Multi Epitope-Targeting Immunoprecipitation Using F(ab') Fragments with High Affinity and Specificity for the Enhanced Detection of a Peptide with Matrix-Assisted Laser Desorption Ionization-Time-of-Flight Mass Spectrometry

Human plasma has been frequently studied using mass spectrometry for new biomarker discovery, although detection of low-abundance biological molecules can be challenging due to sample complexity and dynamic protein concentration ranges of plasma proteins. While immunoprecipitation coupled with mass spectrometric analysis is an essential method for overcoming this difficulty, its sensitivity can be insufficient to detect clinically relevant circulating biomarkers because of limited antibody affinity or specificity. To increase antibody affinity, we developed a strategy using a F(ab') fragment coupled to polyethylene glycol. We produced hetero-F(ab')-(PEG)(24) beads composed of two monoclonal antiamyloid beta antibodies (6E10 and 4G8) that are specific for different epitopes of amyloid beta and assessed the detection limit of amyloid beta(1-28)-spiked human plasma. In human plasma, the detection limit of amyloid beta(1-28) was 6.14 pM, which was 25- to 50-fold more sensitive than single IgG-protein G beads. In addition, an introduction of polyethylene glycol as a linker reduced nonspecific binding, leading to highly specific MS detection. Finally, the present IP method enabled the detection of endogenous amyloid beta(1-40) in 250 mu L of human plasma with matrix-assisted laser desorption ionization-time-of-flight mass spectrometry (MALDI-TOF MS). This technique provides a powerful approach for enhancing the sensitivity and specificity of immunoprecipitation (IP)-MS for detection of low-abundance peptides in plasma and has the potential to accelerate MS-based clinical applications.