Enzyme-less nanopore detection of post-translational modifications within long polypeptides

Abstract

Means to analyse cellular proteins and their millions of variants at the single-molecule level would uncover substantial information previously unknown to biology. Nanopore technology, which underpins long-read DNA and RNA sequencing, holds potential for full-length proteoform identification. We use electro-osmosis in an engineered charge-selective nanopore for the non-enzymatic capture, unfolding and translocation of individual polypeptides of more than 1,200 residues. Unlabelled thioredoxin polyproteins undergo transport through the nanopore, with directional co-translocational unfolding occurring unit by unit from either the C or N terminus. Chaotropic reagents at non-denaturing concentrations accelerate the analysis. By monitoring the ionic current flowing through the nanopore, we locate post-translational modifications deep within the polypeptide chains, laying the groundwork for compiling inventories of the proteoforms in cells and tissues.

Fig. 1. Electro-osmosis-driven translocation of Trx-linker concatemers through a protein nanopore.

a, EOF in a charge-selective αHL nanopore (NN-113R)₇ drives the sequential co-translocational unfolding of Trx units within a polyprotein of >1,000 aa. b, A sodium dodecyl sulfate–polyacrylamide gel showing the Trx-linker dimer (28 kDa), tetramer (55 kDa), hexamer (83 kDa) and octamer (110 kDa). c, Current recordings for the C-terminus-first translocation of a dimer, a tetramer, a hexamer and an octamer without post-acquisition filtering. The repeating features A are indicated by orange and blue bars. d. Zoomed-in view of the repeating feature A boxed in blue in c without post-acquisition filtering. Three levels are assigned as follows: A1, a linker within the pore; A2 and A3, different segments of partly unfolded Trx within the pore. Conditions in c and d are as follows: 750 mM GdnHCl, 10 mM HEPES, 5 mM TCEP at pH 7.2, Trx-linker concatemers (cis) (dimer: 2.23 μM; tetramer: 0.63 μM; hexamer: 0.25 μM; octamer: 0.81 μM), +140 mV (trans), 24 ± 1 °C. Source data

Fig. 2. Chaotrope-facilitated electro-osmotic translocation of the Trx-linker octamers through a nanopore.

a, Translocation of Trx-linker octamers through a weakly anion-selective WT αHL was not observed in the absence of a chaotrope. b–d, Current traces showing the translocation of Trx-linker octamers through the electro-osmotically active nanopore (NN-113R)₇ in the presence of 750 mM KCl (b), 750 mM KCl and 2 M urea (c) or 750 mM GdnHCl (d) with 2 kHz post-acquisition filtering. The use of non-denaturing concentrations of chaotropic agents (urea and GdnHCl) accelerated the co-translocational unfolding of the Trx units. Conditions: 10 mM HEPES at pH 7.2, 0.81 μM Trx-linker octamer (cis), +140 mV (trans), 24 ± 1 °C with 750 mM KCl (a and b); 2 M urea and 750 mM KCl (c); 750 mM GdnHCl (d).

Fig. 3. Detection of PTMs in protein concatemers traversing a nanopore driven by EOF.

a, Trx-linker nonamers tested with a charge-selective nanopore ((NN-113R)₇) containing an RRASAC sequence within the central linker, which was post-translationally phosphorylated (purple), S-glutathionylated (green) or glycosylated (yellow). b, Recordings of C-terminus-first translocation events of Trx-linker nonamers (left), showing a distinct level A1 (boxed in purple, green or yellow) in the presence of a PTM compared with the level A1 of unmodified units (orange dash). Traces have been filtered at 2 kHz; transient A3 levels were truncated by filtering and therefore deviate from ~0 pA. The A3 level produced by the translocation of an unmodified unit before the modified linker is indicated with a blue arrow and each of the features A is indicated by orange and blue bars. The number of repeats of feature A within the polypeptide translocation event shown is specified. Scatter plots of I_r.m.s. and ΔI_res% for individual polypeptide translocation events (right), where ΔI_res% = <I_res%(A1, Trx-linker)> – I_res%(A1, Trx-linker + PTM), where <I_res%(A1, Trx-linker)> is the mean I_res% value of the remaining A1 levels for unmodified repeat units within an individual translocation event. Conditions, 375 mM GdnHCl, 375 mM KCl, 10 mM HEPES at pH 7.2, 1.2 μM Trx-linker nonamer (cis), +140 mV (trans), 24 ± 1 °C. Source data

Extended Data Fig. 1. Repeating current features recorded during electroosmosis-driven concatemer translocation through a nanopore.

a, Two repeating current features, A or B, were recorded with a charge-selective nanopore ((NN-113R)₇) and Trx-linker octamers pre-treated with 5 mM tris(2-carboxyethyl)phosphine (TCEP) for 10 min before their addition to the cis compartment of the recording chamber. Conditions: 750 mM GdnHCl, 10 mM HEPES, 5 mM TCEP, pH 7.2, 0.81 μM Trx-linker octamer (cis), +140 mV (trans), 24 ± 1 °C. b, Without the TCEP pre-treatment, features A were always seen before features B when they occurred together within a single translocation event. The first two levels (B1 and B2) in features B have larger noise and higher I_res% compared with A1 and A2 recorded within a single polypeptide translocation event by the same pore (A1: I_res%= 35 ± 1 %, I_r.m.s. = 1.1 ± 0.1 pA, N = 25; A2: I_res% = 21 ± 1%, I_r.m.s. = 1.5 ± 0.2 pA, N = 25; B1: I_res%= 38 ± 1 %, I_r.m.s. = 1.7 ± 0.4 pA, N = 39; B2: I_res% = 32 ± 1%, I_r.m.s. = 2.0 ± 0.5 pA, N = 39; I_r.m.s. values for each level were reported without subtraction of the noise of the pore; number of individual levels from multiple polypeptide translocation events recorded by the same pore are specified). The translocating molecules, which gave sequential A and B features, were assigned as dimers of octamers linked by a disulfide bond between the two N-terminal cysteines. Therefore, in the unlinked molecules (see ‘a’), C terminus-first translocation occurred when features A were observed and N terminus-first translocation occurred when features B were observed. The recorded repeating features are indicated by orange and blue bars. Conditions: 750 mM GdnHCl, 10 mM HEPES, pH 7.2, 0.81 μM Trx-linker octamer (cis), +140 mV (trans), 24 ± 1 °C. All traces were filtered at 2 kHz for clarity; transient A3 levels were truncated by filtering and therefore deviated from ~0 pA.