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

Abstract

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 (+0.9847 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.

Figure 1

Representative MALDI-TOF spectra from 2-month and 21-year human lens. (A) The major signal in the 2-month lens is full-length AQP0 1-263. Lipid-modified AQP0 (*) is present in both lenses, while major truncation peaks (◊) are apparent only in the 21-year lens. Spectra were generated from regions of interest including the cortex and core of each lens. (B) Theoretical and observed m/z values for modified forms of AQP0. Putative identifications listed are based on accurate mass and previous publications. a.i., arbitrary intensity.

Figure 2

Imaging of AQP0 protein demonstrates age-related truncation. (A) Full-length AQP0 1-263 signal is lost due to extensive C-terminal truncation in older lens cores. Accumulation of 1-259 (B), 1-246 (C), and 1-243 (D) truncated AQP0 is apparent in older lens cores. Images were acquired with 200 μm raster step size on a Bruker Autoflex TOF. Ion intensities are normalized to the TIC for each ion across the tissue. Color scale bars indicate the range of intensities plotted.

Figure 3

Fatty acid–acylated AQP0 protein. Aquaporin-0 is modified with a fatty acid in young fiber cells (A), with a narrow ring of unmodified AQP0 before the lipid is added (B). The fatty acylated form also is C-terminally truncated, predominantly at residue 246 (C). Images were acquired with 200 μm raster step size on a Bruker Autoflex TOF. Ion intensities are normalized to the TIC for each ion across the tissue. Color scale bars indicate the range of intensities plotted.

Figure 4

Deamidation of C-terminal AQP0 peptide 239-263. (A) Undeamidated AQP0 is present in the very young outer cortical fiber cells of a 4-month lens. Deamidation is more abundant in the lens core. Images were acquired with 125-μm raster step size using FTICR MS. All m/z values are plotted ± 0.005 m/z. Ion intensities are normalized to the TIC for each ion across the tissue. Color scale bars indicate the range of intensities plotted. (B) Continuous accumulation of selected ions FTICR spectrum showing un-, singly-, and doubly-deamidated AQP0 peptide. (C) Calculated and observed m/z values for AQP0 C-terminal peptide deamidation. Reported full scan and CASI m/z values were acquired on a Bruker SolariX 15T FTICR MS.

Figure 5

Age-related accumulation of deamidation and truncation. Singly- and doubly-deamidated AQP0 peptide are visible in concentric rings in older lenses. Slightly older fiber cells contain deamidated peptide that has been truncated at residue 259. Images were acquired with 100-μm raster step size on a Bruker SolariX 15T FTICR MS. Ion intensities are normalized to the TIC for each ion across the tissue. Color scale bars indicate the range of intensities plotted.

Figure 6

Tandem mass spectrum identifying deamidation site at N246. High mass resolution CID spectrum of doubly-charged AQP0 peptide 239-259, m/z 1276.13, acquired on a Thermo LTQ Velos Orbitrap. The observed mass corresponds to one deamidated residue on the peptide; starred peaks confirmed deamidation at residue 246. The amino acid sequence showing fragmentation coverage is included above.