubert
Hendrik
H
Drug Control Centre and Department of Pharmacy, King's College London, United Kingdom.
Jacoby
Eli S
ES
Bansal
Sukhvinder S
SS
Iles
Ray K
RK
Cowan
David A
DA
Kicman
Andrew T
AT
D016880:Anisotropy; D001426:Bacterial Proteins; D015415:Biomarkers / Q000032:analysis; D014408:Biomarkers, Tumor / Q000302:isolation & purification; D018997:Chorionic Gonadotropin, beta Subunit, Human / Q000302:isolation & purification; D005260:Female; D006801:Humans; D007074:Immunoglobulin G; D007163:Immunosorbent Techniques / Q000592:standards; D011247:Pregnancy; D011961:Receptors, Fc; D011994:Recombinant Proteins; D019032:Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization / Q000379:methods* / Q000592:standards
D001426:Bacterial Proteins; D015415:Biomarkers; D014408:Biomarkers, Tumor; D018997:Chorionic Gonadotropin, beta Subunit, Human; C034284:IgG Fc-binding protein, Streptococcus; D007074:Immunoglobulin G; D011961:Receptors, Fc; D011994:Recombinant Proteins