Characterization of supramolecular peptide-polymer bioconjugates using multistage tandem mass spectrometry

Abstract

Mass spectrometry (MS) is an essential analytical tool for the characterization of the structure of biological macromolecules, including protein-protein and protein-ligand complexes. One-dimensional MS separates gas-phase analyte ions based on their mass to charge ratio (m/z); however, to obtain more detailed structural information, tandem MS (MS/MS), which involves isolation and subsequent fragmentation of a precursor ion, is required.In this thesis, electrospray ionization multistage tandem mass spectrometry (ESI-MS n ) was employed to examine the non-covalent complexes between poly(styrene sulfonate) (PSS) and poly-L-lysine (PLL). During single-stage ion activation, the PLL peptide chain mainly underwent backbone cleavages without disruption of the noncovalent interaction which could only be broken via sequential application of electron transfer dissociation (ETD) and collisionally activated dissociation (CAD), indicating strong binding interactions between the two polyelectrolyte chains. Such binding properties make PSS a potential "non-covalent (supramolecular) label" for determining the surface accessibility of basic residues on a peptide or protein. To probe this premise, noncovalent complexes of substance P and PSS were characterized by ESI-MS n using different ion activation methods. Both MS 2 and MS 3 experiments on the substance P + PSS complex resulted in the formation of bn (on CAD) or cn (on ETD) fragments attached non-covalently to the intact PSS chain. All peptide fragments containing the intact PSS chain included Arg1, Lys3, and Gln5, pointing out that these residues, which are located near the Nterminus, are most likely involved in the noncovalent interaction with PSS. In contrast, iv Gln6 was excluded from this fragment series, attesting a much weaker interaction with PSS due to lesser accessibility. The strong tendency of PSS to bind peptides noncovalently at sites that can be elucidated by MS n demonstrates a proof-of-concept for the capacity of this approach to unveil higher order structure in proteins.

Sequencing of Side-Chain Liquid Crystalline Copolymers by Matrix-Assisted Laser Desorption/Ionization Tandem Mass Spectrometry

“…MS/MS analysis of multiply charged ions formed by electrospray ionization (ESI) may extend sequencing capabilities beyond this range [39]. Moreover, multiply charged ions can be energized to dissociate not only thermally (as done here), but also by electron-based activation methods such as electron capture dissociation and electron transfer dissociation [40,41], which often improve sequence coverage [42,43]. Finally, MS/MS can be interfaced with ion mobility separation of either the precursor oligomers and/or their fragments to gain more insight into the microstructures and architectures of SCLC materials [14,39,42].…”

Mass spectrometry of polymers: A tutorial review

“…ETD applications have been more widespread and so far have covered polyesters (Katzenmeyer et al, 2015;Prian et al, 2019;Scionti & Wesdemiotis, 2012a), polyacrylamides (Gerișlioǧlu & Wesdemiotis, 2017), and polymer-peptide bioconjugates (B. Wei et al, 2019). The consensus today is that the radical ions emerging after ECD or ETD primarily undergo radical-induced…”

“…ECD has been applied to polyethers (Cerda et al, 2001, 2002) and polyoxazolines (Morgan et al, 2018). ETD applications have been more widespread and so far have covered polyesters (Katzenmeyer et al, 2015; Prian et al, 2019; Scionti & Wesdemiotis, 2012a), polyacrylamides (Gerișlioǧlu & Wesdemiotis, 2017), and polymer–peptide bioconjugates (B. Wei et al, 2019). The consensus today is that the radical ions emerging after ECD or ETD primarily undergo radical‐induced dissociations, promoted by the newly formed radical site (Katzenmeyer et al, 2015; Prian et al, 2019).…”