Mapping Hydrogen Migration Thresholds for Site-Specific HDX-MS

Abstract

A long-standing limitation of Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) has been the difficulty in accurately measuring amide exchange with single amide resolution. Excitation of peptides or proteins during ionization, ion transmission, or collisional activation rapidly induces intermolecular hydrogen migration, leading to a loss of the deuterium-labeled state; a term commonly known as "scrambling." Electron-based fragmentation methods in conjunction with gentle ion transmission settings can minimize scrambling but often not completely. Levels of scrambling have been shown to vary with ion transmission settings, peptide charge, and size, but the general properties that govern the susceptibility of peptides to scrambling are not well understood. Furthermore, it remains unclear whether scrambling is generally a global process or if local scrambling networks commonly exist within peptides. Here, we examine a panel of peptides using gentle electron transfer dissociation and map the activation thresholds of scrambling to define a relationship between peptide charge density and scrambling propensity. This study suggests that by and large, the scrambling process has a single activation threshold and involves all exchangeable sites within a peptide. For some peptides, the activation energy required for scrambling is surprisingly close to that of amide bond dissociation.

Keywords: HDX-MS; hydrogen migration; hydrogen–deuterium exchange; scrambling; site-specific.

Graphical abstract

Fig. 1

Scrambling levels observed for the 3+ charge state of peptide P1 with optimized ion transmission settings for low scrambling (Gentle) or elevated source fragmentation energy (Harsh) to achieve maximal scrambling. A, B, the observed deuterium uptake for the c₇ and z₇ ions are shown. C, D, Theoretical uptake for no scrambling (green) and 100% scrambling (red) are shown for the c and z ion series as dashed lines. The observed deuterium levels with low scrambling and 100% scrambling conditions are shown in blue and orange, respectively. Error bars represent standard deviations from triplicate measurements.

Fig. 2

Scrambling levels for the c and z ion series for peptide P1 using either ETD or EThcD with varying levels of supplemental activation (SA) with HCD for the 3+ precursor (A, B) and the 2+ precursor ions (C, D). Theoretical curves for no scrambling and 100% scrambling are shown as pink and gray dashed lines.

Fig. 3

Determining the activation thresholds for scrambling through source fragmentation ramping.A, the deuterium uptake for the c₅ fragment ion of the 3+ charge state of peptide P1 with ETD is shown for a range of source fragmentation settings. Theoretical values for 0% and 100% scrambling are shown as green and red dashed lines. A sigmoidal fit is used to calculate the source fragmentation energy for the point at where 50% scrambling is achieved (‘scram₅₀’). B, deuterium uptake through varied source energy is shown for all observable c/z ions. The deuterium uptake values were scaled to relative scrambling %, and the overlays are shown in (C). Scram₅₀ curves for 2+ and 4+ charge states are shown in Figure S5.

Fig. 4

Summary of scrambling thresholds.A, Relationship between the source activation energy for 50% scrambling (scram₅₀) for the panel of peptides vs. their m/z. Colors show charge states, and peptides are labeled in the inset. B, the lab-frame energy (scram₅₀ x charge state) is shown for the same set of peptides, indicating that different charge states for several peptides are inherently different with regard to their susceptibility to scrambling. C, relationship between scram₅₀ and the source fragmentation energy for 50% fragmentation (frag₅₀). The dotted line represents the linear trends with statistics shown in the inset. Dashed lines show positions of frag₅₀ = scram₅₀ (orange) and frag₅₀=2·scram₅₀ (purple) for visual reference.