|"Proteins from the laser capture microdissected samples of amyloid from the eyelid, conjunctiva and orbit were extracted using a modified protocol of the Liquid Tissue MS Protein Prep Kit (Expression Pathology Inc., Gaithersburg, MD). LCM films containing captured protein deposits and control tissue samples were stripped from the caps and incubated with 40 Âµl extraction buffer (10 mM Tris, pH 7.8,0.1% RapiGest (Waters)) at 95Â°C for 90 min. Samples were centrifuged at 10.000 Ã g for 1 min and subsequently cooled on ice. Extracted proteins were incubated with 5 Âµg porcine trypsin (Sigma) at 37Â°C for 16 hours, vortexing every 20 min for the first hour. Samples were then centrifuged at 10.000 Ã g for 5 min, supernatant isolated and incubated with 10 mM DTT at 95Â°C for 5 min followed by incubation with 30 mM iodoacetamide for 15 min in the dark. Peptides generated fromeach digest were acidified with 5 Âµl of 5% formic acid and recovered using Stage-tips (Proxeon, Thermo Scientific) . The peptides were eluted with 10 Âµl 70 % acetonitrile, and subsequently lyophilized before being dissolved in 12 Âµl 0.1% formic acid.
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Citation: Nielsen NS, Poulsen ET, Klintworth GK, Enghild JJ (2014) Insight into the
Protein Composition of Immunoglobulin Light Chain Deposits of Eyelid, Orbital and
Conjunctival Amyloidosis. J Proteomics Bioinform S8: 002"