Primary LECs isolated from individual human corneas (n = 4) were transfected with miR-146a mimic (M) and its respective control (MC). Three days after transfection, the cells were collected, and cell lysates were prepared for proteomics analysis. Briefly, the protein lysates were reduced, alkylated, and digested after addition of lys-C and trypsin proteinases, as previously described [34 (link),35 (link)]. Peptides were then labeled using 10-plex TMT isobaric tags [36 (link)]. Labeled samples were mixed, desalted, and then loaded onto a fused silica capillary packed in-house with bulk C18 reversed phase resin (length, 25 cm; inner diameter, 75 µM; particle size, 1.9 µm; pore size, 100 Å; Dr. Maisch GmbH, Germany).
The peptides were separated using a Dionex Ultimate 3000 UHPLC system (Thermo Fisher Scientific). The 140-min water–acetonitrile gradient was delivered at a flow rate of 300 nL/min (Buffer A: water with 3% Dimethyl Sulfoxide (DMSO) and 0.1% formic acid, Buffer B: acetonitrile with 3% DMSO and 0.1% formic acid). Peptides were ionized, introduced into the Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific), and analyzed by tandem mass spectrometry.
The peptides were separated using a Dionex Ultimate 3000 UHPLC system (Thermo Fisher Scientific). The 140-min water–acetonitrile gradient was delivered at a flow rate of 300 nL/min (Buffer A: water with 3% Dimethyl Sulfoxide (DMSO) and 0.1% formic acid, Buffer B: acetonitrile with 3% DMSO and 0.1% formic acid). Peptides were ionized, introduced into the Orbitrap Fusion Lumos mass spectrometer (Thermo Fisher Scientific), and analyzed by tandem mass spectrometry.