Name: analysis of n-glycans from raphanus sativus cultivars using pngase h+
Uploaded: 2018-06-25T15:00:00
Description: Watch this Scientific Journal Video about Analysis of N-glycans from Raphanus sativus Cultivars Using PNGase H+ at JoVE.com

This work was supported in part by the Natural Science Foundation of China (grant numbers 31471703, A0201300537 and 31671854 to J.V. and L.L., grant number 31470435 to G.Y.), and the 100 Foreign Talents Plan (grant number JSB2014012 to J.V.).

1. Sample collection
<ol>
	<li>Purchase different cultivars of fresh radish (Raphanus sativus L.).</li>
</ol>
2. Isolation of Protein from Radish
<ol>
	<li>Homogenize approximately 100 g of fresh radish with a kitchen blender for 10 min.</li>
	<li>Transfer the slurry to a 50 mL centrifuge tube and centrifuge at 14,000 &#215; g at 4 &#176;C for 20 min to remove the insoluble material.</li>
	<li>Transfer the supernatant carefully into a new 50 mL centrifuge tube and add an equa...

<ol>
	<li>Altmann, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Role+of+Protein+Glycosylation+in+Allergy.">The Role of Protein Glycosylation in Allergy.</a> International Archives of Allergy and Immunology. 142 (2), 99-115 (2007).</li><li>Van Ree, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=&#946;+(1,+2)-xylose+and+&#945;+(1,+3)-fucose+residues+have+a+strong+contribution+in+IgE+binding+to+plant+glycoallergens.">&#946; (1, 2)-xylose and &#945; (1, 3)-fucose residues have a strong contribution in IgE binding to plant glycoallergens.</a> Journal of Biological Chemistry. 275 (15), 11451-11458 (2000).</li><li>Biswas, H., Chattopadhyaya, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stability+of+Curcuma+longa+rhizome+lectin:+Role+of+N-linked+glycosylation.">Stability of Curcuma longa rhizome lectin: Role of N-linked glycosylation.</a> Glycobiology. 26 (4), 410-426 (2016).</li><li>Lefebvre, B., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Role+of+N-glycosylation+sites+and+CXC+motifs+in+trafficking+of+Medicago+truncatula+Nod+factor+perception+protein+to+plasma+membrane.">Role of N-glycosylation sites and CXC motifs in trafficking of Medicago truncatula Nod factor perception protein to plasma membrane.</a> Journal of Biological Chemistry. 287 (14), 10812-10823 (2012).</li><li>Severino, V., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+role+of+the+glycan+moiety+on+the+structure-function+relationships+of+PD-L1,+type+1+ribosome-inactivating+protein+from+P.+dioica+leaves.">The role of the glycan moiety on the structure-function relationships of PD-L1, type 1 ribosome-inactivating protein from P. dioica leaves.</a> Molecular BioSystems. 6 (3), 570-579 (2010).</li><li>Lige, B., Ma, S., Van Huystee, R. B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+effects+of+the+site-directed+removal+of+N-glycosylation+from+cationic+peanut+peroxidase+on+its+function.">The effects of the site-directed removal of N-glycosylation from cationic peanut peroxidase on its function.</a> Archives of Biochemistry and Biophysics. 386 (1), 17-24 (2001).</li><li>Ceriotti, A., Duranti, M., Bollini, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+N-glycosylation+on+the+folding+and+structure+of+plant+proteins.">Effects of N-glycosylation on the folding and structure of plant proteins.</a> Journal of Experimental Botany. 49 (324), 1091-1103 (1998).</li><li>Kamerling, J. P., Gerwig, G. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Strategies+for+the+Structural+Analysis+of+Carbohydrates.">Strategies for the Structural Analysis of Carbohydrates.</a> Comprehensive Glycoscience. , 1-68 (2007).</li><li>Huang, Y., Mechref, Y., Novotny, M. V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microscale+nonreductive+release+of+O-linked+glycans+for+subsequent+analysis+through+MALDI+mass+spectrometry+and+capillary+electrophoresis.">Microscale nonreductive release of O-linked glycans for subsequent analysis through MALDI mass spectrometry and capillary electrophoresis.</a> Analytical Chemistry. 73 (24), 6063-6069 (2001).</li><li>Triguero, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chemical+and+enzymatic+N-glycan+release+comparison+for+N-glycan+profiling+of+monoclonal+antibodies+expressed+in+plants.">Chemical and enzymatic N-glycan release comparison for N-glycan profiling of monoclonal antibodies expressed in plants.</a> Analytical Biochemistry. 400 (2), 173-183 (2010).</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+&#8594;+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 &#8594; 3 to the asparagine-linked N-acetylglucosamine residue.</a> European Journal of Biochemistry. 199 (3), 647-652 (1991).</li><li>Fan, J. -. Q., Lee, Y. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Detailed+studies+on+substrate+structure+requirements+of+glycoamidases+A+and+F.">Detailed studies on substrate structure requirements of glycoamidases A and F.</a> Journal of Biological Chemistry. 272 (43), 27058-27064 (1997).</li><li>Altmann, F., Paschinger, K., Dalik, T., Vorauer, K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterisation+of+peptide-N4-(N-acetyl-&#946;-glucosaminyl)asparagine+amidase+A+and+its+N-glycans.">Characterisation of peptide-N4-(N-acetyl-&#946;-glucosaminyl)asparagine amidase A and its N-glycans.</a> European Journal of Biochemistry. 252 (1), 118-123 (1998).</li><li>Altmann, F., Schweiszer, S., Weber, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Kinetic+comparison+of+peptide:+N-glycosidases+F+and+A+reveals+several+differences+in+substrate+specificity.">Kinetic comparison of peptide: N-glycosidases F and A reveals several differences in substrate specificity.</a> Glycoconjugate Journal. 12 (1), 84-93 (1995).</li><li>Wang, T., Voglmeir, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=PNGases+as+valuable+tools+in+glycoprotein+analysis.">PNGases as valuable tools in glycoprotein analysis.</a> Protein and Peptide Letters. 21 (10), 976-985 (2014).</li><li>Wang, T., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Discovery+and+characterization+of+a+novel+extremely+acidic+bacterial+N-glycanase+with+combined+advantages+of+PNGase+F+and+A.">Discovery and characterization of a novel extremely acidic bacterial N-glycanase with combined advantages of PNGase F and A.</a> Bioscience Reports. 34 (6), 00149 (2014).</li><li>Du, Y. M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+sample+preparation+methodology+for+plant+N-.glycan+analysis+using+acid-stable+PNGase+H+.">Rapid sample preparation methodology for plant N-.glycan analysis using acid-stable PNGase H+.</a> Journal of Agricultural and Food Chemistry. 63 (48), 10550-10555 (2015).</li><li>Wang, T., Hu, X. -. C., Cai, Z. -. P., Voglmeir, J., Liu, L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Qualitative+and+Quantitative+Analysis+of+Carbohydrate+Modification+on+Glycoproteins+from+seeds+of+Ginkgo+biloba.">Qualitative and Quantitative Analysis of Carbohydrate Modification on Glycoproteins from seeds of Ginkgo biloba.</a> Journal of Agricultural and Food Chemistry. 65 (35), 7669-7679 (2017).</li><li>Ceroni, A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=GlycoWorkbench:+a+tool+for+the+computer-assisted+annotation+of+mass+spectra+of+glycans.">GlycoWorkbench: a tool for the computer-assisted annotation of mass spectra of glycans.</a> Journal of Proteome Research. 7 (4), 1650-1659 (2008).</li></ol>

