Gas-phase microsolvation of ubiquitin: investigation of crown ether complexation sites using ion mobility-mass spectrometry

Abstract

In this study the gas-phase structure of ubiquitin and its lysine-to-arginine mutants was investigated using ion mobility-mass spectrometry (IM-MS) and electron transfer dissociation-mass spectrometry (ETD-MS). Crown ether molecules were attached to positive charge sites of the proteins and the resulting noncovalent complexes were analysed. Collision induced dissociation (CID) experiments revealed relative energy differences between the wild type and the mutant crown-ether complexes. ETD-MS experiments were performed to identify the crown ether binding sites. Although not all of the binding sites could be revealed, the data confirm that the first crown ether is able to bind to the N-terminus. IM-MS experiments show a more compact structure for specific charge states of wild type ubiquitin when crown ethers are attached. However, data on ubiquitin mutants reveal that only specific lysine residues contribute to the effect of charge microsolvation. A compaction is only observed for one of the investigated mutants, in which the lysine has no proximate interaction partner. On the other hand when the lysine residues are involved in salt bridges, attachment of crown ethers has little effect on the structure.

Noncharged and Charged Monodendronised Perylene Bisimides as Highly Fluorescent Labels and their Bioconjugates

“…Charged PBIs : Mass spectra were measured in positive ion mode on a Synapt G2‐S quadrupole ion‐mobility time‐of‐flight (Q‐IMS‐ToF) mass spectrometer (Waters, Manchester, UK), equipped with a Z‐spray nanoflow ESI (nanoESI) source. The required borosilicate capillaries were prepared in‐house, using a previously described procedure . Sample concentrations were adjusted to 10–20 μ m in mixtures of water and methanol (v/v, 1:1).…”

“…The required borosilicate capillaries were prepared in-house, using ap reviously described procedure. [34] Sample concentrations were adjusted to 10-20 mm in mixtures of water and methanol (v/v,1 :1). ExternaL m/z calibration was performed by means of CsI solutions (ESI + ).…”

Radical solutions: Principles and application of electron‐based dissociation in mass spectrometry‐based analysis of protein structure

“…[193][194][195][196][197][198][199][200] Finally, ECD and ETD have proven to be so little disruptive toward peptide and protein structure that in addition to weak covalent bonds, even noncovalent interactions often survive the process. As a result, dissociation of protein-ligand complexes will often result in the detection of fragments that are still bound to the ligand, to allow identification of, for example, a ligand-binding site, [201][202][203][204][205] residues involved in a specific peptide/peptide interaction, 206 and mapping of a protein/peptide interface. 207 The preservation of noncovalent interactions during the ExD process implies that higher order protein structure should also survive, and have some effect on the observed dissociation pattern.…”

Exploring the Potential of Dendritic Oligoglycerol Detergents for Protein Mass Spectrometry

“…Mass spectra were measured in positive ion mode using a modified Ultima high-mass quadrupole-time of flight (Q-ToF) mass spectrometer (Waters Micromass, Manchester, UK) equipped with a Z-spray nanoflow ESI (nESI) ionization source [ 23 ]. The required borosilicate capillaries were prepared according to a previously described procedure [ 24 ]. Data acquisition and analysis were performed by means of MassLynx (V4.1).…”