Site Specific Characterization of N- and O-Glycosylation in Etanercept by TMT-labeling and LC-MS/MS
Abstract
Improved methods are required for glyco-characterization of biopharmaceuticals and comparison between their corresponding biologics and biosimilar, where they have multiple glycosylation sites such as Etanercept of a fusion protein. In order to enhance sensitivity of discovery and minimize the sample loss, the combination of a TMT labeling method and fractionation using high flow HPLC has contributed to detect low amount of peptide in data dependent mode of LC-MS/MS. Here, we report a new method combined with TMT labeling, fractionation using ZIC-HILIC HPLC, LC-MS/MS using HCD triggered CID/EThcD, and computational software for identification of site-specific N- and O-glycopeptide and comparison of their corresponding biologics and biosimilar. Of three different Etanercept, such as Enbrel (biologics), Etoloce and Eucept biosimilars), a total of 115 N- and O- glycopeptides were identified and quantified using TMT-11plex labeling including triplicates and 22 ZIC-HILIC fractionation in three and 15 of N- and O- glycosylation sites, respectively. Two O-glycopeptides in different O-glycosylation site and seven core 2 type of O-glycopeptides were first reported. In particular, three kinds of O-glycopeptides with NeuGC which could be a foreign agent of immune response. In depth of quantification, our strategy shows site-specific similarity is quite not similar between biologics and biosimilar.
Disruption of Cell Adhesion by Alteration of Fibronectin in the Glycated Extracellular Matrix
Abstract
Glycation is a non-enzymatic process that occurs in the body when sugar molecules interact with long-lived proteins such as extracellular matrix (ECM) components, resulting in the formation of advanced glycation endproducts (AGEs) through repetitive condensation and dehydration. These AGEs are deposited in the ECM as glycotoxins, causing various diseases such as aging, cancer, and atherosclerosis. However, despite abundant evidence that the pathological alterations of ECM proteins induced by AGE accumulation can lead to cellular dysfunction, the mechanisms underlying these biological events remain unclear. Here, we explore mechano-physical and biochemical changes in the matrix caused by glycation using a fibroblast-derived ECM and propose how the changes in the local microenvironment dysregulate cellular behavior based on these analyses. The 14 most glycated proteins in the fibroblast-derived ECM were identified by mass spectrometry-based proteomic analysis. 7 types of AGEs were found, with G-H, G-DH, MG-H, and MG-DH detected at arginine residues and amadori product, CML, and CEL detected at lysine residues. In the case of fibronectin (FN), unlike collagens and other glycoproteins, the arginine residue in the RGD sequence, which is a cell-binding motif, was modified. Consistent with these findings, cells in the glycated ECM had a longer adhesion time to the ECM and a reduced focal adhesion complex, and the expression level of integrin α5β1, which is constitutively expressed by binding to FN, was also downregulated. AGE-mediated modification of the RGD motif in FN contributes to a slippery ECM, providing a conclusive rationale that it may trigger or exacerbate disease by impairing cell function.
Digging Into the Diversity of Mammalian Brain Glycosylation
Abstract
Glycosylation, essential for brain development and function, is indeed a key and common post-translational modification of brain proteins and lipids. However, deciphering molecular mechanism at the glycome level in mammalian brain remains a missing piece of puzzle in molecular neuroscience due to the intrinsic complexity of glycosylation and the lack of analytical tools. Here, in order to understand relationship between spatiotemporal alteration of the brain and glycosylation, we performed a systematic and in-depth analysis of protein-bound glycans and lipid-bound glycans in human and mouse brain samples using a cutting-edge tool based on LC-MS/MS. Regional distribution of glycosylation was examined for nine mouse brain regions (cerebral cortex, prefrontal cortex, striatum, hippocampus, olfactory bulb, diencephalon, midbrain, pons-medulla, and cerebellum). We next targeted the prefrontal cortex (PFC) region, which is particularly vulnerable to lesions because of its protracted developmental process for examining temporal variation in glycomic signatures and their relationships to mammalian PFC development. We further constructed a comprehensive synthesis map using protein-boudn glycans and lipid-bound glycans structurally elucidated by LC-MS/MS. Our findings reveal a high level of diversity in the glycosylation program underlying brain region specificity and age dependency, and our data could be the reference for future mammalian brain glycosylation study. Furthermore, it provides a roadmap for future studies of glycosylation in neurodevelopment and disease.
Room 307, Seoul National University Medical School, 103, Daehak-ro, Jongno-gu, Seoul, Republic of Korea
Phone: (+82)2-393-8328 E-mail : admin@khupo.org Homepage : http://www.khupo.org
Official Secretariat: The Plan B
Room B10, 55, APEC-ro, Haeundae-gu, Busan, Republic of Korea
FAX: +82-51-745-8407 Phone: (+82)2-393-8328 Homepage : https://www.planbgroup.org/