The protocol we have presented here allows the comparison of the N-glycan profiles of various cultivars of radish. A significant advantage of this method compared to existing protocols is that no buffer changes between the enzymatic release of N-glycans and the derivatization reaction with 2-AB are required. The most critical step of this procedure is the purification of N-glycans using the SPE column, as failure to remove salts or other impurities in the reaction mixture may negatively impact ...

N-glycans in plants have drawn increased attention in recent years, as previous research has highlighted N-glycans as a potential source of immunological cross-reactions that may provoke allergic responses1,2. It has been demonstrated previously that N-glycans on plant glycoproteins can affect catalytic activity3,4, thermostability and folding5,6 or subcellular localization and secretion7. In order to correlate the glycan structures with their respective functions, N-glycans must first be released from glycoproteins either chemically or enzymatically. The classical chemical method for releasing both N- and O-glycans is &#946;-elimination, in which alkaline sample treatment is accompanied by reduction with borohydride to yield an alditol8. However, this procedure precludes labeling with a fluorophore and causes significant decay of monosaccharide units from the reducing end of the glycan structure. Chemical deglycosylation based on ammonium hydroxide/carbonate treatment is also a commonly used alternative method9. Neither of these chemical release methods degrades intact proteins, which allows mass spectrometric analysis of unlabeled glycan pools without the interference of peptide fragments in the same mass range. However, one drawback of these methods is the increased degradation rate of &#945;1,3-fucosylated N-glycans, a common carbohydrate structure found in plants10. Alternatively, enzymatic release methods using peptide:N-glycanases (PNGases, EC 3.5.1.52) are also widely applied. Recombinant PNGase F (from Flavobacterium meningosepticum) is the most common choice and permits the release of all types of N-glycans, except the structures bearing a core &#945;1,3 fucose11,12. Therefore, PNGase A (isolated from almond seeds) is usually used for the analysis of plant N-glycans13. However, this enzyme deglycosylates only proteolytically-derived glycopeptides, and is unable to deglycosylate native glycoproteins14. Hence, a multi-step sample workup is required before further analysis, which causes extensive loss of glycans, especially those of low abundance15.The overall goal of the method is to present an optimized workflow for N-glycan release and fluorescence labeling in a simple and robust manner. The underlying rationale is that PNGase H+, which was recently discovered in Terriglobus roseus and can be recombinantly expressed in E. coli, can hydrolyze N-glycans directly from the protein scaffold in acidic conditions16. A key advantage of using PNGase H+ over alternative methods is that fluorescent labeling reactions can be performed in the same reaction tube without changing the reaction buffers17,18. The simple preparation conditions and high recovery of low-abundance oligosaccharides make this method a valuable tool in the analysis of N-glycans. This protocol is suitable for the analysis of N-glycans from various plant species.

