Quantitative MALDI-MS Binding Assays: An Alternative to Radiolabeling

Radiolabeling of ligands is still the gold standard in the study of high‐affinity receptor–ligand interactions. In an effort toward safer and simpler alternatives to the use of radioisotopes, we developed a quantitative and highly sensitive matrix‐assisted laser desorption ionization mass spectrometry (MALDI‐MS) method that relies on the use of chemically tagged ligands designed to be specifically detectable when present as traces in complex biological mixtures such as cellular lysates. This innovative technology allows easy, sensitive detection and accurate quantification of analytes at the sub‐nanomolar level. After statistical validation, we were able to perform pharmacological evaluations of G protein‐coupled receptor (V1A‐R)–ligand interactions. Both saturation and competitive binding assays were successfully processed. Assays with atto‐tude! To perform competitive binding assays and saturation experiments, we developed a non‐radioactive technology that relies on highly specific and sensitive MALDI‐MS detection, based on quantification of a reference‐tagged ligand (α‐cyano‐4‐hydroxycinnamic acid (CHCA)) as an MS‐sensitive tag (attomole detection). Quantification could be directly achieved without any additional processing.