High-Throughput Label- and Immobilization-Free Screening of Human Milk Oligosaccharides Against Lectins

The intense interest in the mechanisms responsible for the beneficial effects of breast-feeding on infant health has created a significant need for analytical methods capable of rapidly identifying interactions between human milk oligosaccharides (HMOs) and their protein receptors. Currently, there are no established, high-throughput assays for the screening libraries of free (unmodified) HMOs against lectins. The present work describes a rapid and label- and immobilization-free assay, based on catch-and-release electrospray ionization mass spectrometry (CaR-ESI-MS), capable of simultaneously screening mixtures of free HMOs of known concentration for binding to lectins in vitro. Ligand identification relies on the molecular weights (MWs), ion mobility separation arrival times, and collision-induced dissociation fingerprints of HMO anions released from the target protein in the gas phase. To establish the reliability of the assay, a library of 31 free HMOs, ranging in size from tri- to octasaccharide, was screened against three human galectin (hGal) proteins (a stable mutant of hGal1 (hGal-1), a C-terminal fragment of hGal-3 (hGal-3C) and hGal-7), with known HMO affinities. When implemented using an equimolar concentration library, the CaR-ESI-MS assay identified 100% of ligands with affinities >500 M^-1 and ≥93% of all HMO ligands (hGal-1-31 of 31 ligands; hGal-3C-25 of 25; hGal-7-28 of 30); no false positives were detected. The assay also successfully identified the majority of the highest affinity HMO ligands (or isomer sets that contain the highest affinity ligands) in the library for each of the three hGal. Notably, for each lectin, CaR-ESI-MS screening required <1 h to complete and consumed <5 ng of each HMO and <0.5 μg of protein.