Efficient Adhesion-Based Plasma Membrane Isolation for Cell Surface N-Glycan Analysis

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.

Differential Site Accessibility Mechanistically Explains Subcellular-Specific N-Glycosylation Determinants

“…The GO terms “ER”, “Golgi/endosome/plasma membrane”, and “extracellular” were used to evaluate the localization/origin of the glycoproteins identified in the subcellular fractions. In agreement with a previous study (23), the GO annotation of the identified proteins showed that the microsomes in general contained a high proportion of ER-residing proteins, Figures 2A–C. Although the proteins are only broadly, and possibly somewhat inaccurately, classified on the basis of GO terms, the trends clearly indicated significant enrichment, although not complete isolation, of the desired proteins in the respective subcellular fractions.…”

“…High-mannose type N -glycans, in particular Man 8–9 structures, were previously reported to be the dominating features of the plasma membrane of human embryonic stem cells (48) and of cancer cells (49, 50). However, cell lysates and total membrane fractions similar to our microsome preparations were used in these studies suggesting significant contributions from intracellular high-mannose-rich ER-residing N -glycoproteins (23). Hence, the actual cell-surface N -glycomes in the previous work may not have been accurately captured.…”

“…Due to the functional implications of both intra- and extracellular N -glycoproteins, we focus on the secreted, cell-surface, and intracellular glycoproteomes, the latter fraction largely represented by microsomal proteins (23). Understanding, how the subcellular glycoproteomes are generated and regulated under normal and altered physiological conditions of the cell is valuable to the understanding of their involvement in immune biology.…”

Rapid Analysis of Cell Surface N-Glycosylation from Living Cells Using Mass Spectrometry

“…In addition, we and others found that the N-glycans obtained by this method are mostly of the high-mannose type, which may originate from intracellular proteins derived from the ER. 4,6,9,[11][12][13] This hinders accurate detection and quantification of the complex-type N-glycans. We recently improved complex-type N-glycan quantification using first Endo H to release high-mannose and hybrid-type N-glycans and subsequently using PNGase F in order to release complex-type N-glycans 14 .…”

“…11 However, these methods cannot be applied to living cells. The cell surface glycome of living cells can be visualized by lectin staining 16 or by incorporating unnatural carbohydrate precursors on cell glycoproteins.…”