1. Enzymatic deglycosylation
<ol>
 <li>Use PCR tubes to minimize water loss due to evaporation. Label one set of tubes 1 to 7.</li>
 <li>Thaw the 10X G7 buffer, the 10X glycoprotein denaturing buffer, and the 10% NP-40 and gently tap the tubes to mix the contents. Keep at room temperature.</li>
 <li>Place the enzyme-containing vials on ice. Try to minimize thaw/freeze cycles.</li>
 <li>Dissolve the contents of the hCG&beta; vial (150 &mu;g) in 600 &mu;l of dH2O and keep on ice.</li>
 <li>Prepare 1 ml of 1X G7 buffer by diluting the 10X stock in dH2O.</li>
 <li>Dilute 0.5 &mu;l of PNGase F in 25 &mu;l 1X G7 buffer and keep on ice.</li>
 <li>Prepare the exoglycosidase mix (EG mix) by combining 2 &mu;l each of &alpha;-N-Acetylgalactosaminidase, &alpha;1-2 Fucosidase, &beta;1-3 Galactosidase, and &alpha;1-3, 6 Galactosidase.</li>
 <li>Set up PCR tubes as indicated:</li>
</ol>




<table border="1">
 <tbody>
 <tr>
 <td>Tube/sample#</td>
 <td>1</td>
 <td>2</td>
 <td>3</td>
 <td>4</td>
 <td>5</td>
 <td>6</td>
 <td>7</td>
 </tr>
 <tr>
 <td>hCG&beta; (0.25mg/ml)</td>
 <td>9&mu;l</td>
 <td>9&mu;l</td>
 <td>9&mu;l</td>
 <td>9&mu;l</td>
 <td>--</td>
 <td>--</td>
 <td>--</td>
 </tr>
 <tr>
 <td>10X Glycoprotein&nbsp;Denaturing&nbsp;Buffer</td>
 <td>1&mu;l</td>
 <td>1&mu;l</td>
 <td>1&mu;l</td>
 <td>1&mu;l</td>
 <td>1&mu;l</td>
 <td>1&mu;l</td>
 <td>1&mu;l</td>
 </tr>
 <tr>
 <td>dH2O</td>
 <td>--</td>
 <td>--</td>
 <td>--</td>
 <td>--</td>
 <td>9&mu;l</td>
 <td>9&mu;l</td>
 <td>9&mu;l</td>
 </tr>
 </tbody>
</table>
<ol start="9">
 <li>Cap the tubes, mix gently and place in the thermocycler, close the lid and denature the proteins by incubating 10 minutes at 94&deg;C, followed by a 4&deg;C hold (using PCR tubes in a thermocycler greatly prevents evaporation in small volume samples).</li>
 <li>Remove the tubes from thermocycler and centrifuge to remove any visible condensation.</li>
 <li>Add the following reagents as indicated:</li>
</ol>
<table border="1">
 <tbody>
 <tr>
 <td>Sample#</td>
 <td>1</td>
 <td>2</td>
 <td>3</td>
 <td>4</td>
 <td>5</td>
 <td>6</td>
 <td>7</td>
 </tr>
 <tr>
 <td>10% NP-40</td>
 <td>2.5&mu;l</td>
 <td>2.5&mu;l</td>
 <td>2.5&mu;l</td>
 <td>2.5&mu;l</td>
 <td>2.5&mu;l</td>
 <td>2.5&mu;l</td>
 <td>2.5&mu;l</td>
 </tr>
 <tr>
 <td>10X G7 Buffer</td>
 <td>2.5&mu;l</td>
 <td>2.5&mu;l</td>
 <td>2.5&mu;l</td>
 <td>2.5&mu;l</td>
 <td>2.5&mu;l</td>
 <td>2.5&mu;l</td>
 <td>2.5&mu;l</td>
 </tr>
 <tr>
 <td>PNGase F 1:50 dil.</td>
 <td>--</td>
 <td>2&mu;l</td>
 <td>--</td>
 <td>--</td>
 <td>2&mu;l</td>
 <td>--</td>
 <td>--</td>
 </tr>
 <tr>
 <td>Deglycosylation Mix</td>
 <td>--</td>
 <td>--</td>
 <td>2&mu;l</td>
 <td>2&mu;l</td>
 <td>--</td>
 <td>2&mu;l</td>
 <td>2&mu;l</td>
 </tr>
 <tr>
 <td>EG mix</td>
 <td>--</td>
 <td>--</td>
 <td>--</td>
 <td>2&mu;l</td>
 <td>--</td>
 <td>--</td>
 <td>2&mu;l</td>
 </tr>
 <tr>
 <td>dH2O</td>
 <td>10&mu;l</td>
 <td>8&mu;l</td>
 <td>8&mu;l</td>
 <td>6&mu;l</td>
 <td>8&mu;l</td>
 <td>8&mu;l</td>
 <td>6&mu;l</td>
 </tr>
 <tr>
 <td>Total reaction vol.</td>
 <td>25&mu;l</td>
 <td>25&mu;l</td>
 <td>25&mu;l</td>
 <td>25&mu;l</td>
 <td>25&mu;l</td>
 <td>25&mu;l</td>
 <td>25&mu;l</td>
 </tr>
 </tbody>
</table>
<ol start="12">
 <li>Close the PCR tubes using new caps (discard the used ones since they do not fit properly after one incubation cycle).</li>
 <li>Mix the tubes by gently tapping 4 times and then spin the contents down.</li>
 <li>Place the tubes in the thermocycler and incubate at 37 &deg;C for 4 hours then cool the samples to 4 &deg;C.</li>
</ol>
2. SDS-PAGE of deglycosylated samples
<ol>
 <li>Prepare 130 &mu;l fresh 3X reducing SDS loading buffer by adding 4 &mu;l of 1.25M DTT.</li>
 <li>Add 12.5 &mu;l of the prepared 3X reducing SDS loading buffer to each sample.</li>
 <li>Close the tubes with new caps and gently tap the tubes to mix.</li>
 <li>Incubate the tubes in a thermocycler at 94&deg;C for 5 minutes and then cool to 4 &deg;C.</li>
 <li>Load 30 &mu;l of each sample and 10 &mu;l of the protein marker on a 10-20% Tris-Glycine gel. Save the remainder of the sample for part 3.1. Load 10&mu;l of ColorPlus Prestained Protein Marker.</li>
 <li>Electrophorese the gel at 130 volts at room temperature until the dye front is near the bottom of the gel.</li>
 <li>When the gel has finished running, remove the gel from the cast and place it in a small plastic box with enough Coomasie blue stain to cover the gel.</li>
 <li>Stain the gel for 1 hour with gentle agitation.</li>
 <li>Wash the gel three times for 30 minutes in 50 ml of destain solution.</li>
 <li>Record the images using a white light transilluminator or scanner. Alternatively, the gel can be dried between sheets of cellophane in a frame.</li>
</ol>
3. Pro-Q Emerald 300 for detection of glycosylated proteins in SDS-PAGE gels
