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Concurrent Atomic Force Spectroscopy ARTICLE https://doi.org/10.1038/s42005-019-0192-y OPEN Concurrent atomic force spectroscopy Carolina Pimenta-Lopes 1,2,3, Carmen Suay-Corredera 1,3, Diana Velázquez-Carreras 1, David Sánchez-Ortiz1 & Jorge Alegre-Cebollada 1 1234567890():,; Force-spectroscopy by atomic force microscopy (AFM) is the technique of choice to measure mechanical properties of molecules, cells, tissues and materials at the nano and micro scales. However, unavoidable calibration errors of AFM probes make it cumbersome to quantify modulation of mechanics. Here, we show that concurrent AFM force measurements enable relative mechanical characterization with an accuracy that is independent of calibration uncertainty, even when averaging data from multiple, independent experiments. Compared to traditional AFM, we estimate that concurrent strategies can measure differences in protein mechanical unfolding forces with a 6-fold improvement in accuracy or a 30-fold increase in throughput. Prompted by our results, we demonstrate widely applicable orthogonal finger- printing strategies for concurrent single-molecule nanomechanical profiling of proteins. 1 Centro Nacional de Investigaciones Cardiovasculares Carlos III (CNIC), 28029 Madrid, Spain. 2Present address: Department of Physiological Sciences, University of Barcelona, Barcelona, Spain. 3These authors contributed equally: Carolina Pimenta-Lopes, Carmen Suay-Corredera. Correspondence and requests for materials should be addressed to J.A.-C. (email: [email protected]) COMMUNICATIONS PHYSICS | (2019) 2:91 | https://doi.org/10.1038/s42005-019-0192-y | www.nature.com/commsphys 1 ARTICLE COMMUNICATIONS PHYSICS | https://doi.org/10.1038/s42005-019-0192-y he development of atomic force microscopy (AFM) has In theory, a manner to overcome calibration errors in relative Tenabled imaging the nanoscale with unprecedented length atomic force spectroscopy is to measure the samples in a single resolution, revolutionizing nanotechnology, materials sci- experiment using one cantilever under the same calibration para- ence, chemistry, and biology1–3. AFM is based on the detection of meters, which we term concurrent atomic force spectroscopy interaction forces between a sample and a microfabricated can- (Fig. 1b)21. Indeed, concurrent mechanical characterization of tilever, the force probe of the technique. Traditionally, low- several proteins in a single experiment has been achieved using scanning speeds have limited the reach of AFM; however, recent microfluidics, on-chip protein patterning, and force spectroscopy developments involving miniaturized cantilevers have achieved measurements in custom-built atomic force/total internal reflection imaging at video frame rates, launching the field of high-speed fluorescence microscopes22,23. However, this advanced technology AFM4. The ability of AFM to measure forces at the nano and is not available to most AFM users, and the extent of improvement micro scale can be exploited to quantify mechanical parameters in performance by concurrent AFM remains unexplored. such as stiffness, viscoelasticity, or adhesion forces, while simul- Here, we use error propagation analysis and Monte Carlo taneously imaging surface topology2,5,6. Due to its force sensi- simulations to examine how force calibration errors impact tivity down to picoNewtons (pN), AFM-based force spectroscopy, determination of mechanical properties of proteins by atomic or simply atomic force spectroscopy, can also be used to examine force spectroscopy, and demonstrate the remarkable improve- single-molecule dynamics and ligand–receptor interactions under ment that stems from concurrent measurements. We find that force7–9 of relevance for cellular stiffness, mechanosensing, and averaging results from multiple concurrent experiments retains mechanotransduction10–13. statistical power, leading to drastically improved accuracy and A key limitation of atomic force spectroscopy stems from throughput in the determination of relative mechanical properties fi uncertain calibration of the spring constant (ksc) of the AFM by AFM. Prompted by our ndings, we have developed ortho- cantilever, which is needed to determine force values14.Different gonal fingerprinting (OFP) as a simple and widely applicable fi calibration methods to estimate ksc have been developed, differing strategy for concurrent