Quantitative MALDI‐MS Binding Assays: An Alternative to Radiolabeling

Abstract

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.

MALDI-TOF-Based Affinity Selection Mass Spectrometry for Automated Screening of Protein–Ligand Interactions at High Throughput

“…Historically, displacement assays, in which the competition between a test compound and a labeled tracer molecule for binding to the target of interest is monitored, have been the method of choice for larger screening campaigns. [2][3][4] Labeling strategies for a tracer ligand of known affinity comprise chemical introduction of functional labels or replacement of specific atoms by their respective radioisotopes for direct or indirect downstream detection. While competitive binding formats usually provide the required throughput, are easy to miniaturize, and can be fully automated, their dependence on a labeled tracer molecule necessitates the availability of a known ligand and constrains the exploration of binding epitopes on the target protein to the respective tracer binding site.…”

Receptor–Ligand Interaction Measured by Inductively Coupled Plasma Mass Spectrometry and Selenium Labeling

“…The synthesis as well as the characterization of the peptides are described in the Sections S1 and S2 of the Supporting Information. The iodinated linear peptidic V 1A antagonist [ 125 I]­HO-Phpa-LVA ( 11 ) was available in the laboratory, and its radioiodination was previously described . Native AVP ( 1 ), CCK-4 ( 7 ), and nonsulfated CCK-8 ( 14 ) ligands were available in the laboratory and characterized prior to their use …”

“…The iodinated linear peptidic V 1A antagonist [ 125 I]HO-Phpa-LVA (11) 52 was available in the laboratory, and its radioiodination was previously described. 53 Native AVP (1), CCK-4 (7), and nonsulfated CCK-8 ( 14) ligands were available in the laboratory and characterized prior to their use. 53 Peptide Purity.…”

Chemical labeling associated to Mass spectrometry as a powerful tool for peptide detection and quantification in biology

“…This original approach allowed to selectively enhance and discriminate the MALDI-MS signals of targeted peptides. This concept was successfully applied to protein structure issues illustrated by the cross-linking of a model protein and peptide quantification for pharmacological studies of receptor/ligand systems [4,5].…”