<ol>
 <li>In parallel with the gel in 2.1), load the remainder of the samples on a 10-20% Tris-Glycine gel. Load 10&mu;l of ColorPlus Prestained Protein Marker.</li>
 <li>While the gel is running dissolve the Pro-Q Emerald reagent with DMF and prepare the stock Fix, Wash and Oxidizing solutions for the Pro-Q Emerald 300 stain following the product manual provided with the kit.</li>
 <li>When the electrophoresis is complete, remove the gel from cast and place it in a plastic box.</li>
 <li>Fix the gel by adding 100 ml of the Fix solution and leaving it overnight at room temperature with gentle agitation.</li>
 <li>Wash the gel with 100 ml of the Wash solution for 10 to 20 minutes at room temperature with gentile agitation. Repeat the wash with fresh Wash solution.</li>
 <li>Oxidize the carbohydrates by incubating the gel with gentle agitation for 30 minutes in 25 ml of Oxidizing solution.</li>
 <li>Wash the gel as described in step 3.5.</li>
 <li>While the gel is washing prepare fresh Pro-Q Emerald 300 stain by adding 500 &mu;l of the Pro-Q Emerald 300 reagent solution dissolved in step 3.2 to 25 ml of staining buffer provided in the kit.</li>
 <li>Stain the gel by adding 25 ml of the stain prepared in step 3.8 and incubating in the dark with gentle agitation for 90 to 120 minutes.</li>
 <li>Repeat the two wash steps described in step 3.5.</li>
 <li>Record the images with an UV transilluminator at 300nm. Use the 80 kDa marker, which is labeled with Pro-Q Emerald Green, to overlap the UV image to a white light picture of the gel showing the prestained ladder.</li>
 <li>Compare the images of the Coomasie stained gel in step 2.10 with the Pro-Q Emerald stained gel from step 3.11.</li>
</ol>
4. Representative results
The changes in protein migration after enzymatic deglycosylation are shown in Figure 2. Compare the control sample (panel A, lane 1) with the PNGase F treatment (removal of N-glycans, lane 2), and Deglycosylation Mix (PNGase F; plus endo-and exoglycosidases to remove O-glycans, lane 3). No further reduction in size is seen after digesting with additional glycosidases (lane 4). Besides a change in mass, bands become sharper as glycans are removed. A band running under the 17 kDa marker (asterisk) represents the fully deglycosylated hCG&beta; polypeptide (MW: 16 kDa). Other bands might derive from incomplete deglycosylation or from the multiple unidentified proteins present in the hCG&beta; sample (see lane 1). Lanes 5 to 7 (controls) show the bands corresponding to the glycosidases.
The glycoprotein staining by Emerald Green is shown in panel B. This reagent oxidizes and stains all glycans present in a protein molecule. Therefore the intensity of the signal decreases as hCG&beta; is enzymatically deglycosylated (lane 1 to lane 4). The residual signal in lanes 3 and 4 indicate the presence of glycan motifs, which are resistant to the enzymes used. The additional glycosidases used in lane 4 remove a few extra sugar residues: the protein migration is the same, but a slight reduction in intensity in staining can be seen. Resistant sugar moieties were not present in all protein species: some bands were not detected by Emerald Green (absent in the UV image, in brackets), indicating they were extensively deglycosylated. Additional data support the conclusion that hCG&beta; is heterogeneously glycosylated. The lower band on lane 2 (arrow) is faint on the Emerald Green image, while the upper band on lane 2 is bright, indicating that many glycans groups are still present. These data support the conclusion that recombinant hCG&beta; expressed in mouse cells contains multiple glycoforms9 . These different glycoforms are due to the inherent heterogeneity of glycosylation where some polypeptides do not receive a glycan in each consensus site and/or some glycans are extended while others even on the same protein are not.
<img alt="Figure 1" src="/files/ftp_upload/3749/3749fig1.jpg" /> 
Figure 1. Typical glycosylation patterns for secreted or cell-surface glycoproteins.
<img alt="Figure 2" src="/files/ftp_upload/3749/3749fig2.jpg" /> 
Figure 2. SDS-PAGE gels showing enzymatic deglycosylation of hCG&beta;. Panel A shows a Coomasie blue staining while Panel B shows the results of Pro-Q Emerald 300 for visualization of glycosylated proteins. Sample number: 1, hCG&beta; control; 2, PNGase F digestion; 3, Deglycosylation Mix digestion; 4, Deglycosylation Mix plus exoglycosidases digestion, samples 5 to 7 are the reagent controls (O-Glyc, O-Glycosidase; GNA, &beta;-N-Acetylglucosaminidase; Gal/Fuc, &beta;1-3 Galactosidase, &alpha;1-3,6 Galactosidase, and &alpha;1-2 Fucosidase; GalNA, &alpha;-N-Acetylglalactosaminidase; Neu, Neuraminidase; PNGase; PNGase F)

