A mass spectrometry-based isotope-coded mass tag method to map thiol accessibility in biological systems

Interactions at biological membranes are important for many cellular functions, but because of the dynamic nature of these interactions, traditional methods of structure investigation are limited. Here, we describe a method that utilizes thiol–maleimide chemistry to monitor the solvent-accessible surface of membrane–protein complexes and membrane dynamics in vitro in real time. This method, called the isotope-coded mass tag (ICMT) method, has been used previously to locate thiols in transmembrane peptides, to elucidate lipid flipping kinetics in bilayers, to determine the oxidation state of disulfide bonds, and to investigate the protein–lipid interface of the peripheral membrane protein cholesterol oxidase. The method requires only microgram quantities of protein or thiolated lipid, uses common laboratory equipment and routine instrumentation, and can be applied to many different systems to investigate macromolecular interactions in the presence or absence of biological membranes.