Placental Growth Factor Regulates the Pentose Phosphate Pathway and Antioxidant Defense Systems in Human Retinal Endothelial Cells

The molecular mechanisms whereby placental growth factor (PlGF) mediates its effects in nonproliferative diabetic retinopathy (DR) are unknown. To better understand the role of PlGF in DR, we used tandem mass tags (TMT)-labeled quantitative proteomics to human retinal endothelial cells (HRECs), treated anti-PlGF antibody as a experimental, PBS as a control. The control and PlGF-ab–treated HRECs of protein quantification was done by bicinchoninic acid (BCA) protein assay kit. The resulting peptide mixtures were labeled with one of the TMT reagents from the 10plex version. The samples were then fractionated by high-pH reverse-phase high-performance liquid chromatography (HPLC) using an Agilent 300 Extend C18 column. Peptide separation was performed using a reversed-phase analytical column. The peptides were subjected to nanospray ionization (NSI) source followed by tandem mass spectrometry (MS/MS) in Q Exactive™ plus (Thermo Fisher Scientific, Waltham, MA) coupled with the ultra high-performance liquid chromatography (UPLC). For MS scans, the m/z scan range was 350–1800. The fixed first mass was set as 100 m/z. Data analysis was performed on Proteome Discoverer 2.4 (Thermo Fisher Scientific) using Sequest and Mascot search engines and MaxQuant tool program. The data were searched against the National Center for Biotechnology Information (NCBI) human reference sequence (NCBI RefSeq) protein database. MS/MS was searched with a precursor mass tolerance of 10 ppm, and the fragment mass tolerance was set to 0.05 Da. The protease used was specified as trypsin, and a maximum of two missed cleavages was allowed. The TMT ratio for each peptide–spectrum match was calculated by the quantitation node, and the probability of phosphorylation for each Ser/Thr/Tyr site was calculated by the phosphoRS3.1 node in the Proteome Discoverer and MaxQuant programs. the KEGG online service tool KAAS (Kregg Automatic Annotation Server) to annotate the protein’s KEGG database description. Protein-protein network analysis was performed for 60 overlap DEPs by using the STRING (Search Tool for the Retrieval of Interacting Genes) database (https://string-db.org/) to disclose possible connections among proteins and to visualize the PPI (protein-protein interaction) network.