<ol>
	<li>Spiro, R. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+glycosylation:+nature,+distribution,+enzymatic+formation,+and+disease+implications+of+glycopeptide+bonds.">Protein glycosylation: nature, distribution, enzymatic formation, and disease implications of glycopeptide bonds.</a> Glycobiology. 12 (4), 43-56 (2002).</li><li>Ohtsubo, K., Marth, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Glycosylation+in+Cellular+Mechanisms+of+Health+and+Disease.">Glycosylation in Cellular Mechanisms of Health and Disease.</a> Cell. 126, 855-867 (2006).</li><li>Arnold, J. N., Wormald, M. R., Sim, R. B., Rudd, P. M., Dwek, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+impact+of+glycosylation+on+the+biological+function+and+structure+of+human+immunoglobulins.">The impact of glycosylation on the biological function and structure of human immunoglobulins.</a> Annual Review of Immunology. 25, 21-50 (2007).</li><li>Mitra, N., Sinha, S., Ramya, T. N. C., Surolia, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=N-linked+oligosaccharides+as+outfitters+for+glycoprotein+folding,+form+and+function.">N-linked oligosaccharides as outfitters for glycoprotein folding, form and function.</a> Trends in Biochemical Sciences. 31, 156-163 (2006).</li><li>Carlsen, R. B., Bahl, O. P., Swaminathan, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Human+chorionic+gonadotropin.+Linear+amino+acid+sequence+of+the+beta+subunit.">Human chorionic gonadotropin. Linear amino acid sequence of the beta subunit.</a> Journal of Biological Chemistry. 248 (19), 6810-6812 (1973).</li><li>Tarentino, A. L., Plummer, T. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Deglycosylation+of+Asparagine-linked+Glycans+by+PNGaseF.">Deglycosylation of Asparagine-linked Glycans by PNGaseF.</a> Trends in Glycoscience and Glycotechnology. 5 (23), 163-170 (1993).</li><li>Tretter, V., Altmann, F., Marz, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Peptide-N4-(N-acetyl-&#946;-glucosaminyl)asparagine+amidase+F+cannot+release+glycans+with+fucose+attached+&#945;1-3+to+the+asparagine-linked+N-acetylglucosamine+residue.">Peptide-N4-(N-acetyl-&#946;-glucosaminyl)asparagine amidase F cannot release glycans with fucose attached &#945;1-3 to the asparagine-linked N-acetylglucosamine residue.</a> European Journal of Biochemistry. 199, 647-652 (1991).</li><li>Koutsioulis, D., Landry, D., Guthrie, E. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Novel+endo-&#945;-N-acetylgalactosaminidases+with+broader+substrate+specificity.">Novel endo-&#945;-N-acetylgalactosaminidases with broader substrate specificity.</a> Glycobiology. 18 (10), 799-805 (2008).</li><li>Thakur, D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Profiling+the+glycoforms+of+the+intact+alpha+subunit+of+recombinant+human+chorionic+gonadotropin+by+high-resolution+capillary+electrophoresis-mass+spectrometry.">Profiling the glycoforms of the intact alpha subunit of recombinant human chorionic gonadotropin by high-resolution capillary electrophoresis-mass spectrometry.</a> Analytical Chemistry. 81 (2), 8900-8907 (2009).</li><li>Park, S., Lee, M. R., Shin, I. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Carbohydrate+microarrays+as+powerful+tools+in+studies+of+carbohydrate-mediated+biological+processes.">Carbohydrate microarrays as powerful tools in studies of carbohydrate-mediated biological processes.</a> Chemical Communications. 37, 4389-4399 (2008).</li><li>Zachara, N. E., Vosseller, K., Hart, G. W. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detection+and+analysis+of+proteins+modified+by+O-linked+N-acetylglucosamine.">Detection and analysis of proteins modified by O-linked N-acetylglucosamine.</a> Current Protocols in Molecular Biology. 95, 17-17 (2011).</li><li>Varki, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Essentials of Glycobiology. , (2008).</li></ol>

The authors are employed by New England Biolabs, which produces many of the reagents used in this article.

