Measuring the hydrodynamic radii of peptides and proteins with an unmodified LC-ESI-MS instrument operating in a Taylor dispersion regime

Native ion mobility - mass spectrometry has been well established in recent years as a structural biology method. However, it is still useful to combine gas-phase techniques with complementary solution-phase measurements, for example hydrodynamic radius (<i>R</i>_h) determination. A common method to measure protein <i>R</i>_h is the use of Taylor dispersion analysis (TDA), which relies on measuring the longitudinal dispersion of an analyte in a laminar flow within a capillary. This type of analysis most often relies on spectroscopic (<i>e.g.</i>, fluorescence-based) detection, although a few studies have reported the use of custom-built or modified fluidics systems coupled with MS-based detection. Here, we have repurposed a standard, unmodified LC-MS system, operated without a column, to measure hydrodynamic radii of peptides and proteins, including noncovalent complexes, ranging from 300 Da to 133 kDa. The <i>R</i>_h values we measured showed excellent correlation with gas-phase collision cross-sections as well as <i>R</i>_h values measured with a conventional TDA instrument with fluorescence-based detection. Notably, due to the mass-selective readout of our method, which is based on analysis of extracted ion chromatograms, we were able to perform multiplexed <i>R</i>_h measurements of several proteins in a single experiment, and also measured apparent <i>R</i>_h values for different oligomeric states of BSA and concanavalin A. Consistent with prior literature, the latter data suggested that concanavalin A primarily exists as a dimer and tetramer in solution, with monomer signals being largely due to dissociation during electrospray ionisation. We have named our method <b>Hyd</b>rodynamic <b>Ra</b>dii from an <b>U</b>nmodified <b>Li</b>quid <b>C</b>hromatography-<b>M</b>ass <b>S</b>pectrometer (HYDRAULIC-MS).