Fig. 1
Atomic force spectroscopy approaches to compare unfolding forces of two proteins. a Traditional strategy. The difference in mean unfolding force, \(\Delta \left\langle {F_{\mathrm{u}}} \right\rangle\), of Protein a and Protein b is estimated from different atomic force microscopy (AFM) experiments done under different calibration parameters (\(k_{{\mathrm{sc1}}}\) and \(k_{{\mathrm{sc2}}}\)). C-terminal cysteine residues used for covalent tethering to gold-covered coverslips are shown. b Concurrent strategy. By sampling Protein a and Protein b in the same experiment, calibration errors affect determination of the unfolding forces of both proteins equally. Hence, the accuracy in determination of \(\Delta \left\langle {F_{\mathrm{u}}} \right\rangle\) is improved. In concurrent strategies, either Protein a or Protein b are probed in different pulling attempts happening at different times within the same AFM experiment (temporal sequence is represented by Δt). Concurrent atomic force spectroscopy can be achieved using protein patterning methods22 or by orthogonal fingerprinting, as described in this report. c Orthogonal fingerprinting is based on concurrent AFM measurements of heteropolyproteins that include marker domains with different fingerprinting unfolding lengths. Both heteropolyproteins are homogeneously distributed on the surface and are picked up randomly throughout the same AFM experiment
