Fig. 1. Principle of MSPT.

a, Schematic displaying the iSCAT-based measurement principle of MSPT. Exemplary structures of three aldolase oligomer states (PDB 4S1F, ref. ⁵¹) are shown in the top panel, and their respective iSCAT images at the bottom. Scale bar, 1 µm. b, Probability density distributions of standard proteins determined using the conventional mass photometry landing assay (left) or using MSPT (right). All data represent pooled distributions of three independent experiments per condition: alcohol dehydrogenase (ADH) (particle number n = 9,828), BSA (n = 11,408), TEV protease (TEV) (n = 1,705), β-amylase (bAm) (n = 10,043), protein A (prA) (n = 12,720); divalent streptavidin (Strep) (n = 16,699 trajectories), divalent streptavidin with biotinylated aldolase (Strep-ALD) (n = 16,727 trajectories), divalent streptavidin with biotinylated BSA (Strep-BSA) (n = 8,842 trajectories) and divalent streptavidin with biotinylated protein A (Strep-prA) (n = 22,424 trajectories). Dashed lines mark peaks not considered for mass calibration (left). Continuous lines represent oligomer states included in the mass calibration. Two-dimensional plots of mass versus diffusion coefficient for the four proteins measured with MSPT (right) are shown in Supplementary Fig. 4. c, Comparison of the contrast-to-mass calibration for mass photometry and MSPT, derived from peak contrasts in b and their assigned sequence masses (Supplementary Tables 2 and 3). Error bars represent the standard error of the peak locations estimated by bootstrapping. d, Two-dimensional KDE of 1.25 nM tetravalent streptavidin bound to biotinylated lipids on a SLB (n = 73,901 trajectories of three independent replicates; particle density: 0.2 µm⁻²). Marginal probability distributions of the molecular mass (top) and the diffusion coefficient (right) are presented.

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