MALDI Imaging Mass Spectrometry Spatially Maps Age-Related Deamidation and Truncation of Human Lens Aquaporin-0

Abstract

Citation: Wenke JL, Rose KL, Spraggins JM, Schey KL. MALDI imaging mass spectrometry spatially maps agerelated deamidation and truncation of human lens Aquaporin-0. Invest Ophthalmol Vis Sci. 2015;56:7398-7405. DOI:10.1167/iovs.15-18117 PURPOSE. To spatially map human lens Aquaporin-0 (AQP0) protein modifications, including lipidation, truncation, and deamidation, from birth through middle age using matrix-assisted laser desorption ionization (MALDI) imaging mass spectrometry (IMS). METHODS.Human lens sections were water-washed to facilitate detection of membrane protein AQP0. We acquired MALDI images from eight human lenses ranging in age from 2 months to 63 years. In situ tryptic digestion was used to generate peptides of AQP0 and peptide images were acquired on a 15T Fourier transform ion cyclotron resonance (FTICR) mass spectrometer. Peptide extracts were analyzed by liquid chromatography-tandem mass spectrometry (LC-MS/MS) and database searched to identify peptides observed in MALDI imaging experiments.RESULTS. Unmodified, truncated, and fatty acid-acylated forms of AQP0 were detected in protein imaging experiments. Full-length AQP0 was fatty acid acylated in the core and cortex of young (2-and 4-month) lenses. Acylated and unmodified AQP0 were C-terminally truncated in older lens cores. Deamidated tryptic peptides (þ0.9847 Da) were mass resolved from unmodified peptides by FTICR MS. Peptide images revealed differential localization of un-, singly-, and doubly-deamidated AQP0 C-terminal peptide (239-263). Deamidation was present at 4 months and increases with age. Liquid chromatography-MS/MS results indicated N246 undergoes deamidation more rapidly than N259. CONCLUSIONS.Results indicated AQP0 fatty acid acylation and deamidation occur during early development. Progressive age-related AQP0 processing, including deamidation and truncation, was mapped in human lenses as a function of age. The localization of these modified AQP0 forms suggests where AQP0 functions may change throughout lens development and aging.

Spatial distributions of glutathione and its endogenous conjugates in normal bovine lens and a model of lens aging

“…While MALDI-IMS has been used extensively to study lens proteins (Anderson et al, 2015; Grey et al, 2009; Grey and Schey, 2009; Wenke et al, 2015) and lipids (Pol et al, 2015; Vidová et al, 2010), IMS of lens metabolites is limited (Grey, 2016). We therefore utilised high dynamic range (HDR) mass spectrometry with the continuous accumulation of selected ions (CASI) to detect metabolites in lens regions with maximum sensitivity (Spraggins et al, 2012), LC-MS/MS to confirm metabolite identities, and MALDI FTICR IMS to spatially localise glutathione and its metabolites throughout bovine lens sections.…”

Advances in MALDI imaging mass spectrometry of proteins in cardiac tissue, including the heart valve

“…Figure 5 shows an example of tryptic peptide imaging used to explore unique protein expression distinct to porcine heart valve and aortic wall. The use of fresh frozen tissue allows detection of modifications on the peptide backbone [54]. Tissue decellularization followed by tryptic digestion has been used to access high molecular weight ECM proteins [55].…”

“…Major PTMs include phosphorylation, glycosylation, acetylation, ubiquitination, deamidation, all of which may be defined by well-developed chromatography coupled mass spectrometry based strategies [61]. For MALDI IMS, PTMs are typically accessed through sequence information from tryptic peptides or intact proteins [54, 62–64]. Enzymatic approaches may improve detection of PTM sites [63].…”

The lipidation profile of aquaporin-0 correlates with the acyl composition of phosphoethanolamine lipids in lens membranes

“…There are two known lipidation sites in AQP0 one at the N-terminus and the other at Lys-238 ( Fig. S1-S3) [5][6][7]. Neither match consensus sequences for enzymatically mediated lipidation events.…”

“…In vitro palmitoylation of AQP0 has been shown to be a post-translational, rather than a co-translational, event [8]. The longevity of lens proteins such as AQP0 in the plasma membrane of lens fiber cells [7], and the fact that all intracellular organelles are removed during lens fiber cell differentiation [9], provides conditions under which modifications by acyl transfer from the membrane lipids will accrue [10]. In this paper we examine the lipidation profile of AQP0 from bovine and human lenses, with the specific objective of determining the acyl group diversity at these known lipidation sites.…”