Structural Characterization of Monomers and Oligomers of D-Amino Acid-Containing Peptides Using T-Wave Ion Mobility Mass Spectrometry

Abstract

The D-residues are crucial to biological function of D-amino acid containing peptides (DAACPs). Previous ion mobility mass spectrometry (IM-MS) studies revealing oligomerization patterns of amyloid cascade demonstrated conversion from native soluble unstructured assembly to fibril ß-sheet oligomers, which has been implicated in amyloid diseases, such as Alzheimer's disease and type 2 diabetes. Although neuropeptides are typically present at very low concentrations in circulation, their local concentrations could be much higher in large dense core vesicles, forming dimers or oligomers. We studied the oligomerization of protonated and metal-adducted achatin I and dermorphin peptide isomers with IM-MS. Our results suggested that dimerization, oligomerization, and metal adduction augment the structural differences between D/L peptide isomers compared to protonated monomers. Dimers and oligomers enhanced the structural differences between D/L peptide isomers in both aqueous and organic solvent system. Furthermore, some oligomer forms were only observed for either D- or L-isomers, indicating the importance of chiral center in oligomerization process. The oligomerization patterns of D/L isomers appear to be similar. Potassium adducts were detected to enlarge the structural differences between D/L isomers. Graphical Abstract ᅟ.

Keywords: CCS; Collision cross-section; Conformational differences; D-amino acid containing peptides; DAACPs; Dimer; IM-MS; Ion mobility mass spectrometry; Metal adducts; Monomer; Native state; Oligomer; Oligomerization pattern; Organic solvent.

Figure 1

Dimerization enhanced structural differences of peptide isomers. A) Achatin I (G(_D/L)FAD, MW 408.41) in 50% methanol/water, the relative CCS difference increased from 0.57% for the monomer to 2.91% for the dimer. B) Dermorphin (Y(_D/L)AFGYPS, MW 802.89) in 50% methanol/water, the relative CCS difference increased from null for the monomer to 1.2 % for the dimer. C) Dermorphin in aqueous solution, the relative CCS difference increased from null for the monomer to 1.1% for the dimer.

Figure 2

Oligomers of dermorphin isomers were detected in both 50% methanol/water for D-dermorphin (A) and L-dermorphin (B) and aqueous solution for D-dermorphin (C) and L-dermorphin (D). Each ion was annotated with its number of subunits and charge state. For example, 2/1 means a dimer at +1 charge state.

Figure 3

Drift time profiles of the dermorphin oligomers in aqueous buffer with some conformers that were unique to D- or L- isomers.

Figure 4

Dynamic conversion of dermorphin oligomer conformations in 50% methanol/water.

Figure 5

(A) Oligomerization pattern of dermorphin isomers in 50% methanol/water as shown in the collision cross section vs. aggregation state distribution. The oligomerization patterns of D/L dermorphin isomers appear to be similar. (B) The collision cross section difference between L- and D-dermorphin oligomers in 50% methanol/water, calculated as the CCS of L-isomers subtract the ones of the corresponding D-isomers. For monomer and oligomer with seven subunits, there was one single measurement and no error bar was obtained for these two data points.

Figure 6

The drift time profiles and their dynamic conversion of dermorphin oligomer conformations of potassium adducts in 50% methanol/water. Monomer and trimer adducts showed obvious difference between D- and L- forms, while the dimer and tetramer adducts did not. The conformational dynamic conversion studies with the ratio of D/L dermorphin changed among 100% L- form, 50/50 D- and L-forms, and 100% D-form showed that the 50/50 mixture preserved conformations from both D- and L-forms. For 100% D-form dermorphin M+K peak, there were two peaks, indicating the presence of at least two conformations.