<table border="0" cellpadding="0" cellspacing="0" style="width:880px;" width="880">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:347px;">Chemicals:</td>
			<td style="width:123px;"></td>
			<td style="width:207px;"></td>
			<td style="width:204px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trichloroacetic acid&#160;</td>
			<td>SCR, Shanghai</td>
			<td>80132618</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetic acid glacial</td>
			<td>Huada, Guangzhou</td>
			<td>64-19-7</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Acetonitrile</td>
			<td>General-reagent</td>
			<td>G80988C</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Trifluoroacetic acid</td>
			<td>Energy chemical</td>
			<td>W810031</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2-aminobenzamide</td>
			<td>Heowns, Tianjin</td>
			<td>A41900</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Sodium cyanoborohydride</td>
			<td>J&#38;K Scientific Ltd</td>
			<td>314162</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Dimethyl sulfoxide</td>
			<td>Huada, Guangzhou</td>
			<td>67-68-5</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">2AB-labeled dextran ladder, 200 pmol</td>
			<td>Agilent Technologies</td>
			<td>AT-5190-6998</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">6-Aza-2-thiothymine&#160;</td>
			<td>Sigma</td>
			<td>275514</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tools/Materials:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Kitchen blender</td>
			<td>Bear, Guangzhou</td>
			<td>LLJ-A10T1</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Centrifuge</td>
			<td>Techcomp</td>
			<td>CT15RT</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Centrifugal Evaporator</td>
			<td>Hualida, Taicang</td>
			<td>LNG-T120</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">SPE column</td>
			<td>Supelco</td>
			<td>Supelclean ENVI Carb SPE column</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MALDI-TOF mass spectrometer</td>
			<td>Bruker</td>
			<td>Autoflex</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HPLC Analysis:</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">High-recovery HPLC vial</td>
			<td>Agilent Technologies</td>
			<td>&#160;# 5188-2788</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HPLC System</td>
			<td>Shimadzu</td>
			<td>Nexera</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Fluorescence Detector for HPLC</td>
			<td>Shimadzu</td>
			<td>RF-20Axs&#160;</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Column oven</td>
			<td>Hengxin</td>
			<td>CO-2000</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">HPLC Column</td>
			<td>Waters</td>
			<td>Acquity UPLC BEH glycan column</td>
			<td>2.1 &#215; 150 mm, 1.7 &#956;m particle size</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">LCMS-grade Water</td>
			<td>Merck Millipore</td>
			<td>#WX00011</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">LCMS-grade Acetonitrile</td>
			<td>Merck Millipore</td>
			<td># 100029</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">Formic acid</td>
			<td>Aladdin</td>
			<td>F112034</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ammonia solution</td>
			<td>Aladdin</td>
			<td>A112080</td>
			<td></td>
		</tr>
	</tbody>
</table>

analysis of n-glycans from raphanus sativus cultivars using pngase h+

In recent years, the carbohydrate moieties of plants have received considerable attention, as they are a potential source of cross-reactive, allergy-provoking immune responses. In addition, carbohydrate structures also play a critical role in plant metabolism. Here, we present a simple and rapid method for preparing and analyzing N-glycans from different cultivars of radish (Raphanus sativus) using an N-glycanase specific for the release of plant-derived carbohydrate structures. To achieve this, crude trichloroacetic acid precipitates of radish homogenates were treated with PNGase H+, and labeled using 2-aminobenzamide as a fluorescent tag. The labeled N-glycan samples were subsequently analyzed by ultra performance liquid chromatography (UPLC) separation and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry for a detailed structural evaluation and to quantify relative abundancies of the radish-derived N-glycan structures. This protocol can also be used for the analysis of N-glycans from various other plant species, and may be useful for further investigation of the function and effects of N-glycans on human health.

Figure 1 shows a schematic overview of the described protocol, including the isolation of (glyco-)proteins from radish, the preparation of N-glycans, the UPLC analysis, and the MALDI-TOF-MS analysis of these components. Figure 2 shows representative UPLC chromatograms of derivatized N-glycans of the analyzed radish cultivars. Figure 3 shows the obtained results of the 2AB-derivatize...

