System-Wide Quantitative N-Glycoproteomic Analysis from K562 Cells and Mouse Liver Tissues.

Abstract

As a key regulator of many biological processes, glycosylation is an essential post-translational modification (PTM) in the living system. Over 50% of human proteins are known to be glycosylated. Alterations in glycoproteins are directly linked to many diseases, making it crucial to understand system-wide glycosylation changes. The majority of known glycoproteins are from plasma membrane; however, glycosylation is a dynamic process that occurs throughout multiple subcellular organelles and involves sets of enzymes, chaperones, transporters, and sugar donor molecules. Many glycoproteins are expressed not only in plasma membranes but also in subcellular organelles. Here, we developed a mass-spectrometry-based quantitative workflow for the system-wide N-glycoproteomic analysis of membrane and cytosolic proteins extracted using a MEM-PER kit. The kit facilitates the extraction and solubilization of both membrane and cytosolic proteins in a simple, efficient, and reproducible manner. We analyzed the K562 cell line and mouse liver tissue to evaluate this approach. A total of 934 glycosites, 5154 glycopeptides, and 536 glycoproteins from the K562 cell line and a total of 1449 glycosites, 7549 glycopeptides, and 660 glycoproteins from mouse liver tissue were identified. This simple and reproducible approach provides a unique way to understand system-wide glycosylation in biological processes and enables the identification and quantitation of glycan profiles at glycosylation sites in proteins.