Sequence and Conformational Analysis of Peptide–Polymer Bioconjugates by Multidimensional Mass Spectrometry

Abstract

The sequence and helical content of two alanine-rich peptides (AQK18 and GpAQK18, Gp: l-propargylglycine) and their conjugates with poly(ethylene glycol) (PEG) have been investigated by multidimensional mass spectrometry (MS), encompassing electrospray ionization (ESI) or matrix-assisted laser desorption ionization (MALDI) interfaced with tandem mass spectrometry (MS) fragmentation and shape-sensitive separation via ion mobility mass spectrometry (IM-MS). The composition, sequence, and molecular weight distribution of the peptides and bioconjugates were identified by MS and MS experiments, which also confirmed the attachment of PEG at the C-terminus of the peptides. ESI coupled with IM-MS revealed the existence of random coil and α-helical conformers for the peptides in the gas phase. More importantly, the proportion of the helical conformation increased substantially after PEG attachment, suggesting that conjugation adds stability to this conformer. The conformational assemblies detected in the gas phase were largely formed in solution, as corroborated by independent circular dichroism (CD) experiments. The collision cross sections (rotationally averaged forward moving areas) of the random coil and helical conformers of the peptides and their PEG conjugates were simulated for comparison with the experimental values deduced by IM-MS in order to confirm the identity of the observed architectures and understand the stabilizing effect of the polymer chain. C-terminal PEGylation is shown to increase the positive charge density and to solvate intramolecular positive charges at the conjugation site, thereby enhancing the stability of α-helices, preserving their conformation and increasing helical propensity.

Ion mobility spectrometry – Mass spectrometry coupling for synthetic polymers

“…In addition to metal adduction, protonation was also envisaged in particular cases where synthetic polymers were conjugated with moieties of high proton affinity, in particular peptides . For example, Wesdemiotis and co‐workers reported that the helical structure of alanine‐rich peptides is further stabilized upon conjugation with PEG via their C‐terminus . The simulated CCS value (1040 Å 2 ) that best matches the experimental TW CCS N2 value (1020 Å 2 ) obtained for the quadruply protonated AQK18‐PEG 70 at m/z 1191.4 is derived from the architecture where two protons are located at glutamine Q17 and lysine K18 while the two other charges are attached on PEG (Figure a): under such conditions, hydrogen‐bonding interactions are evidenced between the PEG chain and the C‐terminal peptide segment, stabilizing positive charges at the C‐terminus and further enhancing the stability of the α‐helical structure.…”

“…In addition to metal adduction, protonation was also envisaged in particular cases where synthetic polymers were conjugated with moieties of high proton affinity, in particular peptides . For example, Wesdemiotis and co‐workers reported that the helical structure of alanine‐rich peptides is further stabilized upon conjugation with PEG via their C‐terminus .…”

“…a, α‐helical structure of [AQK18‐PEG 70 + 4H] 4+ holding one proton at glutamine Q17 and lysine K18 and two protons on PEG. Adapted with permission from Sallam et al Copyright 2018 American Chemical Society. b, Theoretical (open symbols) and experimental TW CCS N2 (filled symbols) values of PEO n ‐PAMAM conjugates when adducted to two H + (blue) or two Li + (orange) as a function of the DP n of the PEO segment.…”

Mass spectrometry in bioresorbable polymer development, degradation and drug‐release tracking

“…Both MALDI and ESI MS, in some cases with the support of MS/MS and/or IM‐MS, have been used in the characterization of polymer conjugates, to confirm the effective functionalization and the success of the synthetic approch . The transesterification reaction of PHAs has been used as a strategic and simple tool for the synthesis of delivery systems for selected bioactive compounds, holding carboxyl or hydroxyl functionalities.…”

“…MALDI or ESI) with IM‐MS and MS/MS fragmentation, relevant insights into the composition, structure, and architecture of bioconjugates and other complex biomacromolecules have been gained. Sallam et al highlighted the usefulness of combining MALDI, ESI, MS/MS and IM‐MS experiments for the comprehensive characterization of the primary structure and architecture of a polyether dendron conjugated with two different bioactive peptides . In addition, MS/MS and IM‐MS/MS techniques were applied by Alawiat et al in the determination of the sequence, derivatization site, and conformation of two alanine‐rich peptides (AQK18 and GpAQK18, Gp: Lpropargylglycine) and their conjugates with PEG.…”