Interaction between alpha and upsilon‐crystallin, common to the eye of the Australian platypus, by radical probe mass spectrometry

Abstract

The interaction between alpha-crystallin and upsilon-crystallin, a class recently discovered in the eye of the Australian platypus, has been shown by native shift gel assay and examined by radical probe mass spectrometry in the context of the ability of alpha-crystallin to protect upsilon-crystallin from oxidation and oxidative damage through radical-based oxidative stress mechanisms. Residues 22-41, 132-148, 212-227 and 245-264 of upsilon-crystallin display the greatest protection when interacted with alpha-crystallin at a ratio of 2 : 1 observed for the complex, which is commensurate with their levels measured in the eye of the platypus. Across each domain, a delay in the onset of oxidative damage is observed as the time of exposure to radicals is increased. The results are discussed in the context of the structure of the porcine homologue of upsilon-crystallin.

Oxidation Artifacts in the Electrospray Mass Spectrometry of Aβ Peptide

“…The converse approach of swamping the effect by using O 2 sheath gas (as in "radical probe MS" [6][7][8][9][10]) may complicate interpretation of H/D exchange data. Electron micrographs of emitter A and mass spectra acquired with it in the triaxial mode after 300 h use: (a) before polishing; (b) after polishing.…”

Impact of limited oxidation on protein ion mobility and structure of importance to footprinting by radical probe mass spectrometry

“…Particular advantages of this structural elucidation approach, over other experimental methods to study protein structures and their complexes that employ mass spectrometry (MS), include the ability to probe the structure of proteins in solution using hydroxyl radicals. These can be produced with relative ease from the bulk solvent using a variety of methods including radiolysis and electrical discharge .…”

Ability of N‐acetylcarnosine to protect lens crystallins from oxidation and oxidative damage by radical probe mass spectrometry (RP‐MS)

“…Each crystallin solution (50 µM, 100 µL) was infused using a model 11 syringe pump from Harvard Apparatus (Holliston, MA, USA) at a flow rate of 5 µL/min into an electrical discharge source as previously described 15, 19, 20, 30–32. The discharge needle was maintained at a stable voltage of 8 kV (35 µA) and an oxygen sheath gas was used at an approximate flow rate of 10 L/min.…”

“…This demonstrated that certain regions of the protein subunits were more susceptible to oxidative damage than others 30. Furthermore, the impact of this damage on the protein's interaction with υ‐crystallin, a taxon‐specific crystallin recently discovered in the eye of the Australian platypus, was examined 31. The study reported herein investigates the impact of NAC on the oxidation and damage to both α‐ and β‐crystallin,32 the most abundant proteins of the mammalian lens.…”