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

Bu eser kısmen Çin doğal Bilim Vakfı tarafından desteklenmiştir (sayı 31471703, A0201300537 ve 31671854 J.V. ve ll, G.Y. için sayı 31470435 izni grant) ve 100 yabancı yeteneklerini planı (grant sayıya JSB2014012 J.V.).

1. örnek koleksiyonu
<ol><li>Taze turp (Raphanus sativus L.) farklı çeşitlerin satın.</li>
</ol>2. izolasyon-in Protein turp
<ol><li>10 dakika için bir mutfak blender ile yaklaşık 100 gr taze turp lunaparkçı.</li>
	<li>Bulamaç 50 mL santrifüj tüpü ve santrifüj 14.000 × g çözünmez malzeme çıkarmak 20 dk için 4 ° C'de de aktarın.</li>
	<li>Süpernatant dikkatli bir şekilde yeni bir 50 mL santrifüj tüpüne aktarmak ve 2 M trichloroacetic asit (TCA) ç?...

<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>

Biz burada sundu iletişim kuralı turp çeşitli çeşitlerin N- glycan profilleri karşılaştırmasını sağlar. Varolan iletişim kurallarına göre bu yöntemin önemli bir avantaj enzimatik serbest bırakmak-in N- glukanlardir ve derivatization reaksiyon 2-AB ile arasında hiçbir arabellek değişiklikleri gerekli olmasıdır. N- glukanlardir tuzları kaldırmak için başarısızlık olarak SPE sütun kullanarak arıtma, bu yordamın en önemli adımdır veya diğer kirleri tepki karı...

Önceki araştırma N- glukanlardir Alerjik yanıt-e doğru1,2 kışkırtmak immünolojik cross-reactions potansiyel bir kaynak vurgulanmış bulunmaktadır olarak N- glukanlardir tesislerinde son yıllarda artış dikkat çekiyor . N- glukanlardir bitki glikoproteinlerin üzerinde katalitik aktivitesi3,4, thermostability ve katlama5,6 veya hücre altı yerelleştirme etkileyebilir daha önce kanıtlanmıştır ve salgı7. Glycan yapıları ile ilgili işlevleri ilişkilendirmek için N- glukanlardir ilk glikoproteinlerin kimyasal veya enzimatik yayımlanması gerekir. N- ve O- glukanlardir serbest bırakmak için klasik kimyasal yöntem β-hangi azaltma borhidrür alditol8verim ile alkali örnek tedavi eşlik eder, ortadan kaldırılmasıdır. Ancak, bu yordamı bir fluorophore ile etiketleme önler ve yaşam üniteleri önemli çürüme azaltmak glycan yapısı sonundan itibaren neden olur. Kimyasal deglycosylation amonyum hidroksit/karbonat tedavisine göre de yaygın olarak kullanılan alternatif yöntem9' dur. Bu kimyasal ürün yöntemlerin hiçbiri sağlam proteinler, aynı kitle aralıkta etiketlenmemiş glycan havuzları peptid parçaları müdahale olmadan kitle spektrometrik analizi sağlayan alçaltır. Ancak, bu yöntemlerin bir dezavantajı artan bozulma α1, 3-fucosylated N- glukanlardir, ortak bir karbonhidrat yapısı bitkiler10içinde bulunan oranıdır. Alternatif olarak, peptid kullanarak tekrar tekrar enzimatik yayın yöntemleri:N- glycanases (PNGases, EC 3.5.1.52) de yaygın olarak uygulanan. Rekombinant PNGase F (dan Flavobacterium meningosepticum) en yaygın seçim ve N- glukanlardir, taşıyan bir çekirdek α1, 3 fucose11,12yapıları dışında her türlü yayın izin verir. Bu nedenle, PNGase A (badem tohumları izole) genellikle bitki N- glukanlardir13analizi için kullanılır. Ancak, bu enzim deglycosylates sadece proteolytically elde edilen glycopeptides ve deglycosylate yerel glikoproteinlerin14' e değiştiremiyor. Bu nedenle, bir çok adımlı örnek testleri daha geniş glukanlardir, özellikle de düşük bereket15kaybolmasına neden analiz daha önce gereklidir. Yöntem genel amacı basit ve sağlam bir şekilde etiketleme floresans ve N- glycan sürümü için en iyi duruma getirilmiş bir iş akışı sunmaktır. PNGase H+, geçtiğimiz günlerde Terriglobus çevresi keşfedilmiştir ve recombinantly E. coliifade edilebilir, N- glukanlardir doğrudan doğruya--dan içinde asidik protein iskele hidrolize temel mantığı olduğunu koşullar16. PNGase H+ üzerinde alternatif yöntemler kullanarak bir anahtar tepki arabellekleri17,18değiştirmeden floresan etiketleme tepkiler aynı tepki tüp içinde gerçekleştirilebilir bir avantajdır. Bu yöntem basit hazırlık koşulları ve yüksek düşük-bereket oligosakkaritler kurtarılması N- glukanlardir analizinde değerli bir araç olun. Bu iletişim kuralı, N- glukanlardir çeşitli bitki türleri üzerinden analizi için uygundur.

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

Onlar cross-reactive, Anti kışkırtan bağışıklık yanıtı potansiyel bir kaynak olarak son yıllarda büyük ilgi, bitkilerin karbonhidrat moieties aldık. Buna ek olarak, karbonhidrat yapıları da bitki metabolizmasında kritik bir rol oynamaktadır. Burada, hazırlama ve N- glukanlardir turp (Raphanus sativus) farklı çeşitlerin üzerinden bir N- glycanase özel bitki kaynaklı karbonhidrat yapılarının sürülmesi kullanarak analiz için basit ve hızlı bir yöntem mevcut. Bunu başarmak için ham trichloroacetic asit precipitates turp homogenates, PNGase H+ile tedavi ve 2-aminobenzamide floresan etiket olarak kullanarak etiketli. Etiketli N- glycan örnekleri daha sonra ultra performans sıvı kromatografi (UPLC) ayırma ve matris yardımlı Lazer Desorpsiyon iyonlaşma-uçuş (MALDI-TOF) kütle spektrometresi detaylı yapısal için süreyi tarafından analiz edildi değerlendirme ve N- glycan turp elde edilen yapıların göreli abundancies ölçmek için. Bu protokolü de N- glukanlardir diğer çeşitli bitki tür analizi için kullanılabilir ve daha fazla araştırma yapılması işlev ve N- glukanlardir insan sağlığı üzerindeki etkileri için yararlı olabilir.

Şekil 1 açıklanan protokol yalıtım proteinlerin (glyco-) turp, N- glukanlardir hazırlanması, UPLC analiz ve bu bileşenleri MALDI-TOF-Bayan analizi de dahil olmak üzere, şematik bir dökümünü gösterir. Şekil 2 derivatized N- glukanlardir analiz turp çeşitlerin temsilcisi UPLC chromatograms gösterir. Şekil 3 2AB-derivatized N- glycan yapıları MALDI-TOF-K?...

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+

N- glukanlardir Raphanus sativus çeşitlerin kullanarak PNGase h+ Analizi

Biz hazırlık ve N- glukanlardir turp (Raphanus sativus) farklı çeşitlerin üzerinden analizi için basit ve hızlı bir yöntem açıklanmaktadır.

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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