Oriented Functionalization of Magnetic Beads with <i>in Vivo</i> Biotinylated Nanobodies for Rapid MALDI-TOF MS Ultrasensitive Quantitation of Microcystins in Biological Samples

Abstract

Here we present a new analytical method where immunoconcentration of the analyte is coupled to quantitative matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) analysis allowing in minutes the identification and highly sensitive quantitation of microcystins (MCs) as model targets. The key element is a site-specific in vivo biotinylated nanobody of broad cross-reactivity with microcystins. The single biotin moiety at the C-terminus and the small size of the nanobody (15 kDa) enable its oriented and tightly packed immobilization on magnetic beads, providing a highly efficient capture of the toxin. The binding capacity of the bioadsorbent is partially loaded with an easily synthesized internal standard for MS quantitation. After capture, the beads are directly dispensed on the MALDI-TOF MS target enabling the identification and sensitive quantitation of the microcystin (MC) congeners. Since salts and contaminants are removed during the concentration step, no cleanup or other sample treatments are needed. The method was validated with a large number of water and serum samples with excellent precision and recovery at quantitation limits of 0.025 μg/L of MC.