Rat liver membrane glycoproteome: enrichment by phase partitioning and glycoprotein capture.
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.
Lee
Albert
A
Department of Chemistry and Biomolecular Sciences, Macquarie University, Sydney Australia.
Kolarich
Daniel
D
Haynes
Paul A
PA
Jensen
Pia H
PH
Baker
Mark S
MS
Packer
Nicolle H
NH
D000818:Animals; D002846:Chromatography, Affinity / Q000295:instrumentation / Q000379:methods; D006023:Glycoproteins / Q000737:chemistry / Q000302:isolation & purification; D006031:Glycosylation; D006801:Humans; D037102:Lectins / Q000737:chemistry; D008099:Liver / Q000737:chemistry* / Q000166:cytology; D008565:Membrane Proteins / Q000737:chemistry / Q000302:isolation & purification; D008969:Molecular Sequence Data; D020543:Proteome / Q000032:analysis*; D051381:Rats
D006023:Glycoproteins; D037102:Lectins; D008565:Membrane Proteins; D020543:Proteome