Selective Enrichment of Cell Surface Proteins Using ProMTag to Detect O‐glycosylation‐Dependent Changes

Abstract

O‐linked glycosylation, which is regulated by the enzyme GalNAc transferase, plays a vital role in cellular function by regulating membrane protein composition and sorting. Changes in O‐linked glycosylation are very common in diseases such as cancers, diabetes, and Alzheimer’s. It is important to study the effects of changes in O‐link glycosylation on cell membrane composition and function to understand how these changes affect the onset, phenotype, and progression of these diseases. However, membrane proteins are notoriously challenging to analyze at the proteomic level due to their hydrophobicity and the inability to separate the membrane proteins from other proteins in the cell.Here we introduce a new sample preparation workflow that enriches membrane proteins for mass spectrometry analysis to understand changes in protein composition of the plasma membrane as the result of changes in O‐linked glycosylation. To do this, we used the bifunctional tag ProMTag to label and capture plasma membrane proteins via their extracellular domains in cells treated with GalNAc Transferase inhibitors. One end of the ProMTag forms a reversible, covalent bond with protein the other end of the ProMTag is capable of forming an irreversible, covalent bond with a solid bead support via the click chemistry pair of methyltetrazine and trans‐Cyclooctene (TCO). The extracellular domains of proteins from intact wild type HEK cells and HEK cells treated with GalNAc Transferase inhibitors were ProMTagged, captured, cleaned up, and eluted for comparison by mass spectrometry (MS) analysis. MS confirmed enrichment of plasma membrane proteins after sample preparation and protein changes in the plasma membrane as a result of GalNAc Transferase inhibition were characterized. This work establishes a method for enrichment of plasma membrane proteins and reveals how changes in O‐linked glycosylation via GalNAc Transferase inhibition leads to changes in plasma membrane protein composition due to altered membrane protein stability and sorting.