Single-molecule site-specific detection of protein phosphorylation with a nanopore

Abstract

We demonstrate single-molecule, site-specific detection of protein phosphorylation with protein nanopore technology. A model protein, thioredoxin, was phosphorylated at two adjacent sites. Analysis of the ionic current amplitude and noise, as the protein unfolds and moves through an α-hemolysin pore, enables the distinction between unphosphorylated, monophosphorylated and diphosphorylated variants. Our results provide a step toward nanopore proteomics.

Figure 1

Single-molecule nanopore detection of phosphorylation of a model substrate. (a) Current signature of the unfolding and translocation of TrxS112^−P-oligo(dC)₃₀ through an αHL pore showing the four characteristic levels: (1) open pore; (2) oligonucleotide leader threaded into the pore; (3) C terminus of the protein substrate in the pore; (4) unfolding of the remainder of the protein and diffusion through the pore. (b) Sequences of the C termini of Trx mutants used in this work. Phosphorylatable Ser residues, red; non-phosphorylatable Ala residues, black. (c) Model of TrxS112^−P: phosphorylatable Ser-112, red ball. (d) Current signature of TrxS112^−P-oligo(dC)₃₀. (e) Signature of TrxS112^+P-oligo(dC)₃₀. (f) Representative scatter plot of the residual current (I_RES%) and noise (I_n) in levels 3 of TrxS112^−P-oligo(dC)₃₀ and TrxS112^+P-oligo(dC)₃₀ and the associated histograms (200 events were recorded). (g) Model of TrxS107^−P, phosphorylatable Ser-107, red ball. (h) Current signature of TrxS107^−P-oligo(dC)₃₀. (i) Signature of TrxS107^+P-oligo(dC)₃₀. (j) Representative scatter plot of I_RES% and I_n in levels 3 of TrxS107^−P-oligo(dC)₃₀ and TrxS107^+P-oligo(dC)₃₀ and the associated histograms (199 events). (k) Model of TrxS95^−P, phosphorylatable Ser-95, red ball. (l) Current signature of TrxS95^−P-oligo(dC)₃₀. (m) Signature of TrxS95^+P-oligo(dC)₃₀. (n) Representative scatter plot of I_RES% and I_n in levels 3 of TrxS95^−P-oligo(dC)₃₀ and TrxS95^+P-oligo(dC)₃₀ and the associated histograms (250 events). All measurements were done at +140 mV. The experiments in (f), (j) and (n) were repeated three times.

Figure 2

Single-molecule nanopore detection of four phosphorylation states. (a–f) Representative ionic current traces at +140 mV for TrxS107^−P/S112^−P (a), TrxS107^+P/S112^+P (b), TrxA107/S112^−P (c), TrxA107/S112^+P (d), TrxS107^−P/A112 (e) and TrxS107^+P/A112 (f). The Trx constructs were fused at the C terminus to oligo(dC)₃₀. (g) Representative scatter plot and histograms of the residual currents (I_RES%) and noise (I_n) at +140 mV in level 3 (Fig. 1) for the four phosphorylation states in a, b, d and f. Only the low conductance substate is analyzed (see Supplementary Fig. 10). The same pore was used throughout and the cis compartment was perfused before the addition of each Trx variant. In total, 342 events were recorded, and a few events may be due to carry over because of incomplete perfusion. The ability of the same pore to distinguish the four constructs was verified twice. (h–i) Single-molecule nanopore detection of a sample containing a mixture of phosphorylation states. (h) Fraction of TrxS107^−P/S112^−P converted over time to the doubly phosphorylated TrxS107^+P/S112^+P as determined by IEF. (i) Representative scatter plot and histograms of I_RES% and I_n in level 3 after 2 h of phosphorylation, followed by conjugation to oligo(dC)₃₀ and αHL nanopore analysis at +140 mV. In total, 205 events recorded, of which three were discarded as too short (<30 ms) for analysis. The boxes delimit areas obtained as shown in Supplementary Figure 11. The ability to distinguish the components of the mixture was verified with a second pore.

Figure 3