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

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.