The method described here using enzymatic deglycosylation and SDS-PAGE can provide valuable information about the glycosylation state of a protein of interest, while glycan-specific reagents facilitate the interpretation of the data. This protocol is intended for the initial studies of protein glycosylation and it is particularly suited for secretory and membrane glycoproteins from mammalian cells: the enzymes chosen in this case will specifically remove all N-glycans, and or N-glycans plus and the most common sugars extending and forming the core of O-glycans. Glycosidases have the additional advantage of being mild, compared with chemical deglycosylation methods, preserving the integrity of both sugars and protein backbone.
To elucidate the rate of occupancy (which amino acids are glycosylated), extent of glycosylation, or to determine the fine structure of glycans, more sophisticated techniques such as mass spectrometry, liquid chromatography or NMR are required.
Because of its simplicity, several steps in this protocol can be adjusted, substituted, and/or combined to accommodate various experimental needs. However, in order to obtain results that can be clearly interpreted it is important to understand its strengths and limitations. First, the specificity and purity of the glycosidases are crucial: only well-characterized enzymes tested to be free of proteases and other contaminating activities should be used. Unfortunately, there is no standard enzyme unit definition for glycosidases; the user should determine the appropriate substitution according to the manufacturer's specifications. Second, a careful choice of detection is necessary: a) Protein staining reagents are useful only if deglycosylation results in a significant shift in molecular mass. Such a clear result as shown here is not always obtained. In other cases, we have seen abnormal migration after deglycosylation (far from the predicted molecular weight, or even slower migration). This phenomenon is not well understood, but it can be said that any change in migration is evidence that the protein has been deglycosylated. b) Sugar detection with antibodies presents unique challenges limiting their applicability. It has been very difficult to generate general anti-glycan antibodies; they are usually raised against a complex glycan target, which restricts their use. Moreover, several monoclonal anti-glycan antibodies display undesired cross reactivity10. c) Lectins (proteins with intrinsic sugar affinity) are well suited for sugar detection, plus they give insight on glycan structure. However, not all of them have a narrow specificity; and many are only partially characterized (meaning that they could have unknown affinities). As a consequence positive lectin staining provides indication, not proof, of the presence of a given sugar. d) Chemical labeling kits (based on periodate oxidation of sugars) are the method of choice to stain all glycoproteins, and thus well suited to follow deglycosylation.
When processing an unknown sample, it is a good practice to include glycoprotein controls. Fetuin is a readily available N- and O-glycoprotein; chorionic gonadotropin (both subunits) is also a good choice. Bovine serum albumin (BSA) can be used as a negative control. However, it should be noted that some non-glycosylated proteins react slightly with the Pro-Q Emerald 300, particularly when used at high concentrations. Non-glycosylated molecular weight standards, such as the prestained protein marker used in this protocol, have the advantage of displaying sharp bands. Only by chance is one of these proteins (80 kDa) reactive to Pro-Q Emerald 300. Therefore, the user might want to run a glycoprotein standard ladder instead, such as Candy-Cane from Invitrogen.
Finally, simple in-gel detection of nucleocytosolic glycoproteins (which are modified with a single O-GlcNAc) is possible with the use of a monoclonal antibody, along &beta;-N-Acetylglucosaminidase for specificity control 11.The description of this technique is beyond the scope of this article, but it should be mentioned as glycosidases are useful tools for the study of many other glycoproteins and glycoconjugates present in cells. A comprehensive treatment of all known forms of glycosylation can be found in the second edition of the "Essentials of Glycobiology"; available free online at the NCBI Bookshelf (Bookshelf ID: NBK1908; PMID: 20301239)12.

<table><tbody><tr><td>Human chorionic gonadotropin &amp;#946;</td><td>Sigma-Aldrich</td><td>C6572</td><td> </td></tr><tr><td>PCR Tubes</td><td>VWR international</td><td>20170-004</td><td> </td></tr><tr><td>PCR Thermocycler</td><td>Applied Biosystems</td><td>4359659</td><td> </td></tr><tr><td>PNGase F</td><td>New England Biolabs</td><td>P0704</td><td>Supplied with buffers </td></tr><tr><td>Protein Deglycosylation Mix</td><td>New England Biolabs</td><td>P6039</td><td>Supplied with buffers</td></tr><tr><td>&amp;alpha;-N-Acetylglalactosaminidase</td><td>New England Biolabs</td><td>P0734</td><td>Supplied with buffer</td></tr><tr><td>&amp;alpha;1-2 Fucosidase</td><td>New England Biolabs</td><td>P0724</td><td>Supplied with buffer</td></tr><tr><td>&amp;alpha;1-3,6 Galactosidase</td><td>New England Biolabs</td><td>P0731</td><td>Supplied with buffer</td></tr><tr><td>&amp;#946;1-3 Galactosidase</td><td>New England Biolabs</td><td>P0726</td><td>Supplied with buffer</td></tr><tr><td>10X Buffer G7 (500 mM Sodium Phosphate)</td><td>New England Biolabs</td><td>---</td><td>Supplied with PNGaseF or Degl Mix</td></tr><tr><td>10X Glycoprotein Denaturing Buffer (5% SDS, 400 mM DTT)</td><td>New England Biolabs</td><td>---</td><td>Supplied with PNGaseF or Degl Mix</td></tr><tr><td>10% NP-40</td><td>New England Biolabs</td><td>---</td><td>Supplied with PNGaseF or Degl Mix</td></tr><tr><td>3X SDS loading buffer (187.5 mM Tris-HCl pH 6.8, 6% SDS, 30% glycerol, 0.03% bromophenol blue)</td><td>New England Biolabs</td><td>B7703</td><td>Supplied with 1.25M DTT</td></tr><tr><td>ColorPlus Prestained Protein Marker</td><td>New England Biolabs</td><td>P7709</td><td> </td></tr><tr><td>10-20% Tris-Glycine Multigel</td><td>Cosmo Bio Co.</td><td>DCB-414893</td><td> </td></tr><tr><td>Cassette Electrophoresis Unit</td><td>Cosmo Bio Co.</td><td>DCB-303111</td><td> </td></tr><tr><td>Electrophoresis Power Supply EPS 301</td><td>GE Healthcare</td><td>18-1130-01</td><td> </td></tr><tr><td>Pro-Q Emerald 300 Glycoprotein Stain Kit</td><td>Invitrogen</td><td>P-21857</td><td> </td></tr><tr><td>Brilliant Blue R</td><td>Sigma-Aldrich</td><td>B0149</td><td> </td></tr><tr><td>AlphaImager HP System</td><td>Cell Biosciences</td><td>92-13823-00</td><td> </td></tr></tbody></table>

identification and characterization of protein glycosylation using specific endo- and exoglycosidases