Watch this Scientific Journal Video about Analysis of N-glycans from Raphanus sativus Cultivars Using PNGase H+ at JoVE.com

Analysis of N-glycans from Raphanus sativus Cultivars Using PNGase H+

We describe a simple and rapid method for the preparation and analysis of N-glycans from different cultivars of radish (Raphanus sativus).

Analysis of N-glycans from Raphanus sativus Cultivars Using PNGase H+

In recent years, the carbohydrate moieties of plants have received considerable attention, as they are a potential source of cross-reactive, ...

This method can help answer questions in the field of plant Glycoscience such as how N-glycans of plant origin can be quickly analyzed and characterized. The main advantage of this technique is that it doesn't require any buffer changes between the enzymatic release of N-glycans and the derivatization reactions with 2-aminobenzamide. Demonstrating the procedure will be miss Ya-Min Du, and miss Shen-Li Zhen;two graduate students from the Glycomics and Glycan Bioengineering Research Center at Nanjing Agricultural University.To begin isolating the proteins, use a kitchen blender to homogenize approximately 100 grams of fresh radish for ten minutes. Transfer the resulting slurry to a 50 milliliter centrifuge tube. Centrifuge at 14, 000 times gravity at four degrees Celsius for 20 minutes to remove the insoluble material.After this, carefully transfer the supernatant into a new 50 milliliter centrifuge tube. Add an equal amount of a two molar trichloroacetic acid solution. Centrifuge at 14, 000 times gravity at four degrees Celsius for 30 minutes.And then remove the supernatant. Wash the pellet with 20 milliliters of deionized water. Centrifuge again at 14, 000 times gravity at four degrees Celsius for five minutes to remove soluble oligosaccharides and polysaccharides.Then, re-suspend the pellet with one milliliter of deionized water. And transfer the protein solution to a fresh 1.5 milliliter centrifuge tube. To begin preparing the N-glycans, mix 50 microliters of the protein solution with 0.2 milliunits of recombinant PNGase H+in ten millimolar acetic acid.Incubate at 37 degrees Celsius for 12 hours. Then, centrifuge at 14, 000 times gravity for five minutes to remove the extra protein and enzyme. Transfer the supernatant to a fresh 1.5 milliliter centrifuge tube until ready to purify the N-glycans.To begin purifying the N-glycans, wash the solid phase extraction column with three milliliters of water. Activate the column by adding three milliliters of an 80 percent acetonitrile solution containing trifluoroacetic acid. Then, three milliliters of deionized water to equilibrate the column.Transfer the sample onto the column and discard the flow through. Wash the column by adding 1.5 milliliters of deionized water. Failure to remove salts and add impurities in the sample mixture may negatively impact the efficiency of the fluorescence derivatization reaction.Then, add 1.5 milliliters of an aqueous 20 percent acetonitrile solution containing 0.1 percent TFA to elute the N-glycans into a two milliliter tube. Remove the solvent by centrifugal evaporation at room temperature, evaporating until the sample is completely dry. To begin fluorescence derivatization of N-glycans, prepare the 2-AB solution as outlined in the text protocol.Add five microliters of the prepared 2-AB solution to the dried samples. Vortex until each sample is completely dissolved. Incubate at 65 degrees Celsius for two hours.After this, let the sample cool to room temperature for five minutes. Add five microliters of deionized water and 40 microliters of acetonitrile to the cooled sample. Centrifuge at 14, 000 times gravity for three minutes.Then, transfer 48 microliters of the resulting supernatant into a 300 microliter high recovery HPLC vial. Store at negative 20 degrees Celsius for up to one month or when ready to use. To begin profiling, use a standard UPLC system equipped with a hydrophobic interaction liquid chromatography UPLC glycan column and connect it to an online fluorescence detector to analyze the samples.Set the excitation and emission wavelengths to 330 nanometers and 420 nanometers, respectively. Set the column temperature to 60 degrees Celsius. Next, inject 45 microliters of the sample into the UPLC system.Elute the N-glycans by UPLC using retention times of 15 and 40 minutes. Using two milliliters centrifuge tubes collect each observed UPLC peak fraction. Then, use centrifugal evaporation to dry the samples.Inject approximately one picomole of 2-AB labeled dextran standard to calibrate the plant N-glycan profiling and to assign their elution times and to standardize glucose units. To begin MALDI-TOF analysis, dissolve the dried samples in five microliters of LCMS grade water. Mix one microliter of this solution with one microliter of ATT solution.Then, pipet this mixture into the MALDI-TOF sample carrier. After this, use a MALDI-TOF mass spectrometer instrument to analyze the samples as outlined in the text protocol. In this study, N-glycan profiles are compared from various cultivars of radish.Representative UPLC chromatograms show that derivatized N-glycans and the relative abundance of each and different radishes can be successfully analyzed through this process. After this, MALDI-TOF mass spectroscopy is used to analyze the structures of 2-AB derivatized N-glycans. Though this method can provide insight into the composition of N-glycans from different Raphanus sativus cultivars, it can also be applied to other systems such as peanuts or other allergenic plant species.Once mastered, samples can be prepared within two working days which already includes the UPLC analysis procedure. When attempting this procedure, it's important to remember that the solid phase extraction columns used to isolate the N-glycans are sufficiently moistened with distilled water prior to sample application. Otherwise, binding of N-glycans to the column may be hampered.For additional verification of N-glycan structures, other methods can also be used. This could be, for example, sequential exoglycosidase treatments or MALDI-TOF MS N based fragmentation techniques. After its development, this technique paved the way for researchers in the field of Glycoscience to explore the composition and function of plant N-glycans more rapidly.After watching this video, you should have a good understanding of how to prepare N-glycans from different cultivars of radish using PNGase H+and how to analyze the 2-aminobenzamide derivatives by UPLC. Don't forget that working with trifluoroacetic acid and trichloroacetic acid can be extremely hazardous and precautions such as wearing protective gear should always be taken while performing this procedure.

