The cellular processes underpinning life are orchestrated by proteins and their interactions. The associated structural and dynamic heterogeneity, despite being key to function, poses a fundamental challenge to existing analytical and structural methodologies. We used interferometric scattering microscopy to quantify the mass of single biomolecules in solution with 2% sequence mass accuracy, up to 19-kilodalton resolution, and 1-kilodalton precision. We resolved oligomeric distributions at high dynamic range, detected small-molecule binding, and mass-imaged proteins with associated lipids and sugars. These capabilities enabled us to characterize the molecular dynamics of processes as diverse as glycoprotein cross-linking, amyloidogenic protein aggregation, and actin polymerization. Interferometric scattering mass spectrometry allows spatiotemporally resolved measurement of a broad range of biomolecular interactions, one molecule at a time.

Background: Three genes encode human nonmuscle myosin II (NM II) heavy chains, and the proteins have different intracellular roles and localizations. Results: NM II paralogs form bipolar filaments, but there are important differences in filament structure, enzymatic, and actin binding behavior. Conclusion: NM II filaments show diverse interactions with actin. Significance: NM II filaments are adapted to work in cytoskeletal networks.

Perrault syndrome is a genetically and clinically heterogeneous autosomal-recessive condition characterized by sensorineural hearing loss and ovarian failure. By a combination of linkage analysis, homozygosity mapping, and exome sequencing in three families, we identified mutations in CLPP as the likely cause of this phenotype. In each family, affected individuals were homozygous for a different pathogenic CLPP allele: c.433A>C (p.Thr145Pro), c.440G>C (p.Cys147Ser), or an experimentally demonstrated splice-donor-site mutation, c.270+4A>G. CLPP, a component of a mitochondrial ATP-dependent proteolytic complex, is a highly conserved endopeptidase encoded by CLPP and forms an element of the evolutionarily ancient mitochondrial unfolded-protein response (UPR(mt)) stress signaling pathway. Crystal-structure modeling suggests that both substitutions would alter the structure of the CLPP barrel chamber that captures unfolded proteins and exposes them to proteolysis. Together with the previous identification of mutations in HARS2, encoding mitochondrial histidyl-tRNA synthetase, mutations in CLPP expose dysfunction of mitochondrial protein homeostasis as a cause of Perrault syndrome.

Optical detection of individual proteins requires fluorescent labeling. Cavity and plasmonic methodologies enhance single molecule signatures in the absence of any labels but have struggled to demonstrate routine and quantitative single protein detection. Here, we used interferometric scattering microscopy not only to detect but also to image and nanometrically track the motion of single myosin 5a heavy meromyosin molecules without the use of labels or any nanoscopic amplification. Together with the simple experimental arrangement, an intrinsic independence from strong electronic transition dipoles and a detection limit of <60 kDa, our approach paves the way toward nonresonant, label-free sensing and imaging of nanoscopic objects down to the single protein level.