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes such as cell adhesion, molecular trafficking, clearance, and signal transduction1-4. In eukaryotes, the most common glycosylation modifications in the secretory pathway are additions at consensus asparagine residues (N-linked); or at serine or threonine residues (O-linked) (Figure 1). Initiation of N-glycan synthesis is highly conserved in eukaryotes, while the end products can vary greatly among different species, tissues, or proteins. Some glycans remain unmodified ("high mannose N-glycans") or are further processed in the Golgi ("complex N-glycans"). Greater diversity is found for O-glycans, which start with a common N-Acetylgalactosamine (GalNAc) residue in animal cells but differ in lower organisms1.
The detailed analysis of the glycosylation of proteins is a field unto itself and requires extensive resources and expertise to execute properly. However a variety of available enzymes that remove sugars (glycosidases) makes possible to have a general idea of the glycosylation status of a protein in a standard laboratory setting. Here we illustrate the use of glycosidases for the analysis of a model glycoprotein: recombinant human chorionic gonadotropin beta (hCG&beta;), which carries two N-glycans and four O-glycans 5. The technique requires only simple instrumentation and typical consumables, and it can be readily adapted to the analysis of multiple glycoprotein samples.
Several enzymes can be used in parallel to study a glycoprotein. PNGase F is able to remove almost all types of N-linked glycans6,7. For O-glycans, there is no available enzyme that can cleave an intact oligosaccharide from the protein backbone. Instead, O-glycans are trimmed by exoglycosidases to a short core, which is then easily removed by O-Glycosidase. The Protein Deglycosylation Mix contains PNGase F, O-Glycosidase, Neuraminidase (sialidase), &beta;1-4 Galactosidase, and &beta;-N-Acetylglucosaminidase. It is used to simultaneously remove N-glycans and some O-glycans8 . Finally, the Deglycosylation Mix was supplemented with a mixture of other exoglycosidases (&alpha;-N-Acetylgalactosaminidase, &alpha;1-2 Fucosidase, &alpha;1-3,6 Galactosidase, and &beta;1-3 Galactosidase ), which help remove otherwise resistant monosaccharides that could be present in certain O-glycans.
SDS-PAGE/Coomasie blue is used to visualize differences in protein migration before and after glycosidase treatment. In addition, a sugar-specific staining method, ProQ Emerald-300, shows diminished signal as glycans are successively removed. This protocol is designed for the analysis of small amounts of glycoprotein (0.5 to 2 &mu;g), although enzymatic deglycosylation can be scaled up to accommodate larger quantities of protein as needed.

Watch this Scientific Journal Video about Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases at JoVE.com

Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases

Using specific glycosidases to remove sugars from glycoproteins followed by SDS-PAGE is a valuable method to detect glycan modifications on protein samples and is a good choice for initial glycobiology studies. Changes following deglycosylation can be detected as shifts in gel mobility or by staining with glycan sensitive reagents.

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes ...

identification-characterization-protein-glycosylation-using-specific

Nanyang Technological University

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36.5K Views.  New England Biolabs. Using specific glycosidases to remove sugars from glycoproteins followed by SDS-PAGE is a valuable method to detect glycan modifications on protein samples and is a good choice for initial glycobiology studies. Changes following deglycosylation can be detected as shifts in gel mobility or by staining with glycan sensitive reagents.

Video: Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases

Improved In-gel Reductive &#946;-Elimination for Comprehensive O-linked and Sulfo-glycomics by Mass Spectrometry

Separation of proteins by SDS-PAGE followed by in-gel proteolytic digestion of resolved protein bands has produced high-resolution proteomic analysis of biological samples. Similar approaches, that would allow in-depth analysis of the glycans carried by glycoproteins resolved by SDS-PAGE, require special considerations in order to maximize recovery and sensitivity when using mass spectrometry (MS) as the detection method. A major hurdle to be overcome in achieving high-quality data is the removal of gel-derived contaminants that interfere with MS analysis. The sample workflow presented here is robust, efficient, and eliminates the need for in-line HPLC clean-up prior to MS. Gel pieces containing target proteins are washed in acetonitrile, water, and ethyl acetate to remove contaminants, including polymeric acrylamide fragments. O-linked glycans are released from target proteins by in-gel reductive &#946;-elimination and recovered through robust, simple clean-up procedures. An advantage of this workflow is that it improves sensitivity for detecting and characterizing sulfated glycans. These procedures produce an efficient separation of sulfated permethylated glycans from non-sulfated (sialylated and neutral) permethylated glycans by a rapid phase-partition prior to MS analysis, and thereby enhance glycomic and sulfoglycomic analyses of glycoproteins resolved by SDS-PAGE.

In order to comprehensively explore the diversity of O-linked glycans, a new procedure for in-gel reductive &#946;-elimination, combined with permethylation and a rapid phase-partition method, is applied to the analysis of O-linked glycans directly released from glycoproteins resolved by SDS-PAGE and amenable to subsequent glycomic analysis by mass spectrometry.

Separation of proteins by SDS-PAGE followed by in-gel proteolytic digestion of resolved protein bands has produced high-resolution proteomic analysis of ...

Chemistry

Glycoproteomics of the Extracellular Matrix: A Method for Intact Glycopeptide Analysis Using Mass Spectrometry

Fibrosis is a hallmark of many cardiovascular diseases and is associated with the exacerbated secretion and deposition of the extracellular matrix (ECM). Using proteomics, we have previously identified more than 150 ECM and ECM-associated proteins in cardiovascular tissues. Notably, many ECM proteins are glycosylated. This post-translational modification affects protein folding, solubility, binding, and degradation. We have developed a sequential extraction and enrichment method for ECM proteins that is compatible with the subsequent liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis of intact glycopeptides. The strategy is based on sequential incubations with NaCl, SDS for tissue decellularization, and guanidine hydrochloride for the solubilization of ECM proteins. Recent advances in LC-MS/MS include fragmentation methods, such as combinations of higher-energy collision dissociation (HCD) and electron transfer dissociation (ETD), which allow for the direct compositional analysis of glycopeptides of ECM proteins. In the present paper, we describe a method to prepare the ECM from tissue samples. The method not only allows for protein profiling but also the assessment and characterization of glycosylation by MS analysis.