single-molecule nanomechanical pro ling in their simplicity, damage to the cantilever tip, experimental of proteins (Fig. 1c). compatibility, and associated uncertainty. Estimates of calibration uncertainty (CU) up to 25% are usually reported;15 however, even higher inaccuracies can result from defects in individual canti- Results levers, and from unpredictable changes in ksc during experi- Interexperimental errors in traditional force spectroscopy.To mentation due to material deposition, mechanical drift, and understand how errors in calibration of AFM lead to inaccurate wear7,16. determination of mechanical properties, we have measured pro- Force calibration uncertainties in AFM lead to inaccurate tein mechanical stability by single-molecule force spectroscopy quantification of mechanical properties, a situation that is AFM7 (Fig. 2a, Supplementary Fig. 1). This AFM mode is very worsened in modes where the elusive geometry of the cantilever well suited to examine propagation of calibration errors since tip is required to extract mechanical information17. Indeed, protein unfolding forces are obtained directly from experimental relative AFM studies to characterize mechanical modulation of data and do not depend on further modelling or approximations. proteins, materials, and cells are challenging, since they We first measured the resistance to mechanical unfolding of necessitate multiple experiments, each one affected by different the same protein in different, independently calibrated atomic calibration errors (Fig. 1a)18,19. As a result, there is a pressing force spectroscopy experiments. We produced a polyprotein need to develop methods that can overcome inaccurate AFM containing eight repetitions of the C3 domain of human cardiac force calibration18. Averaging results from several experiments myosin-binding protein C (Supplementary Fig. 2, Supplementary obtained with different cantilevers can reduce the systematic Note 1) and subjected individual (C3)8 polyproteins to a linear error, since individual calibration errors are more probable to increase in force of 40 pN s−1 using a force-clamp AFM. Results be averaged out as more experiments are included in the ana- from two independent experiments are shown in Fig. 2b, c. In any lysis20. The drawback of this method, which we refer to as given AFM experiment, several single-molecule events coming traditional atomic force spectroscopy, is a considerable loss of from different molecules are recorded with a single cantilever, throughput of the technique. under the same calibration parameters. In these single-molecule abc TRADITIONAL CONCURRENT Experiment 1 Experiment 2 Same experiment Marker 1 Marker 2 k k Methods: sc 1 sc 2 Δt Δ - Orthogonal t fingerprinting - Patterning Cys Cys Cys Cys Δ Protein a <Fu>? Protein b Protein a & Protein b Markers with different unfolding lengths Fig. 1 Atomic force spectroscopy approaches to compare unfolding forces of two proteins. a Traditional strategy. The difference in mean unfolding force, ΔhiF k u , of Protein a and Protein b is estimated from different atomic force microscopy (AFM) experiments done under different calibration parameters ( sc1 k and 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 ΔhiF determination of u 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 2 COMMUNICATIONS PHYSICS | (2019) 2:91 | https://doi.org/10.1038/s42005-019-0192-y | www.nature.com/commsphys COMMUNICATIONS PHYSICS | https://doi.org/10.1038/s42005-019-0192-y ARTICLE acb Δ Experiment 1 <Fu> (k ) C3 1.0 sc 1 C3 C3 Lehtgn C3 0.8 C3 0.6 <<FFu>22 <Fu>1 Experiment 2 0.4 C3 (ksc 2) C3 0.2 C3 Lehtgn C3 C3 Probability of unfolding 0.0 25 nm 0.25 s 50 100 150 150 Force Force Force (pN) 0 Force (pN) d e f 70 10 6 60 Traditional Traditional Traditional Concurrent 8 Concurrent 5 Concurrent 50 3.6% CU 3.6% CU 4 3.6% CU 40 6 3 30 4 2 20 # of observations Relative SD (%) 3.2% RSD Relative SD (%) 2 10 1 5% RSD 0 0 0 –20 –10 0 10 20 0 100 200 300 400 500 0 5 10 15 20 25 30 Δ <Fu> (pN) Total number of events Number of experiments g h 3.6% CU 25 Traditional 2 20 Concurrent 1.8 15 1.6 10 1.4 Relative SD (%) 5 RSD (Traditional) RSD RSD (Concurrent) RSD 1.2 0 500 4 6 8 10 12 14 16 18 30 400 20 300 Calibration uncertainty (%) # of experiments10 200 0 100 # of events per traditional experiment Fig. 2 Concurrent mechanical profiling by atomic force spectroscopy. a
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