We report an advanced chemoenzymatic strategy for the direct fluorescence detection, proteomic analysis, and cellular imaging of O-GlcNAc-modified proteins. O-GlcNAc residues are selectively labeled with fluorescent or biotin tags using an engineered galactosyltransferase enzyme and [3+2] azide-alkyne cycloaddition chemistry. We demonstrate that this approach can be used for direct ingel detection and mass spectrometric identification of O-GlcNAc proteins, identifying 146 novel glycoproteins from the mammalian brain. Furthermore, we show that the method can be exploited to quantify dynamic changes in cellular O-GlcNAc levels and to image O-GlcNAc-glycosylated proteins within cells. As such, this strategy enables studies of O-GlcNAc glycosylation that were previously inaccessible and provides a new tool for uncovering the physiological functions of OGlcNAc.Understanding posttranslational modifications to proteins is critical for elucidating the functional roles of proteins within the dynamic environment of cells. O-Linked β-Nacetylglucosamine (O-GlcNAc) glycosylation has emerged as important for the regulation of diverse cellular processes, including transcription, cell division, and glucose homeostasis. 1 While new chemical tools have provided rapid, sensitive methods for detecting the modification and enabled better control over the activity of O-GlcNAc enzymes, 1a,2 significant challenges remain with regard to elucidating the functions of O-GlcNAc in cells. For instance, a robust method for the direct fluorescence detection of O-GlcNAc proteins in gels would permit monitoring of changes in glycosylation levels in response to cellular stimuli and greatly extend the reach of existing technologies. Furthermore, new tools for imaging OGlcNAc-glycosylated proteins would enable the expression and dynamics of the modification to be monitored in cells and tissues. Here, we report an advanced chemoenzymatic labeling strategy that addresses these important needs. HHMI Author Manuscript HHMI Author Manuscript HHMI Author ManuscriptPrevious studies have shown that an engineered β-1,4-galactosyltransferase enzyme (Y289L GalT) efficiently transfers a ketogalactose moiety from an unnatural UDP substrate selectively onto O-GlcNAc-modified proteins. 2a However, treatment of cell lysates with an aminooxy fluorescein derivative resulted in some nonspecific labeling of proteins. We therefore investigated whether Y289L GalT would accept the UDP-azidogalactose substrate 1 (UDPGalNAz), which would allow for labeling of O-GlcNAc proteins using [3+2] azide-alkyne cycloaddition chemistry ( Figure 1A). 3 In addition to providing alternative dyes to potentially reduce nonspecific interactions, this Cu(I)-catalyzed cycloaddition reaction would have the advantage of being performed more rapidly and at physiological pH.We tested the approach using α-crystallin, a known O-GlcNAc-modified protein with a low extent (~10%) of glycosylation. α-Crystallin was treated with 1 and Y289L GalT, followed by reaction with CuSO 4 , sodium ascor...

Pro-Q Emerald 488 glycoprotein stain reacts with periodic acid-oxidized carbohydrate groups, generating a bright green-fluorescent signal on glycoproteins. The stain permits detection of less than 5-18 ng of glycoprotein per band, depending upon the nature and the degree of protein glycosylation, making it roughly 8-16-fold more sensitive than the standard colorimetric periodic acid-Schiff base method using acidic fuchsin dye (pararosaniline). The green-fluorescent signal from Pro-Q Emerald 488 stain may optimally be visualized using charge-coupled device/xenon arc lamp-based imaging systems or 470-488 nm laser-based gel scanners. Though glycoprotein detection may be performed on transfer membranes, direct detection in gels avoids electroblotting and the specificity of staining is better in gels. After detecting glycoproteins with Pro-Q Emerald 488 dye, total protein profiles may subsequently be detected using SYPRO Ruby protein gel stain. Using computer-assisted registration techniques, images may then be merged to generate differential display maps.

A recently developed proteomic strategy, the “GG-azide”-labeling approach, is described for the detection and proteomic analysis of geranylgeranylated proteins. This approach involves metabolic incorporation of a synthetic azido-geranylgeranyl analog and chemoselective derivatization of azido-geranylgeranyl-modified proteins by the “click” chemistry, using a tetramethylrhodamine-alkyne. The resulting conjugated proteins can be separated by 1-D or 2-D and pH fractionation, and detected by fluorescence imaging. This method is compatible with downstream LC-MS/MS analysis. Proteomic analysis of conjugated proteins by this approach identified several known geranylgeranylated proteins as well as Rap2c, a novel member of the Ras family. Furthermore, prenylation of progerin in mouse embryonic fibroblast cells was examined using this approach, demonstrating that this strategy can be used to study prenylation of specific proteins. The “GG-azide”-labeling approach provides a new tool for the detection and proteomic analysis of geranylgeranylated proteins, and it can readily be extended to other post-translational modifications.