This paper describes a methodology to prepare cardiovascular tissue samples for MS analysis that allows for (1) the analysis of ECM protein composition, (2) the identification of glycosylation sites, and (3) the compositional characterization of glycan forms. This methodology can be applied, with minor modifications, to the study of the ECM in other tissues.

Fibrosis is a hallmark of many cardiovascular diseases and is associated with the exacerbated secretion and deposition of the extracellular matrix (ECM). ...

Biochemistry

A Quantitative Glycomics and Proteomics Combined Purification Strategy

There is a growing desire in the biological and clinical sciences to integrate and correlate multiple classes of biomolecules to unravel biology, define pathways, improve treatment, understand disease, and aid biomarker discovery. N-linked glycosylation is one of the most important and robust post-translational modifications on proteins and regulates critical cell functions such as signaling, adhesion, and enzymatic function. Analytical techniques to purify and analyze N-glycans have remained relatively static over the last decade. While accurate and effective, they commonly require significant expertise and resources. Though some high-throughput purification schemes have been developed, they have yet to find widespread adoption and often rely on the enrichment of glycopeptides. One promising method, developed by Thomas-Oates et al., filter aided N-glycan separation (FANGS), was qualitatively demonstrated on tissues. Herein, we adapted FANGS to plasma and coupled it to the individuality normalization when labeling with glycan hydrazide tags strategy in order to achieve accurate relative quantification by liquid chromatography mass spectrometry and enhanced electrospray ionization. Furthermore, we designed new functionality to the protocol by achieving tandem, shotgun proteomics and glycosylation site analysis on hen plasma. We showed that N-glycans purified on filter and derivatized by hydrophobic hydrazide tags were comparable in terms of abundance and class to those by solid phase extraction (SPE); the latter is considered a gold standard in the field. Importantly, the variability in the two protocols was not statistically different. Proteomic data that was collected in-line with glycomic data had the same depth compared to a standard trypsin digest. Peptide deamidation is minimized in the protocol, limiting non-specific deamidation detected at glycosylation motifs. This allowed for direct glycosylation site analysis, though the protocol can accommodate 18O site labeling as well. Overall, we demonstrated a new in-line high-throughput, unbiased, filter based protocol for quantitative glycomics and proteomics analysis.

A high-throughput protocol was developed for combined proteomics and glycomics purification and LC-MS/MS quantification in plasma. Deamidation analysis of N-linked glycosylation motifs was specific to deglycosylated sites. Accurate quantitation of N-glycans was achieved by coupling filter aided N-glycan separation to the individuality normalization when labeling with glycan hydrazide tags strategy.

There is a growing desire in the biological and clinical sciences to integrate and correlate multiple classes of biomolecules to unravel biology, define ...

Mass Spectrometric Analysis of Glycosphingolipid Antigens

Glycosphingolipids (GSL's) belong to the glycoconjugate class of biomacromolecules, which bear structural information for significant biological processes such as embryonic development, signal transduction, and immune receptor recognition1-2. They contain complex sugar moieties in the form of isomers, and lipid moieties with variations including fatty acyl chain length, unsaturation, and hydroxylation. Both carbohydrate and ceramide portions may be basis of biological significance. For example, tri-hexosylceramides include globotriaosylceramide (Gal&alpha;4Gal&beta;4Glc&beta;1Cer) and isoglobotriaosylceramide (Gal&alpha;3Gal&beta;4Glc&beta;1Cer), which have identical molecular masses but distinct sugar linkages of carbohydrate moiety, responsible for completely different biological functions3-4. In another example, it has been demonstrated that modification of the ceramide part of alpha-galactosylceramide, a potent agonist ligand for invariant NKT cells, changes their cytokine secretion profiles and function in animal models of cancer and auto-immune diseases5. The difficulty in performing a structural analysis of isomers in immune organs and cells serve as a barrier for determining many biological functions6.
Here, we present a visualized version of a method for relatively simple, rapid, and sensitive analysis of glycosphingolipid profiles in immune cells7-9. This method is based on extraction and chemical modification (permethylation, see below Figure 5A, all OH groups of hexose were replaced by MeO after permethylation reaction) of glycosphingolipids10-15, followed by subsequent analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) and ion trap mass spectrometry. This method requires 50 million immune cells for a complete analysis. The experiments can be completed within a week. The relative abundance of the various glycosphingolipids can be delineated by comparison to synthetic standards. This method has a sensitivity of measuring 1% iGb3 among Gb3 isomers, when 2 fmol of total iGb3/Gb3 mixture is present9.
Ion trap mass spectrometry can be used to analyze isomers. For example, to analyze the presence of globotriaosylceramide and isoglobtriaosylceramide in the same sample, one can use the fragmentation of glycosphingolipid molecules to structurally discriminate between the two (see below Figure 5). Furthermore, chemical modification of the sugar moieties (through a permethylation reaction) improves the ionization and fragmentation efficiencies for higher sensitivity and specificity, and increases the stability of sialic acid residues. The extraction and chemical modification of glycosphingolipids can be performed in a classic certified chemical hood, and the mass spectrometry can be performed by core facilities with ion trap MS instruments.

