Fig. 6
Orthogonal-fingerprinting-based concurrent atomic force spectroscopy. In the traditional approach, comparison of the mechanical stability of Protein a and Protein b involves independent purification and several atomic force microscopy (AFM) experiments to compensate for inaccurate force calibration. Orthogonal fingerprinting (OFP) is based on the production of heteropolyproteins composed of the proteins of interest fused to marker domains. Since the markers provide unequivocal fingerprints in single-molecule pulling experiments, OFP enables simultaneous purification and concurrent mechanical measurements, circumventing errors in force calibration. Concurrent measurements can achieve the same accuracy in \(\Delta \left\langle {F_{\mathrm{u}}} \right\rangle\) as conventional single-molecule atomic force spectroscopy with much better throughput, as shown in the left part of the figure. Alternatively, by keeping the speed of data acquisition constant, concurrent measurements considerably improve the accuracy in the determination of \(\Delta \left\langle {F_{\mathrm{u}}} \right\rangle\), as exemplified in the right part of the figure. Improvements in throughput and accuracy are estimated from Monte Carlo simulations at 10.8% calibration uncertainty (100 events per experiment and protein)
