Native Mass Spectrometry and Surface Induced Dissociation Provide Insight into the Post-Translational Modifications of Tetrameric AQP0 Isolated from Bovine Eye Lens

Abstract

Aquaporin-0 (AQP0) is a tetrameric membrane protein and the most abundant membrane protein in the eye lens. Interestingly, there is little to no cellular turnover once mature lens fiber cells are formed, and hence, age-related modifications accumulate with time. While bottom-up mass spectrometry-based approaches can provide identification of post-translational modifications, they cannot provide information on how these modifications coexist in a single chain or complex. Native mass spectrometry, however, enables the transfer of the intact complex into the gas-phase allowing modifications to be identified at the tetramer level. Here, we present the use of native mass spectrometry and surface-induced dissociation to study the post-translational modifications of AQP0 isolated and purified from bovine eye lens, existing as multiple forms due to the different modification states naturally present.

Insights into the biochemical and biophysical mechanisms mediating the longevity of the transparent optics of the eye lens

“…It is estimated to account for 66% of the total PTMs tabulated when water-soluble fractions and WIF were analyzed (134). Modern technological advances in mass spectrometry will continue to develop our appreciation of PTM abundance and protein profiles in the aging lens (158,159). To follow dynamic changes in lens transparency, a measure of the collective and diverse protein interactions is needed, matched to the age-related PTMs for lens proteins.…”

“…The cytoskeleton is anchored to and stabilizes the cell membranes (124,327,328). Cytoplasmic proteins partition along the lens membranes as the fiber cells mature (68,214,216,230,(329)(330)(331), and protein PTMs accumulate (155,159,230,332), affecting lipid organization (333,334) and regulating membrane protein diffusion (78,(335)(336)(337)(338). In the absence of a vascular system, these organizational cues become important for the development of the microcirculation system, which underpins lens homeostasis and SRO and therefore preserves optical function over the lifetime of the lens.…”

Lens Aquaporins in Health and Disease: Location is Everything!

“…Early crystallographic studies confirmed previous reports that physiologically, aquaporins adopt a tetrameric structure ( Mitra et al, 1994 ; Daniels et al, 1999 ). Recent native mass spectrometry results have confirmed this tetrameric structure of AQP0 in solution ( Harvey et al, 2022 ).…”

Native Ambient Mass Spectrometry of an Intact Membrane Protein Assembly and Soluble Protein Assemblies Directly from Lens Tissue

“…At 113 kDa, the Aqp0 assembly is one of the largest protein assemblies to have been analysed by native ambient MS. We also observed signals indicating phosphorylation of Aqp0 within intact tetramers (Figure S2, Supporting Information). Evidence of phosphorylation has previously been reported by bottom‐up proteomics and native MS of purified Aqp0 [25, 26] . In the latter, Aqp0 subunits were determined to be singly‐phosphorylated by using surface‐induced dissociation (SID) to dissociate doubly‐phosphorylated tetrameric Aqp0 and leave post‐translational modifications intact.…”