High-efficiency labeling of sialylated glycoproteins on living cells

Abstract

We describe a simple method for efficiently labeling cell surface glycans on virtually any living animal cell. The method employs mild Periodate oxidation to generate an aldehyde on sialic acids, followed by Aniline-catalyzed oxime Ligation with a suitable tag (PAL). Aniline catalysis dramatically accelerates oxime ligation, allowing use of low concentrations of aminooxy-biotin at neutral pH to label the majority of cell surface glycoproteins while maintaining high cell viability. Keywordsglycoprotein; sialic acid; oxime ligation; aniline; periodate oxidation; metabolic labeling; live cell labelingThe expanding interest in glycoproteomics and the biological roles of glycoconjugates has increased efforts to develop efficient tools to label cell surface glycoproteins. Several elegant approaches have exploited metabolic labeling of cells, and even whole model organisms, using analogs of glycan precursors that carry bio-orthogonal groups (e.g. azide, alkyne, ketone or aldehyde), allowing the chemical ligation of reporter groups onto cell surface glycoconjugates 1, 2.The chemistries used for conjugation with these functional groups each have both advantages and disadvantages for use with living cells. The conjugation of azides with substituted triphenylphosphines using the Staudinger-Bertozzi ligation can be performed on living cells, but suffers from slow reaction kinetics 3 . Conjugation of azides with substituted-alkynes (or vice versa) with the Huisgen cycloaddition, or 'click chemistry', has rapid reaction kinetics, but requires a copper catalyst that is toxic to living cells4 , 5. The newly described ligation of azides with ring-strained alkynes is compatible with living cells and has rapid reaction kinetics, but requires reagents that are not currently commercially available 2 . Finally, imine (oxime or A limitation of all these methods is the need for culturing cells with a glycan precursor containing a bio-orthogonal group prior to labeling. As an alternative, aldehydes can be readily introduced into cell surface glycans by mild periodate oxidation, known for nearly 40 years to selectively oxidize the polyhydroxy side chain of sialic acids 7,8 . The recent demonstration that oxime ligation on complex biomolecules is dramatically accelerated using aniline as a nucleophilic catalyst 9-12 inspired us to explore the efficiency of this reaction with aldehydes on living cells introduced by metabolic labeling or periodate oxidation. While aniline can be efficiently used as a nucleophilic catalyst for labeling biomolecules in solution by oxime and hydrazone ligations, we chose to employ the oxime ligation, which gives a more stable product than the hydrazone ligation 13 . NIH Public AccessThe two approaches used for the introduction of aldehydes onto cell surface sialic acids for subsequent ligation with aminooxy-biotin are illustrated in Fig. 1a. Cells were subjected to mild periodate oxidation (1 mM NaIO 4 at 4 °C for 30 min; Supplementary Fig. 1) to selectively introduce an aldehyde at C-7 of sialic acid...

A Combined Omics Approach to Generate the Surface Atlas of Human Naive CD4+ T Cells during Early T-Cell Receptor Activation

“…Therefore an omics approach was performed, combining quantitative proteomic and transcriptomic methods. For the identification of surface proteins of CD4 ϩ /CD45RA ϩ naive T cells by massspectrometry the recently described PAL (periodate oxidation and aniline-catalyzed oxime ligation) approach (18) was applied and adapted for the use with primary T cells and combined with quantitative LC-MS/MS (19). This method allows an efficient labeling of cell surface sialic acid-containing glycans on living cells.…”

“…Cell Surface Labeling, Protein Digestion, and N-glycopeptide Enrichment-Periodate oxidation and aniline-catalyzed oxime ligation (PAL)-based cell surface labeling procedure was performed as described previously (18) and adapted for primary human naive CD4 ϩ T cells. All following steps were performed on ice.…”

Functional Organization of Golgi N- and O-Glycosylation Pathways Involves pH-dependent Complex Formation That Is Impaired in Cancer Cells

“…Functional Relevance of the Complexes-To assess the functional relevance of the glycosyltransferase complexes, we measured first the in vivo cell surface sialylation state in transfected COS7 cells by using the recently established biotin-streptavidin fluorescence-based protocol (20). In comparison to non-transfected cells (Fig.…”

Fluorescent oxygen sensitive microbead incorporation for measuring oxygen tension in cell aggregates

“…The strategy applied here was to 19 precede cell surface biotinylation with a periodate treatment. This procedure has been shown to yield a 1,000-fold increase in total biotin expression [45].…”