Quantifying the heterogeneity of macromolecular machines by mass photometry

Sample purity is central to in vitro studies of protein function and regulation, as well as to the efficiency and success of structural studies requiring crystallization or computational alignment of individual molecules. Here, we show that mass photometry (MP) accurately reports on sample heterogeneity using minimal volumes with molecular resolution within minutes. We benchmark our approach by negative stain electron microscopy (nsEM), including workflows involving chemical crosslinking and multi-step purification of a multi-subunit ubiquitin ligase. When applied to proteasome stability, we detect and quantify assemblies invisible to nsEM. Our results illustrate the unique advantages of MP for rapid sample characterization, prioritization and optimization.