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Kevin Schey, Kristie Rose, David Anderson; Identification and Spatial Localization of Ocular Lens Age- and Cataract-Specific Protein Signatures. Invest. Ophthalmol. Vis. Sci. 2013;54(15):4962.
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© ARVO (1962-2015); The Authors (2016-present)
To image and identify age- and cataract-specific peptide and protein signals in human lenses.
Frozen human lenses from 7-78 years old including a nuclear cataract lens were sectioned equatorially at twenty micron thickness. Lens sections were washed with 50% acetonitrile to remove highly soluble proteins and subsequently coated with MALDI matrix, sinapinic acid, in 50% acetonitrile. MALDI imaging experiments were conducted using a Bruker AutoFlex Speed MALDI-TOF instrument at a spatial resolution of 300 micron and images compared using FlexImaging software. Peptide identification was accomplished by extraction of peptides from tissue sections with 1.5 uL 20% acetonitrile. Extracted peptides were separated over a C18 packed nanoLC column and eluted into a Velos linear ion trap mass spectrometer. Tandem mass spectra were acquired in data dependent fashion using both electron transfer dissociation (ETD) and collision induced dissociation (CID) to produce peptide sequence information. Manual interpretation of tandem mass spectra was accomplished to positively identify peptide signals.
Age-specific signals were observed in distinct locations (shells) within human lens tissues of different ages. Cataract-specific signals were observed in the nuclear cataract region and not in age-matched transparent lens tissue. A large majority of signals corresponded to peptides from the C-terminus of gammaS-crystallin. Additional peptides were observed from betaA3-crystallin and gammaD-crystallin.
MALDI imaging mass spectrometry revealed age-and cataract-specific peptide signals in human lens sections. Identification of the cataract-specific signals as primarily gammaS-crystallin peptides suggests its possible role in cataractogenesis.
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