A specific and sensitive method to gain insight into the expression profile of glycosphingolipid antigens in immune organs and cells is described. The method takes advantage of the ion trap mass spectrometry allowing step-wise fragmentation of glycosphingolipid molecules for structural analysis in comparison to chemically synthesized standards.

Glycosphingolipids (GSL's) belong to the glycoconjugate class of biomacromolecules, which bear structural information for significant biological processes ...

Immunology and Infection

N-glycan Profiling of Glycoproteins by Hydrophilic Interaction Liquid Chromatography with Fluorescence and Mass Spectrometric Detection

Glycosylation is a vital modification found in proteins. N-glycan profiling of glycoproteins is required to detect novel biomarker candidates and determine glycan alterations in diseases. Most commercially available biopharmaceutical proteins are glycoproteins. The efficacy of these drugs is affected by glycosylation patterns. Therefore, an in-depth characterization method for the N-glycans is necessary. Here, we present a comprehensive approach for qualitative and quantitative analysis of N-glycans using hydrophilic interaction liquid chromatography equipped with fluorescence detection and tandem mass spectrometry (HILIC-FLD-MS/MS). N-glycans were released from glycoproteins with a facile method and labeled by a procainamide fluorophore tag in the strategy. Subsequently, the procainamide labeled N-glycans were analyzed by a HILIC-FLD-MS/MS technique. In this approach, N-glycan structures were confirmed by the tandem mass spectrometric analysis, whereas fluorescence detection was used for the quantitative analysis. An application for data analysis of the detected N-glycan peaks is described in the study. This protocol can be applied to any glycoprotein extracted from various species.

N-glycan Profiling of Glycoproteins by Hydrophilic Interaction Liquid Chromatography with Fluorescence and Mass Spectrometric Detection

N-glycan profiling of glycoproteins is essential for discovering novel biomarkers and understanding glycan functions in cellular events. Additionally, N-glycan analysis of protein biopharmaceuticals is very important for human use. In this current article, a high-throughput strategy for identifying and quantifying N-glycan structures was presented using the HILIC-FLD-MS/MS technique.

Glycosylation is a vital modification found in proteins. N-glycan profiling of glycoproteins is required to detect novel biomarker candidates and ...

Recombinant Production of Bifidobacterial Endoglycosidases for N-glycan Release

Protein glycosylation is a diverse and common post-translational modification that has been associated with many important roles such as protein function, including protein folding, stability, enzymatic protection, and biological recognition. N-glycans attached to glycoproteins (such as lactoferrin, lactadherin, and immunoglobulins) cannot be digested by the host and reach the large intestine, where they are consumed by certain beneficial microbes. Therefore, they are considered next-generation prebiotic compounds that can selectively stimulate the gut microbiome's beneficial microorganisms. However, the isolation of these new classes of prebiotics requires novel enzymes. Here, we describe the recombinant production of novel glycosidases from different Bifidobacteria strains (isolated from infants, rabbits, chicken, and bumblebee) for improved N-glycan isolation from glycoproteins. The method presented in this study includes the following steps: molecular cloning of Bifidobacterial genes by an in vivo recombinational cloning strategy, control of transformation success, protein induction, and protein purification.

Recombinant Production of Bifidobacterial Endoglycosidases for N-glycan Release

Bifidobacteria possess a unique genomic capability for N-glycan cleavage. Recombinantly producing these enzymes would be a promising novel tool to release bioactive N-glycans from glycoprotein-rich substrates such as colostrum.

Protein glycosylation is a diverse and common post-translational modification that has been associated with many important roles such as protein function, ...

The Application of Open Searching-based Approaches for the Identification of Acinetobacter baumannii O-linked Glycopeptides

Protein glycosylation is increasingly recognized as a common modification within bacterial organisms, contributing to prokaryotic physiology and optimal infectivity of pathogenic species. Due to this, there is increasing interest in characterizing bacterial glycosylation and a need for high-throughput analytical tools to identify these events. Although bottom-up proteomics readily enables the generation of rich glycopeptide data, the breadth and diversity of glycans observed in prokaryotic species make the identification of bacterial glycosylation events extremely challenging.
Traditionally, the manual determination of glycan compositions within bacterial proteomic datasets made this a largely bespoke analysis restricted to field-specific experts. Recently, open searching-based approaches have emerged as a powerful alternative for the identification of unknown modifications. By analyzing the frequency of unique modifications observed on peptide sequences, open searching techniques allow the identification of common glycans attached to peptides within complex samples. This article presents a streamlined workflow for the interpretation and analysis of glycoproteomic data, demonstrating how open searching techniques can be used to identify bacterial glycopeptides without prior knowledge of the glycan compositions.
Using this approach, glycopeptides within samples can rapidly be identified to understand glycosylation differences. Using Acinetobacter baumannii as a model, these approaches enable the comparison of glycan compositions between strains and the identification of novel glycoproteins. Taken together, this work demonstrates the versatility of open database-searching techniques for the identification of bacterial glycosylation, making the characterization of these highly diverse glycoproteomes easier than ever before.

The Application of Open Searching-based Approaches for the Identification of Acinetobacter baumannii O-linked Glycopeptides

Open searching enables the identification of glycopeptides decorated with previously unknown glycan compositions. Within this article, a streamlined approach for undertaking open searching and subsequent glycan-focused glycopeptide searches are presented for bacterial samples using Acinetobacter baumannii as a model.

Protein glycosylation is increasingly recognized as a common modification within bacterial organisms, contributing to prokaryotic physiology and optimal ...

