Rat Liver Membrane Glycoproteome: Enrichment by Phase Partitioning and Glycoprotein Capture

Abstract

Past proteomic studies of membrane proteins have often been hampered by the low abundance and relatively high hydrophobicity of these proteins. Proteins are often glycosylated, particularly on the extracellular surface of the plasma membrane, and this characteristic was targeted as an enrichment strategy for identifying membrane proteins. Here, we report a strategy for identifying the tissue membrane glycoproteome, which involves (1) Triton X-114 phase partitioning, (2) isolation of glycosylphosphatidylinositol (GPI)-anchored proteins, and (3) glycoprotein capture using lectin affinity or hydrazine chemistry. Surprisingly, the capture of membrane proteins by lectin affinity and hydrazine chemistry resulted in mostly different populations of enriched glycoproteins. Lectins enriched high molecular weight functional membrane proteins with more potential glycosylation such as those involved in signal transduction and cell adhesion. Conversely, hydrazine chemistry isolated a higher proportion of smaller, enzymatic and peripheral membrane proteins such as solute carrier transporters and cytochrome p450s. We have applied our strategy to characterize the rat liver membrane glycoproteome and identified four new predicted GPI-anchored proteins and two that have not previously been seen in the liver. We also identified 424 nonredundant membrane proteins, of which 335 had potential N-linked glycosylation sites.

Liver Membrane Proteome Glycosylation Changes in Mice Bearing an Extra-hepatic Tumor

“…Membrane Protein Enrichment by Triton X-114 Phase Partitioning-Approximately 100 l (equivalent to ϳ0.5-1 mg) of the mouse liver membrane pellet was homogenized in 4 volumes Tris-buffered saline buffer containing 1% (v/v) Triton X-114 and chilled on ice with intermittent vortexing for 20 min (37). The samples were heated to 37°C for 20 min and phase partitioned followed by centrifugation at 300 g for 5 min.…”

Specific Glycosylation of Membrane Proteins in Epithelial Ovarian Cancer Cell Lines: Glycan Structures Reflect Gene Expression and DNA Methylation Status

“…The supernatant was collected and diluted with 2 ml of Tris binding buffer (20 mM Tris-HCl, and 100 mM NaCl at pH 7.4) and sedimented by ultracentrifugation at 120,000 ϫ g for 80 mins at 4°C. The supernatant was discarded and 140 l of Tris binding buffer was added into each sample to re-suspend the membrane pellet [modified from (32)]. A volume of 450 l of Tris binding buffer containing 1% (v/v) Triton X-114 was added to the suspended mixture, homogenized by pipetting and chilled on ice for 10 mins.…”

“…The lower detergent layer containing the membrane proteins was mixed with 1 ml of ice-cold acetone and left overnight at Ϫ20°C. Precipitated membrane proteins were pelleted by centrifugation at 1000 ϫ g for 3 mins and solubilized in 10 l of 8 M urea (32).…”

Quantitative N-linked Glycoproteomics of Myocardial Ischemia and Reperfusion Injury Reveals Early Remodeling in the Extracellular Environment

“…The glycoproteome represents an attractive subproteome for investigation because cell surface proteins are generally glycosylated and these proteins are at the interface that mediates the relationship between the cell and environment. Glycoproteomics tools offer an effective means for enriching this typically difficult set of proteins that are predominantly hydrophobic and often of low abundance (7)(8)(9)(10)(11).…”