Best practice mass photometry: a guide to optimal single-molecule mass measurement
Jiří Kratochvíl, Raman van Wee, Jan Christoph Thiele, Dan Loewenthal, Jack Bardzil, Kishwar Iqbal, Justin L. P. Benesch, Stephen Thorpe, Philipp Kukura
Abstract
Mass photometry (MP) has emerged as a powerful approach to study quaternary biomolecular structure, dynamics and interactions. The capabilities of the method ultimately hinge on the ability to accurately measure the tiny optical contrast generated by individual molecules at a glass–water interface, which enables mass-resolved quantification of biomolecular mixtures. Ideally, this capability is limited only by photon shot noise, but in practice depends on additional parameters and details of the assay. Here, we focus on the key factors affecting MP performance and present simple steps that can be taken to achieve optimal MP measurements in terms of mass resolution, quantitative detection limit, reproducibility and analyte concentration range without compromising the speed and simplicity of the technique. Each sample takes <10 min to analyse, with an additionial 2 h if amination of the glass surface is desired. In mass photometry, the optical contrast generated by individual molecules at a glass–water interface enables mass-resolved quantification of biomolecular mixtures. This protocol describes how to optimize and validate this method.