Mertz, J. L. | bioRxiv Science

bioRxiv2020

Comparison of three glycoproteomic methods for the analysis of CHO cells treated with 1,3,4-O-Bu3ManNAc

Comprehensive analysis of the glycoproteome is critical due to the widespread importance of this post-translational modification to protein function, and difficult because of the tremendous complexity it exhibits. Here we compared three glycoproteomic analysis methods, a recently described chemoenzymatic glycoproteome analysis methods, N-linked glycans and glycosite containing peptides (NGAG), Solid-phase extraction of N-linked glycoproteins (SPEG), and hydrophilic interaction liquid chromatography (HILIC), for the analysis of N-linked glycosites of Chinese hamster ovarian (CHO) cells treated with 1,3,4-O-Bu3ManNAc. The NGAG protocol resulted in substantially increased glycosite identifications over both SPEG and HILIC. Interestingly, while the glycosites identified by SPEG and HILIC overlapped strongly, NGAG identified many glycosites not observed in either of the other two methods. Further, utilizing the enhanced intact glycopeptide identification afforded by the NGAG workflow, we also found that of the sugar analog 1,3,4-O-Bu3ManNAc increases sialylation of proteins secreted by CHO cells, including an ectopically expressed human proteins.

biochemistry↗

bioRxiv2020

Proteomic and phosphoproteomic analysis identifies novel liver-related signaling in retinal pigment epithelial cells during epithelial-mesenchymal transition

Epithelial-mesenchymal transition (EMT) of the retinal pigment epithelium (RPE) is associated with several potentially blinding retinal diseases. Proteomic and phosphoproteomic studies were performed on human pluripotent stem cell-derived RPE (hPSC-RPE) monolayers to better understand the pathways mediating RPE EMT. EMT was induced by enzymatic dissociation or by co-treatment with transforming growth factor beta (TGF{beta}) and tumor necrosis factor alpha (TNF; TGNF). The global and phosphoproteomes were analyzed at 1 hr post EMT induction to capture early events in kinase/phosphatase signaling cascades and at 12 hrs to define early changes in protein abundance. Pathway enrichment analysis revealed that TGNF and Dissociation rapidly perturbed signaling in many of the same pathways, with striking similarity in the phosphoproteome at 1 hr. Surprisingly, functions related to liver cell proliferation and hyperplasia were strongly enriched in the phosphosites altered by both treatments at 1 hr and in protein abundance changes at 12 hrs. Hepatocyte Growth Factor-cMET signaling exhibited the strongest overall enrichment in both treatments. These signaling pathways may serve as suitable targets for the development of therapeutic strategies for the inhibition of RPE EMT, and thus progression of several debilitating visual diseases.

cell biology↗