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

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.


Table of Contents:
Table S1

Trx-linker octamer
Trx-linker octamer [c]   Trx-linker octamer [d]   Ires% (A1) [b] Trx-linker units that produced a Level A3 with a dwell time <1 ms were discarded during analysis.The associated spikey appearance suggested under-sampling and therefore an inaccurate Ires% value.Trx-linker units that generated a Level A3 with a dwell time >1 ms and a square shape were included in the Ires% analysis.
[d] Conditions: 750 mM GdnHCl, 10 mM HEPES, 0.81 μM octamer (trans), pH 7.2, -140 mV (trans), 24 ± 1 °C.A sub-conductance level was seen at Level A, which might be attributed to the folded thioredoxin unit adopting two conformations as a stopper under the electroosmotic force at the trans opening of the pore.

Table S3 Frequency of C terminus-first or N terminus-first translocation events recorded with
Trx-linker concatemers [a] .[a] If the initial A3 level appeared as a spike to ~0 pA and was followed by at least one complete iteration of feature A, it was counted as one repeat.
[c] Root-mean-square noise values (IRMS) were measured from current traces after an applied postrecording filter at 2 kHz.IRMS was normalised by the noise of each pore (IRMS 2 = IRMS(A1, Trx-linker+PTM) 2 -IRMS(open pore) 2 ).3b).Given that the applied potential drops mostly across the transmembrane β barrel 2 , the current difference between 14S/16C+PTM and 24S/26C+PTM is likely to be larger in c than in d.

Fig. S6
Fig.S6Positions of modification sites during translocation through an αHL pore.a, The Trxlinker nonamers contained a RRASAC sequence within the central linker, which was posttranslationally modified (hexagon).In a C-terminus-first threading configuration, as shown, the 14S/16C modification sites would be located closer to the cis opening of the αHL pore than the 24S/26C pair, when translocation is paused with a Trx unit at the cis mouth of the pore.b-d, Depending on the degree of extension of the polypeptide chain under the EOF (3.5 Å per aa when fully extended, 1.7-2.2Å per aa under ~5-10 pN 1 ), the 14S/16C and 24S/26C sites would be located at different positions within an αHL pore.Assuming that the N-terminal residue of the linker is at the cis opening of the pore when the translocation is arrested by a folded Trx unit, the modified linker (red) might fully span the αHL pore (b) or occupy only a part of the nanopore (c,d).When the 24S/26C sites are located nearer the central constriction of the αHL pore (c,d), a PTM at 24S/26C would produce a larger current blockade than that at 14S/16C (PTM = Ser-P, Cys-GSH, Cys-SLN), which is what is observed (Fig.3b).Given that the applied potential drops mostly across the transmembrane β barrel 2 , the current difference between 14S/16C+PTM and 24S/26C+PTM is likely to be larger in c than in d.