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 Gâ protein-coupled receptor (V1A-R)-ligand interactions. Both saturation and competitive binding assays were successfully processed.
Subject(s)
Chemistry Techniques, Analytical/methods , Spectrometry, Mass, Matrix-Assisted Laser Desorption-Ionization , Animals , Binding, Competitive , CHO Cells , Coumaric Acids/chemistry , Coumaric Acids/metabolism , Cricetinae , Cricetulus , Isotope Labeling , Ligands , Peptides/chemical synthesis , Peptides/chemistry , Receptors, G-Protein-Coupled/metabolismABSTRACT
We report on the synthesis and use of a new supported reagent consisting in tris(2-carboxyethyl)phosphine (TCEP) immobilized on hydrophilic PEG based resin beads. Used in conjunction with a 5 min microwave (MW) irradiation, "supported TCEP" reduced disulfide bridges in free thiols in peptides having two or more cysteine residues. Separation of reaction products from reducing agent was easily performed by simple filtration.
Subject(s)
Disulfides/chemistry , Peptides/chemistry , Phosphines/chemistry , Reducing Agents/chemistry , Amino Acid Sequence , Microwaves , Molecular Sequence Data , Oxidation-Reduction , Phosphines/chemical synthesis , Reducing Agents/chemical synthesisABSTRACT
The cross-linking approach combined with MS for protein structure determination is one of the most striking examples of multidisciplinary success. Indeed, it has become clear that the bottleneck of the method was the detection and the identification of low-abundance cross-linked peptides in complex mixtures. Sample treatment or chromatography separation partially addresses these issues. However, the main problem comes from over-represented unmodified peptides, which do not yield any structural information. A real breakthrough was provided by high mass accuracy measurement, because of the outstanding technical developments in MS. This improvement greatly simplified the identification of cross-linked peptides, reducing the possible combinations matching with an observed m/z value. In addition, the huge amount of data collected has to be processed with dedicated software whose role is to propose distance constraints or ideally a structural model of the protein. In addition to instrumentation and algorithms efficiency, significant efforts have been made to design new cross-linkers matching all the requirements in terms of reactivity and selectivity but also displaying probes or reactive systems facilitating the isolation, the detection of cross-links, or the interpretation of MS data. These chemical features are reviewed and commented on in the light of the more recent strategies.
