Basement Membranes from Human Retinal Blood Vessels, Inner Limiting Membranes, and Lens Capsules
Three types of BMs from adult human eyes, the inner limiting membrane, the retinal vascular BMs and the lens capsule, were isolated for analysis by 1D-SDS-PAGE and LC-MS/MS.
Sample Processing Protocol
BMs from adult human eyes, the inner limiting membrane, the retinal vascular BMs and the lens capsule, were manually dissected from normal human cadaver eyes and incubated in 2% Triton X100 overnight. Deoxycholate was added, and the detergent-insoluble BMs were transferred multiple times into new detergent. The tube-like blood vessel BMs (BV) were manually separated from the sheet-like inner limiting membranes (ILMs) under a dissecting microscope using dark-field illumination. Lens capsule (LCs) were dissected from the isolated lenses. The BMs were then deglycosylated with heparitinase and chondroitinase ABC lyase (100U/ml; Seikagaku America, East Falmouth, MA) for 2 hours, then solubilized in 8M urea/mercaptoethanol. The solubilized sample was separated using a low percentage gradient (3-15%) 1D SDS PAGE gel under reducing conditions. SDS PAGE gel lanes were cut into 14-48 bands, reduced with TCEP, alkylated with iodoacetamide, and digested with trypsin. Digests were analyzed on a Thermo Fisher LTQ Velos Orbitrap.
Data Processing Protocol
Protein and peptide identification along with relative quantification by iBAQ values were done by MaxQuant.
Uechi G, Sun Z, Schreiber EM, Halfter W, Balasubramani M. A proteomic view of basement membranes from human retinal blood vessels, inner limiting membranes, and lens capsules. J Proteome Res. 2014 Jul 2 PubMed: 24990792