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

この作品は自然科学基金、中国のによって部分的に支えられた (許可番号 31471703、A0201300537、J.V.、l. l.、31671854 許可番号 31470435 G.Y.)、および 100 外国の才能計画 (合弁会社許可番号 JSB2014012)。

1. サンプル コレクション
<ol><li>新鮮な大根 (ダイコン) の異なる品種を購入します。</li>
</ol>2. 大根から蛋白質の分離
<ol><li>10 分間キッチン ミキサーで新鮮な大根の約 100 g をホモジナイズしてください。</li>
	<li>50 mL の遠心管と不溶性物質を削除する 20 分のための 4 ° C で 14,000 × gで遠心スラリーに転送します。</li>
	<li>新しい 50 mL 遠心チューブに上清を慎?...

<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>

我々 はここで提示しているプロトコルは、さまざまな品種の大根のN型糖鎖のプロファイルの比較をできます。既存のプロトコルと比較してこの方法の重要な利点は、 N型糖鎖の酵素的リリースと 2 AB と誘導体化反応のバッファーの変更が必要ないことです。この手順の最も重要なステップは塩を削除する失敗として SPE カラムを用いたN型糖鎖の精製や反応混合物の他の不...

植物におけるN型糖鎖が注目増加近年、前の研究がアレルギー反応1,2 を引き起こすかもしれない免疫の内の潜在的なソースとしてN型糖鎖をハイライト表示します。.それが実証されて以前植物糖タンパク質糖鎖のNは、触媒活性3,4、耐熱性と折りたたみ式5,6または細胞レベル下のローカリゼーションに影響を与えることができ、分泌7。糖鎖構造をそれぞれの機能に関連付ける、するためにN型糖鎖最初から解放しなければならない糖タンパク質化学的にまたは酵素によって。N- とO型糖鎖を解放するための古典的な化学方法は β-除去するアルカリ サンプル治療と一緒に伴われる還元糖アルコール8を生成する水素化ホウ素。ただし、この手順は、蛍光でラベルを排除、糖鎖構造の還元末端から単糖類の単位の重要な崩壊を引き起こします。水酸化アンモニウム炭酸塩治療に基づいて化学糖鎖切り出しも一般的に使用される代替方法9です。これらの化学物質の放出方法のどちらも同じ質量範囲にラベルのない糖ペプチド フラグメントの干渉なしプールの質量分析が可能そのまま蛋白質が低下します。ただし、これらのメソッドの 1 つの欠点である α 1, 3 次N型糖鎖の増加の分解速度は共通の炭水化物構造植物10。また、ペプチドを用いた酵素リリース方法:N- 多糖分解酵素 (PNGases、EC 3.5.1.52) も広く適用されています。(病菌 meningosepticum) から組換え PNGase F は最も一般的な選択であり、コア α 1, 3 フコース11,12軸受構造を除いて、 N型糖鎖のすべてのタイプのリリースを許可します。したがって、PNGase A (アーモンドの種子から分離された) は通常植物13 N型糖鎖の解析に使用されます。しかし、この酵素 deglycosylates のみ生由来ペプチド、deglycosylate ネイティブ糖14ことができないと。したがって、詳細な分析、糖、低豊富15の特にそれらの広汎な損失を引き起こす前にマルチ ステップ サンプル精密検査が必要です。メソッドの全体的な目標は、 N型糖鎖のリリースとシンプルで堅牢な方法で蛍光のために最適化されたワークフローを提示することです。基になる根拠は PNGase H+ Terriglobus ニチニチソウの最近発見された recombinantly , 大腸菌で発現することができます、ことができる酸性のタンパク質足場から直接N型糖鎖を加水分解条件16。PNGase H+を使った代替方法の主な利点は、反応バッファー17,18を変更することがなく同じ反応管内の蛍光標識反応が可能です。簡単な調製条件と低豊富なオリゴ糖の高回収率このメソッドは、 N型糖鎖の解析で貴重なツールを作る。このプロトコルは、さまざまな植物の種からN型糖鎖の解析に適しています。

<table border="0" cellpadding="0" cellspacing="0" style="width:880px;" width="880">
	<colgroup>
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	<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+

近年、植物の糖鎖は、十字反応、アレルギー誘発の免疫応答の潜在的なソースは、かなりの注目を受けた。さらに、炭水化物の構造はまた植物の代謝に重要な役割を果たします。簡便・迅速な準備と、 N型糖鎖大根 (ダイコン) の品種から植物由来の炭水化物構造の解放のためのN- glycanase 固有を使用しての分析法を紹介します。これを達成するには、大根ホモジネートの原油のトリクロロ酸析出された PNGase H+で扱われ、蛍光タグとして 2 aminobenzamide を使用してラベル付け。ラベル付きのN型糖鎖のサンプルを超高性能液体クロマトグラフィー (高精度) 分離して飛行 (MALDI-TOF) 質量分析法による詳細な構造のマトリックス支援レーザー脱離イオン化時間行った後評価と大根由来のN型糖鎖構造の相対的な abundancies を定量化します。このプロトコルは、他の各種の植物の種からN型糖鎖の解析も使用でき、関数のさらなる調査と人間の健康に及ぼすN型糖鎖の役に立つかもしれません。

図 1は、(糖-) 大根、 N型糖鎖の調製、高精度分析とこれらのコンポーネントの MALDI TOF MS 分析からの蛋白質の分離を含む記述のプロトコルの概要を示しています。誘導体N型分析ダイコン品種の糖鎖の代表的な高精度のクロマト グラムを図 2に示します。図 3は、MALDI TOF 質量分析を用いた 2...

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+

ダイコン品種使用 PNGase H+からN型糖鎖の解析

簡便・迅速な準備と大根 (ダイコン) の品種間におけるN型糖鎖の解析法について述べる。

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.

Research

JoVE Journal

Biochemistry

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

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