Diazirine-tagged D-and L-adrenaline derivatives formed abundant noncovalent gas-phase ion complexes with peptides N-Ac-SSIVSFY-NH 2 (peptide S) and N-Ac-VYILLNW-IGY-NH 2 (peptide V) upon electrospray ionization. These peptide sequences represent the binding motifs in the β 2 -adrenoreceptor. The structures of the gas-phase complexes were investigated by selective laser photodissociation of the diazirine chromophore at 354 nm, which resulted in a loss of N 2 and formation of a transient carbene intermediate in the adrenaline ligand without causing its expulsion. The photolyzed complexes were analyzed by collisioninduced dissociation (CID-MS 3 and CID-MS 4 ) in an attempt to detect cross-links and establish the binding sites. However, no cross-linking was detected in the complexes regardless of the peptide and D-or L-configuration in adrenaline. Cyclic ion mobility measurements were used to obtain collision cross sections (CCS) in N 2 for the peptide S complexes. These showed identical values, 334 ± 0.9 Å 2 , for complexes of the L-and D-adrenaline derivatives, respectively. Identical CCS were also obtained for peptide S complexes with natural L-and D-adrenaline, 317 ± 1.2 Å 2 , respectively. Born−Oppenheimer molecular dynamics (BOMD) in combination with full geometry optimization by density functional theory calculations provided structures for the complexes that were used to calculate theoretical CCS with the ion trajectory method. A close match (337 Å 2 ) was found for a single low Gibbs energy structure that displayed a binding pocket with Ser 2 and Ser 5 residues forming hydrogen bonds to the adrenaline catechol hydroxyls. Analysis of the BOMD trajectories revealed a small number of contacts between the incipient carbene carbon atom in the ligand and X−H bonds in the peptide, which was consistent with the lack of cross-linking. Temperature dependence of the internal dynamics of peptide S-adrenaline complexes as well as the specifics of the adrenaline carbene reactions are discussed. In particular, peptide amide hydrogen transfer to the carbene carbon atom was calculated to require crossing a potential energy barrier, which may hamper cross-linking in competition with carbene internal rearrangements.
Alzheimer’s disease (AD) is a neurodegenerative disorder of increasing concern. It belongs to diseases termed tauopathies which are characterized by inclusions of abnormally hyperphosphorylated and truncated forms of the protein tau. Studies of tauopathies often focus on detection and characterization of these aberrant tau proteoforms, in particular the phosphorylation sites, which represent a significant analytical challenge for example when several phosphosites can be present on the same peptide. Such isomers can even be difficult to fully separate chromatographically. Since recently introduced cyclic ion mobility–mass spectrometry can offer different selectivity, we have investigated the closely positioned phosphorylation sites S214, T212, and T217 of a tryptic peptide from proline rich region of tau–TPSLPTPPTREPK. The conformational heterogeneity of the isomeric peptides in the gas phase hindered their separation due to their overlapping arrival time distributions. Increasing the resolution of the analysis alone is insufficient to distinguish the peptides in a mixture typical of patient samples. We therefore developed a method based on a combination of collision-induced dissociation, isomeric product ions (m/z 677) mobility separation and post-mobility dissociation to aid in analyzing the isomeric phosphopeptides of tau in diseased brain extract. For all three isomers (T212, S214, and T217), the ion mobility signal of the ion at m/z 677 was still observable at the concentration of 0.1 nmol/L. This work not only offers insights into the phosphorylation of tau protein in AD but also provides an analytical workflow for the characterization of challenging pathological protein modifications in neurodegenerative diseases.
The efficiency of desorption/ionization becomes more critical as the sampled surface area decreases. Desorption electrospray and desorption nanoelectrospray belong to ambient ionizations and enable direct surface analysis including mass spectrometric imaging. Lateral resolution in tens of micrometers was demonstrated for desorption
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