analysis-of-n-glycans-from-raphanus-sativus-cultivars-using-pngase-h

Research

JoVE Journal

Biochemistry

High-resolution Single Particle Analysis from Electron Cryo-microscopy Images Using SPHIRE

SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single particle electron cryo-microscopy (cryo-EM) data. The protocol presented here describes in detail how to obtain a near-atomic resolution structure starting from cryo-EM micrograph movies by guiding users through all steps of the single particle structure determination pipeline. These steps are controlled from the new SPHIRE graphical user interface and require minimum user intervention. Using this protocol, a 3.5 &#197; structure of TcdA1, a Tc toxin complex from Photorhabdus luminescens, was derived from only 9500 single particles. This streamlined approach will help novice users without extensive processing experience and a priori structural information, to obtain noise-free and unbiased atomic models of their purified macromolecular complexes in their native state.

This paper presents a protocol for processing cryo-EM images using the software suite SPHIRE. The present protocol can be applied for nearly all single particle EM projects that target near-atomic resolution.

SPHIRE (SPARX for High-Resolution Electron Microscopy) is a novel open-source, user-friendly software suite for the semi-automated processing of single ...

Isolation of Intermediate Filament Proteins from Multiple Mouse Tissues to Study Aging-associated Post-translational Modifications

Intermediate filaments (IFs), together with actin filaments and microtubules, form the cytoskeleton &#8211; a critical structural element of every cell. Normal functioning IFs provide cells with mechanical and stress resilience, while a dysfunctional IF cytoskeleton compromises cellular health and has been associated with many human diseases. Post-translational modifications (PTMs) critically regulate IF dynamics in response to physiological changes and under stress conditions. Therefore, the ability to monitor changes in the PTM signature of IFs can contribute to a better functional understanding, and ultimately conditioning, of the IF system as a stress responder during cellular injury. However, the large number of IF proteins, which are encoded by over 70 individual genes and expressed in a tissue-dependent manner, is a major challenge in sorting out the relative importance of different PTMs. To that end, methods that enable monitoring of PTMs on IF proteins on an organism-wide level, rather than for isolated members of the family, can accelerate research progress in this area. Here, we present biochemical methods for the isolation of the total, detergent-soluble, and detergent-resistant fraction of IF proteins from 9 different mouse tissues (brain, heart, lung, liver, small intestine, large intestine, pancreas, kidney, and spleen). We further demonstrate an optimized protocol for rapid isolation of IF proteins by using lysing matrix and automated homogenization of different mouse tissues. The automated protocol is useful for profiling IFs in experiments with high sample volume (such as in disease models involving multiple animals and experimental groups). The resulting samples can be utilized for various downstream analyses, including mass spectrometry-based PTM profiling. Utilizing these methods, we provide new data to show that IF proteins in different mouse tissues (brain and liver) undergo parallel changes with respect to their expression levels and PTMs during aging.

In this method, we present biochemical procedures for rapid and efficient isolation of intermediate filament (IF) proteins from multiple mouse tissues. Isolated IFs can be used to study changes in post-translational modifications by mass spectrometry and other biochemical assays.

Intermediate filaments (IFs), together with actin filaments and microtubules, form the cytoskeleton &#8211; a critical structural element of every cell. ...

