Purchase this article with an account.
Kevin L Schey, Jamie L Wenke; Imaging the Distribution of Modified AQP0 in Human Lenses as a Function of Age. Invest. Ophthalmol. Vis. Sci. 2015;56(7 ):3562.
Download citation file:
© ARVO (1962-2015); The Authors (2016-present)
The lens water channel Aquaporin 0 (AQP0) is extensively modified with age, but the precise location of the modified forms is not known. The purpose of this work is to use novel MALDI imaging mass spectrometry methods to spatially map truncated, phosphorylated, lipidated, and deamidated forms of AQP0 protein in human lenses as a function of age.<br />
Human lenses (4 month to 56 yr, N=5) obtained from NDRI were stored at -80°C until use. Twenty micron thick sections were methanol soft-landed onto MALDI targets. Tissue was washed ten times with water, followed by three 1% formic acid washes. Sinapinic acid matrix was spotted on tissue using an acoustic reagent spotter at a 200µm final spacing. Spatially-resolved protein mass spectra were acquired on a Bruker Autoflex Speed and images were compiled using Bruker FlexImaging software. For in situ digestion and peptide analysis, washed sections were further washed using 4M and 7M urea. Trypsin was applied using an HTX Imaging TM Sprayer and sections were incubated in a humidified petri dish at 37°C for two hours, followed by CHCA matrix application. Peptide mass spectra were collected on a Bruker SolariX 15T FTICR mass spectrometer. Peptide microextractions were analyzed by LC-MS/MS to confirm peptide identification.
MALDI imaging of human lens AQP0 highlights the significant modification of AQP0 protein with age. Using an extensive washing protocol, we could detect and image truncated and lipid-modified (palmitate and oleate) forms of the protein. Our results confirm progressive truncation of AQP0 with fiber cell age and demonstrate that AQP0 is lipidated in young fiber cells. Truncation occurred in lenses as young as 4 months. Additional experiments confirmed that the N-terminus is the major site of lipid addition. Unprecedented high mass resolution peptide images show the distribution of a deamidated AQP0 C-terminal peptide for the first time. The images indicate that AQP0 is extensively deamidated in all but the very youngest fiber cells of a 4 month lens.
This study provides spatial maps of age-related AQP0 modifications in human lenses. The spatial distribution of AQP0 protein and representative peptide fragments, suggests programmed lipid modification and truncation. Moreover, deamidated AQP0 was mapped for the first time showing extensive C-terminal deamidation in most fiber cells.
This PDF is available to Subscribers Only