Fig. 3
Orthogonal fingerprinting (OFP) enables concurrent measurement of proteins. a In traditional experiments, (C3)8 and (C3-L)4 are measured in different atomic force microscopy (AFM) experiments, while in OFP, either one or the other protein are measured in single pulling attempts within the same AFM experiment (temporal sequence is represented by Δt; the protein being pulled at each time is highlighted). b Mechanical unfolding of (C3)8 (red) and (C3-L)4 (black) were measured in separate experiments. Individual traces were classified according to the number of 16 and 24 nm steps they contain. We show representative unfolding traces of one (C3)8 molecule (red), in which five 24 nm unfolding events are detected, and one (C3-L)4 polyprotein (black), comprising two 24 nm and three 16 nm unfolding events. The graph plot shows the frequency of the traces that have different combinations of unfolding events, as indicated by the size of the dots. The rectangles represent gating strategies that identify traces coming from (C3)8 or (C3-L)4. Number of traces: (C3)8, 59; (C3-L)4, 129. c Experimental cumulative unfolding probability distribution of the C3 domain in the context of (C3)8 (11 experiments, 1334 events, red) and (C3-L)4 (6 experiments, 177 events, black), following traditional AFM. d (C3)8 and (C3-L)4 polyproteins were measured concurrently by orthogonal fingerprinting (OFP). 468 traces were classified according to the number of 16 and 24 nm events. The plot shows the frequency of the traces that have different combinations of unfolding events, as indicated by the size of the dots. The gating criterion defined in b allows the classification of individual traces as resulting from (C3)8 (red rectangle) or (C3-L)4 (black rectangle). e Experimental cumulative unfolding probability distribution of the C3 domain in the context of (C3)8 (625 events, red) and (C3-L)4 (311 events, black), resulting from unfolding data obtained in five independent OFP experiments. Relative standard deviation (RSD) values in the insets in c, e are estimated using Monte Carlo simulations that consider extreme values of calibration uncertainty (CU). The pulling rate in all experiments was 40 pN s−1