Nonspecific Proteolysis-Based Glycomics Strategy for Bacterial Glycoproteins

Protein glycosylation is one of the most common and complex post-translational modifications. Many techniques have been developed to characterize the specific roles of glycans, the relationship between their structures and their impact on the functions of proteins. A common method for glycan analysis is to employ exoglycosidase cleavage to release N-linked glycans from glycoproteins or glycopeptides using Peptide-N-Glycosidase F (PNGase F). However, the glycan-protein linkages in bacteria are different and there is no enzyme available to release glycans from bacterial glycoproteins. In addition, free glycans have also been described in mammalian cells, bacteria, yeast, plants, and fish. In this article, we present a method that can characterize the N-linked glycosylation system in Campylobacter jejuni by detecting asparagine (Asn)-linked and free glycans that are not attached to their target proteins. In this method, total proteins from C. jejuni were digested by Pronase E with a higher enzyme to protein ratio (2:1&#8722;3:1) and a longer incubation time (48&#8722;72 h). The resulted Asn-glycans and free glycans were then purified using porous graphitic carbon cartridges, permethylated, and analyzed by mass spectrometry.

This study presents a method for glycomics analysis of glycoproteins by a combination of pronase E digestion, permethylation, and mass spectrometry analysis. This method is capable of analyzing all types of N-linked glycans, including bacterial N-glycans.

Protein glycosylation is one of the most common and complex post-translational modifications. Many techniques have been developed to characterize the ...

Profiling of Colorectal Cancer Tissues Using Glycomics-Guided Glycoproteomics

Glycomics-Guided Glycoproteomics Facilitates Comprehensive Profiling of the Glycoproteome in Complex Tumor Microenvironments

Glycosylation is a common and structurally diverse protein modification that impacts a wide range of tumorigenic processes. Mass spectrometry-driven glycomics and glycoproteomics have emerged as powerful approaches to analyze liberated glycans and intact glycopeptides, respectively, offering a deeper understanding of the heterogeneous glycoproteome in the tumor microenvironment (TME). This protocol details the glycomics-guided glycoproteomics method, an integrated omics technology, which firstly employs porous graphitized carbon-LC-MS/MS-based glycomics to elucidate the glycan structures and their quantitative distribution in the glycome of tumor tissues, cell populations, or bodily fluids being investigated. This allows for a comparative glycomics analysis to identify altered glycosylation across patient groups, disease stages, or conditions, and, importantly, serves to enhance the downstream glycoproteomics analysis of the same sample(s) by creating a library of known glycan structures, thus reducing the data search time and the glycoprotein misidentification rate. Focusing on N-glycoproteome profiling, the sample preparation steps for the glycomics-guided glycoproteomics method are detailed in this protocol, and key aspects of the data collection and analysis are discussed. The glycomics-guided glycoproteomics method provides quantitative information on the glycoproteins present in the TME and their glycosylation sites, site occupancy, and site-specific glycan structures. Representative results are presented from formalin-fixed paraffin-embedded tumor tissues from colorectal cancer patients, demonstrating that the method is sensitive and compatible with tissue sections commonly found in biobanks. Glycomics-guided glycoproteomics, therefore, offers a comprehensive view into the heterogeneity and dynamics of the glycoproteome in complex TMEs, generating robust biochemical data required to better understand the glycobiology of cancers.

This protocol details the glycomics-guided glycoproteomics method, an integrated omics technology that offers comprehensive insights into the heterogeneous glycoproteome in complex tumor microenvironments required to better understand the glycobiology of cancers.

Glycosylation is a common and structurally diverse protein modification that impacts a wide range of tumorigenic processes. Mass spectrometry-driven ...

Cancer Research

Glycopeptide Capture for Cell Surface Proteomics

Cell surface proteins, including extracellular matrix proteins, participate in all major cellular processes and functions, such as growth, differentiation, and proliferation. A comprehensive characterization of these proteins provides rich information for biomarker discovery, cell-type identification, and drug-target selection, as well as helping to advance our understanding of cellular biology and physiology. Surface proteins, however, pose significant analytical challenges, because of their inherently low abundance, high hydrophobicity, and heavy post-translational modifications. Taking advantage of the prevalent glycosylation on surface proteins, we introduce here a high-throughput glycopeptide-capture approach that integrates the advantages of several existing N-glycoproteomics means. Our method can enrich the glycopeptides derived from surface proteins and remove their glycans for facile proteomics using LC-MS. The resolved N-glycoproteome comprises the information of protein identity and quantity as well as their sites of glycosylation. This method has been applied to a series of studies in areas including cancer, stem cells, and drug toxicity. The limitation of the method lies in the low abundance of surface membrane proteins, such that a relatively large quantity of samples is required for this analysis compared to studies centered on cytosolic proteins.

Cell surface proteins are biologically important and widely glycosylated. We introduce here a glycopeptide-capture approach to solubilize, enrich, and deglycosylate these proteins for facile LC-MS based proteomic analyses.

Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases

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Improved In-gel Reductive β-Elimination for Comprehensive O-linked and Sulfo-glycomics by Mass Spectrometry

Glycoproteomics of the Extracellular Matrix: A Method for Intact Glycopeptide Analysis Using Mass Spectrometry

A Quantitative Glycomics and Proteomics Combined Purification Strategy

Mass Spectrometric Analysis of Glycosphingolipid Antigens

N-glycan Profiling of Glycoproteins by Hydrophilic Interaction Liquid Chromatography with Fluorescence and Mass Spectrometric Detection

Recombinant Production of Bifidobacterial Endoglycosidases for N-glycan Release

The Application of Open Searching-based Approaches for the Identification of Acinetobacter baumannii O-linked Glycopeptides

Nonspecific Proteolysis-Based Glycomics Strategy for Bacterial Glycoproteins

Glycomics-Guided Glycoproteomics Facilitates Comprehensive Profiling of the Glycoproteome in Complex Tumor Microenvironments

Glycopeptide Capture for Cell Surface Proteomics