Measurement of Mitochondrial Oxygen Consumption in Permeabilized Fibers of Drosophila Using Minimal Amounts of Tissue

The fruit fly, Drosophila melanogaster, represents an emerging model for the study of metabolism. Indeed, drosophila have structures homologous to human organs, possess highly conserved metabolic pathways and have a relatively short lifespan that allows the study of different fundamental mechanisms in a short period of time. It is, however, surprising that one of the mechanisms essential for cellular metabolism, the mitochondrial respiration, has not been thoroughly investigated in this model. It is likely because the measure of the mitochondrial respiration in Drosophila usually requires a very large number of individuals and the results obtained are not highly reproducible. Here, a method allowing the precise measurement of mitochondrial oxygen consumption using minimal amounts of tissue from Drosophila is described. In this method, the thoraxes are dissected and permeabilized both mechanically with sharp forceps and chemically with saponin, allowing different compounds to cross the cell membrane and modulate the mitochondrial respiration. After permeabilization, a protocol is performed to evaluate the capacity of the different complexes of the electron transport system (ETS) to oxidize different substrates, as well as their response to an uncoupler and to several inhibitors. This method presents many advantages compared to methods using mitochondrial isolations, as it is more physiologically relevant because the mitochondria are still interacting with the other cellular components and the mitochondrial morphology is conserved. Moreover, sample preparations are faster, and the results obtained are highly reproducible. By combining the advantages of Drosophila as a model for the study of metabolism with the evaluation of mitochondrial respiration, important new insights can be unveiled, especially when the flies are experiencing different environmental or pathophysiological conditions.

In this paper, a method to measure oxygen consumption using high-resolution respirometry in permeabilized thoraxes of Drosophila is described. This technique requires a minimal amount of tissue compared to the classic mitochondrial isolation technique and the results obtained are more physiologically relevant.

The fruit fly, Drosophila melanogaster, represents an emerging model for the study of metabolism. Indeed, drosophila have structures homologous to human ...

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea (Camellia Sinensis L.) Leaves

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form loose callus on Murashige and Skoog (MS) basal media with the plant hormones 2,4-dichlorophenoxyacetic acid (2,4-D, 1.0 mg L-1) and kinetin (KT, 0.1 mg L-1). Callus formed after 3 or 4 rounds of subculturing, each lasting 28 days. Loose callus (about 3 g) was then inoculated into B5 liquid media containing the same plant hormones and was cultured in a shaking incubator (120 rpm) in the dark at 25 &#177; 1 &#176;C. After 3&#8722;4 subcultures, a cell suspension derived from tea leaf was established at a subculture ratio ranging between 1:1 and 1:2 (suspension mother liquid: fresh medium). Using this platform, six insecticides (5 &#181;g mL-1 each thiamethoxam, imidacloprid, acetamiprid, imidaclothiz, dimethoate, and omethoate) were added into the tea leaf-derived cell suspension culture. The metabolism of the insecticides was tracked using liquid chromatography and gas chromatography. To validate the usefulness of the tea cell suspension culture, the metabolites of thiamethoxan and dimethoate present in treated cell cultures and intact plants were compared using mass spectrometry. In treated tea cell cultures, seven metabolites of thiamethoxan and two metabolites of dimethoate were found, while in treated intact plants, only two metabolites of thiamethoxam and one of dimethoate were found. The use of a cell suspension simplified the metabolic analysis compared to the use of intact tea plants, especially for a difficult matrix such as tea.

Study on the Metabolism of Six Systemic Insecticides in a Newly Established Cell Suspension Culture Derived from Tea Camellia Sinensis L. Leaves

This work presents a protocol for establishing a cell suspension culture derived from tea (Camellia sinensis L.) leaves that can be used to study the metabolism of external compounds that can be taken up by the whole plant, such as insecticides.

A platform for studying insecticide metabolism using in vitro tissues of tea plant was developed. Leaves from sterile tea plantlets were induced to form ...

Isolation of Histone from Sorghum Leaf Tissue for Top Down Mass Spectrometry Profiling of Potential Epigenetic Markers

Histones belong to a family of highly conserved proteins in eukaryotes. They pack DNA into nucleosomes as functional units of chromatin. Post-translational modifications (PTMs) of histones, which are highly dynamic and can be added or removed by enzymes, play critical roles in regulating gene expression. In plants, epigenetic factors, including histone PTMs, are related to their adaptive responses to the environment. Understanding the molecular mechanisms of epigenetic control can bring unprecedented opportunities for innovative bioengineering solutions. Herein, we describe a protocol to isolate the nuclei and purify histones from sorghum leaf tissue. The extracted histones can be analyzed in their intact forms by top-down mass spectrometry (MS) coupled with online reversed-phase (RP) liquid chromatography (LC). Combinations and stoichiometry of multiple PTMs on the same histone proteoform can be readily identified. In addition, histone tail clipping can be detected using the top-down LC-MS workflow, thus, yielding the global PTM profile of core histones (H4, H2A, H2B, H3). We have applied this protocol previously to profile histone PTMs from sorghum leaf tissue collected from a large-scale field study, aimed at identifying epigenetic markers of drought resistance. The protocol could potentially be adapted and optimized for chromatin immunoprecipitation-sequencing (ChIP-seq), or for studying histone PTMs in similar plants.

