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Title 

Efficient adhesion-based plasma membrane isolation for cell surface N-glycan analysis

Authors 

Ji Young MunK J LeeHoon SeoMin-Sun SungYee Sook ChoSeung Goo LeeOh Suk KwonDoo-Byoung Oh

Publisher 

American Chemical Society

Issue Date 

2013

Citation 

Analytical Chemistry, vol. 85, no. 15, pp. 7462-7470

Keywords 

Complex-type glycansEfficient detectionEndoplasmic reticulumFluorescence imagingIntracellular organelleMALDI-TOF mass spectrometryMannosidase inhibitorsMembrane fraction

Abstract 

Glycans, which decorate cell surfaces, play crucial roles in various physiological events involving cell surface recognition. Despite the importance of surface glycans, most analyses have been performed using total cells or whole membranes rather than plasma membranes due to difficulties related to isolation. In the present study, we employed an adhesion-based method for plasma membrane isolation to analyze N-glycans on cell surfaces. Cells were attached to polylysine-coated glass plates and then ruptured by hypotonic pressure. After washing to remove intracellular organelles, only a plasma membrane fraction remained attached to the plates, as confirmed by fluorescence imaging using organelle-specific probes. The plate was directly treated with trypsin to digest and detach the glycoproteins from the plasma membrane. From the resulting glycopeptides, N-glycans were released and analyzed using MALDI-TOF mass spectrometry and HPLC. When N-glycan profiles obtained by this method were compared to those by other methods, the amount of high-mannose type glycans mainly contaminated from the endoplasmic reticulum was dramatically reduced, which enabled the efficient detection of complex type glycans present on the cell surface. Moreover, this method was successfully used to analyze the increase of high-mannose glycans on the surface as induced by a mannosidase inhibitor treatment.

ISSN 

0003-2700

Link 

http://dx.doi.org/10.1021/ac401431u

Appears in Collections

1. Journal Articles > Journal Articles

Registered Date

2017-04-19


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