Isobaric quantitative proteomics reveals altered extracellular matrix, cytoskeletal, and degradation pathways in glaucomatous trabecular meshwork cells

Abstract Glaucoma trabecular meshwork (GTM) cells cultured in vitro retain many characteristics of their in situ phenotype. Here, we used isobaric tandem mass tags (TMTpro) to label peptides from glaucomatous and non-glaucomatous TM (NTM) cells to identify differentially regulated proteins. Confluent NTM (n = 5) and GTM (n = 5) cells were lysed, proteins were trypsin digested, and peptides were labeled with 18-plex TMTpro. TMT-labeled peptides were fractionated on an Orbitrap Fusion mass spectrometer and data were processed using the PAW/Comet pipeline and EdgeR with Benjami–Hochberg multiple correction testing. Isobaric multiplexed quantitative proteomics identified 206 proteins that were significantly (FDR < 0.1) upregulated in GTM cells, 42 proteins that were downregulated, with 5270 non-candidates. Significant regulated pathways included extracellular matrix (DCN, COL4A1, CHI3L1), Wnt signaling (FZD1, FZD7, GSK3B), cytoskeletal regulation (ROCK2, MSN, TPM2, VIM, NF2), protein degradation (USP9X, LAMP1, SYNV1, UBE2L3), and nuclear proteins (LMNA, DFFA, CHMP3, RAD21). Western immunoblotting studies confirmed the TMTpro data. Immunofluorescence showed that the SNX7-stained nucleoli of GTM cells were significantly (p < 0.05) larger, and the DIAPH2 immunostaining was more distended into the cytosol than in NTM cells. This study identified many significantly regulated proteins in cultured GTM cells, and the results revealed several new avenues for developing clinical therapies for glaucoma patients.