Fig. 2
Concurrent mechanical profiling by atomic force spectroscopy. a Schematic representation of traditional single-molecule atomic force spectroscopy, in which data are obtained in multiple, independent experiments under different force calibration parameters. b Results from two independent atomic force microscopy (AFM) experiments in which a (C3)8 polyprotein is pulled. Due to uncertain calibration of the cantilever’s spring constant (\(k_{{\mathrm{sc}}}\)), force values are affected by errors that differ between experiments. We show two individual unfolding traces of the (C3)8 polyprotein, in which mechanical unfolding events of individual C3 domains are detected by increases of 24 nm in the length of the polyprotein. c Experimental cumulative unfolding probability distributions obtained from 117 (Experiment 1) and 191 (Experiment 2) C3 unfolding events. The corresponding \(\left\langle F_{\mathrm{{u}}} \right\rangle\) values are 98.7 and 82.9 pN, respectively. d Distributions of \(\Delta \left\langle {F_{\mathrm{u}}} \right\rangle\) obtained by Monte Carlo simulations, considering the same total number of experiments and unfolding events for both traditional (blue) and concurrent measurements (black). We considered two experiments, 200 total unfolding events per protein, and a 3.6% force calibration uncertainty (CU). e Relative standard deviation (RSD) of the distribution of \(\Delta \left\langle {F_{\mathrm{u}}} \right\rangle\) decreases with the total number of unfolding events both in traditional (blue) and in concurrent measurements (black). Number of experiments is 2. f Keeping the same number of events per experiment (nevents per experiment = 200, in the traditional approach, and 100 in the concurrent strategy), the RSD of the distribution of \(\Delta \left\langle {F_{\mathrm{u}}} \right\rangle\) decreases with the number of experiments (blue: traditional; black: concurrent). g The relative improvement in the RSD of \(\Delta \left\langle {F_{\mathrm{u}}} \right\rangle\) distributions by the concurrent strategy increases with the number of events per experiment, and remains fairly insensitive to the number of averaged experiments. h RSD of the distributions of \(\Delta \left\langle {F_{\mathrm{u}}} \right\rangle\) at increasing CU for the traditional (blue) and the concurrent (black) strategies. The remaining simulation parameters are as in d. In d–h, the number of events per experiment and protein in the concurrent approach was half of the number of events in the traditional strategy so that RSDs are compared between conditions with equal total number of events