The protocol has been developed to effectively extract intact histones from sorghum leaf materials for profiling of histone post-translational modifications that can serve as potential epigenetic markers to aid engineering drought resistant crops.

Histones belong to a family of highly conserved proteins in eukaryotes. They pack DNA into nucleosomes as functional units of chromatin. ...

Preparation of Sample Support Films in Transmission Electron Microscopy using a Support Floatation Block

Structure determination by cryo-electron microscopy (cryo-EM) has rapidly grown in the last decade; however, sample preparation remains a significant bottleneck. Macromolecular samples are ideally imaged directly from random orientations in a thin layer of vitreous ice. However, many samples are refractory to this, and protein denaturation at the air-water interface is a common problem. To overcome such issues, support films-including amorphous carbon, graphene, and graphene oxide-can be applied to the grid to provide a surface which samples can populate, reducing the probability of particles experiencing the deleterious effects of the air-water interface. The application of these delicate supports to grids, however, requires careful handling to prevent breakage, airborne contamination, or extensive washing and cleaning steps. A recent report describes the development of an easy-to-use floatation block that facilitates wetted transfer of support films directly to the sample. Use of the block minimizes the number of manual handling steps required, preserving the physical integrity of the support film, and the time over which hydrophobic contamination can accrue, ensuring that a thin film of ice can still be generated. This paper provides step-by-step protocols for the preparation of carbon, graphene, and graphene oxide supports for EM studies.

Sample preparation for cryo-electron microscopy (cryo-EM) is a significant bottleneck in the structure determination workflow of this method. Here, we provide detailed methods for using an easy-to-use, three-dimensionally printed block for the preparation of support films to stabilize samples for transmission EM studies.

Structure determination by cryo-electron microscopy (cryo-EM) has rapidly grown in the last decade; however, sample preparation remains a significant ...

Extraction and Purification of FAHD1 Protein from Swine Kidney and Mouse Liver

Fumarylacetoacetate hydrolase domain-containing protein 1 (FAHD1) is the first identified member of the FAH superfamily in eukaryotes, acting as oxaloacetate decarboxylase in mitochondria. This article presents a series of methods for the extraction and purification of FAHD1 from swine kidney and mouse liver. Covered methods are ionic exchange chromatography with fast protein liquid chromatography (FPLC), preparative and analytical gel filtration with FPLC, and proteomic approaches. After total protein extraction, ammonium sulfate precipitation and ionic exchange chromatography were explored, and FAHD1 was extracted via a sequential strategy using ionic exchange and size-exclusion chromatography. This representative approach may be adapted to other proteins of interest (expressed at significant levels) and modified for other tissues. Purified protein from tissue may support the development of high-quality antibodies, and/or potent and specific pharmacological inhibitors.

This protocol describes how to extract fumarylacetoacetate hydrolase domain-containing protein 1 (FAHD1) from swine kidney and mouse liver. The listed methods may be adapted to other proteins of interest and modified for other tissues.

Fumarylacetoacetate hydrolase domain-containing protein 1 (FAHD1) is the first identified member of the FAH superfamily in eukaryotes, acting as ...

Routine Collection of High-Resolution cryo-EM Datasets Using 200 KV Transmission Electron Microscope

Cryo-electron microscopy (cryo-EM) has been established as a routine method for protein structure determination during the past decade, taking an ever-increasing share of published structural data. Recent advances in TEM technology and automation have boosted both the speed of data collection and quality of acquired images while simultaneously decreasing the required level of expertise for obtaining cryo-EM maps at sub-3 &#197; resolutions. While most of such high-resolution structures have been obtained using state-of-the-art 300 kV cryo-TEM systems, high-resolution structures can be also obtained with 200 kV cryo-TEM systems, especially when equipped with an energy filter. Additionally, automation of microscope alignments and data collection with real-time image quality assessment reduces system complexity and assures optimal microscope settings, resulting in increased yield of high-quality images and overall throughput of data collection. This protocol demonstrates the implementation of recent technological advances and automation features on a 200 kV cryo-transmission electron microscope and shows how to collect data for the reconstruction of 3D maps that are sufficient for de novo atomic model building. We focus on best practices, critical variables, and common issues that must be considered to enable the routine collection of such high-resolution cryo-EM datasets. Particularly the following essential topics are reviewed in detail: i) automation of microscope alignments, ii) selection of suitable areas for data acquisition, iii) optimal optical parameters for high-quality, high-throughput data collection, iv) energy filter tuning for zero-loss imaging, and v) data management and quality assessment. Application of the best practices and improvement of achievable resolution using an energy filter will be demonstrated on the example of apo-ferritin that was reconstructed to 1.6 &#197;, and Thermoplasma acidophilum 20S proteasome reconstructed to 2.1-&#197; resolution using a 200 kV TEM equipped with an energy filter and a direct electron detector.

