Protein conformations can be probed in top-down HDX MS experiments utilizing electron transfer dissociation of protein ions without hydrogen scrambling

Abstract

Electron-transfer dissociation (ETD) is evaluated as a technique to provide local information on higher order structure and dynamics of a whole protein molecule. Isotopic labeling of highly flexible segments of a model 18 kDa protein is carried out in solution under mildly denaturing conditions by means of hydrogen/deuterium exchange (HDX), followed by transfer of intact protein ions to the gas phase by means of electrospray ionization, and mass-selection of a precursor ion for subsequent reactions with fluoranthene radical anions. The ETD process gives rise to abundant fragment ions, whose deuterium content can be measured as a function of duration of the HDX reaction in solution. No backbone protection is detected for all protein segments spanning the 25-residue long N-terminal part of the protein, which is known to lack structure in solution. At the same time, noticeable protection is evident for segments representing the structured regions of the protein. The results of this work suggest that ETD of intact protein ions is not accompanied by detectable hydrogen scrambling and can be used in tandem with HDX to probe protein conformation in solution.

Figure 1

Tertiary structure and sequence of wt*-CRABP I showing the His-tag (red), the unstructured N-terminal segment of the wild-type protein (orange) and the first N-terminal segment forming a stable element of its secondary (strand β1, blue).

Figure 2

Evolution of isotopic distributions of c₂₃ ³⁺ and c₃₁ ⁴⁺ fragment ions in ETD mass spectra of wt*-CRABP I undergoing HDX in solution (black traces). The gray traces represent the end-point of the exchange reaction

Figure 3

Blue bars: distribution of deuterium atoms retained by ETD fragments of fully deuterated wt*-CRABP I exposed to protiated buffer (room temperature, pH 3.0) for 30 sec obtained by deconvoluting the isotopic distributions of each fragment ion at the end-point of the exchange reaction from that at following 30 sec of exchange. Brown bars (insets): distribution of retained deuterium atoms within short protein segments obtained by deconvoluting isotopic distributions of overlapping fragment ions.