High-resolution cryo-EM maps of macromolecules can be also achieved by using 200 kV TEM microscopes. This protocol shows the best practices for setting accurate optics alignments, data acquisition schemes, and selection of imaging areas that are all essential for the successful collection of high-resolution datasets using a 200 kV TEM.

Cryo-electron microscopy (cryo-EM) has been established as a routine method for protein structure determination during the past decade, taking an ...

MyDoBID- A Novel Technique for Identifying Muscle Fiber Types Efficiently

Fiber Type Identification of Human Skeletal Muscle

The technique described here can be used to identify specific myosin heavy chain (MHC) isoforms in segments of individual muscle fibers using dot blotting, hereafter referred to as Myosin heavy chain detection by Dot Blotting for IDentification of muscle fiber type (MyDoBID). This protocol describes the process of freeze-drying human skeletal muscle and isolating segments of single muscle fibers. Using MyDoBID, type I and II fibers are classified with MHCI- and IIa-specific antibodies, respectively. Classified fibers are then combined into fiber type-specific samples for each biopsy.
The total protein in each sample is determined by Sodium Dodecyl-Sulfate Polyacrylamide Gel Electrophoresis (SDS-PAGE) and UV-activated gel technology. The fiber type of samples is validated using western blotting. The importance of performing protein loading normalization to enhance target protein detection across multiple western blots is also described. The benefits of consolidating classified fibers into fiber type-specific samples compared to single-fiber western blots, include sample versatility, increased sample throughput, shorter time investment, and cost-saving measures, all while retaining valuable fiber type-specific information that is frequently overlooked using homogenized muscle samples. The purpose of the protocol is to achieve accurate and efficient identification of type I and type II fibers isolated from freeze-dried human skeletal muscle samples.
These individual fibers are subsequently combined to create type I and type II fiber type-specific samples. Furthermore, the protocol is extended to include the identification of type IIx fibers, using Actin as a marker for fibers that were negative for MHCI and MHCIIa, which are confirmed as IIx fibers by western blotting. Each fiber type-specific sample is then used to quantify the expression of various target proteins using western blotting techniques.

Author Spotlight: Deciphering the Mysteries of Skeletal Muscle Fiber Types Using the MyDoBID Technique 

This protocol demonstrates single-fiber isolation from freeze-dried human skeletal muscle and fiber-type classification according to Myosin heavy chain (MHC) isoform using the dot blotting technique. Identified MHC I and II fiber samples can then be further analyzed for fiber type-specific differences in protein expression using western blotting.

The technique described here can be used to identify specific myosin heavy chain (MHC) isoforms in segments of individual muscle fibers using dot ...

Accelerating Macromolecular Structural Analysis Using Smart Leginon Autoscreen

Cryo-Electron Microscopy Screening Automation Across Multiple Grids Using Smart Leginon

Advancements in cryo-electron microscopy (cryoEM) techniques over the past decade have allowed structural biologists to routinely resolve macromolecular protein complexes to near-atomic resolution. The general workflow of the entire cryoEM pipeline involves iterating between sample preparation, cryoEM grid preparation, and sample/grid screening before moving on to high-resolution data collection. Iterating between sample/grid preparation and screening is typically a major bottleneck for researchers, as every iterative experiment must optimize for sample concentration, buffer conditions, grid material, grid hole size, ice thickness, and protein particle behavior in the ice, amongst other variables. Furthermore, once these variables are satisfactorily determined, grids prepared under identical conditions vary widely in whether they are ready for data collection, so additional screening sessions prior to selecting optimal grids for high-resolution data collection are recommended. This sample/grid preparation and screening process often consumes several dozen grids and days of operator time at the microscope. Furthermore, the screening process is limited to operator/microscope availability and microscope accessibility. Here, we demonstrate how to use Leginon and Smart Leginon Autoscreen to automate the majority of cryoEM grid screening. Autoscreen combines machine learning, computer vision algorithms, and microscope-handling algorithms to remove the need for constant manual operator input. Autoscreen can autonomously load and image grids with multi-scale imaging using an automated specimen-exchange cassette system, resulting in unattended grid screening for an entire cassette. As a result, operator time for screening 12 grids may be reduced to ~10 min with Autoscreen compared to ~6 h using previous methods which are hampered by their inability to account for high variability between grids. This protocol first introduces basic Leginon setup and functionality, then demonstrates Autoscreen functionality step-by-step from the creation of a template session to the end of a 12-grid automated screening session.

Author Spotlight: Enhancing Cryo-Electron Microscopy by Automated Data Collection and Analysis Techniques

Cryo-electron microscopy (cryoEM) multi-grid screening is often a tedious process that demands hours of attention. This protocol shows how to set up a standard Leginon collection and Smart Leginon Autoscreen to automate this process. This protocol can be applied to the majority of cryoEM holey foil grids.

Advancements in cryo-electron microscopy (cryoEM) techniques over the past decade have allowed structural biologists to routinely resolve macromolecular ...