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

이 작품은 중국의 자연 과학 재단에 의해 부분적으로 지원 (부여 번호 31471703, A0201300537 및 31671854 J.V. 하 L.L., 번호 31470435 G.Y.에 부여), 그리고 100 외국 재능 계획 (보조금 번호 JSB2014012 J.V.).

1. 샘플 컬렉션
<ol><li>신선한 무 (Raphanus sativus L.)의 다른 cultivars을 구입.</li>
</ol>2입니다. 무에서 단백질의 격리
<ol><li>10 분 동안 부엌 믹서와 신선한 무의 약 100 g을 균질.</li>
	<li>50 mL 원심 분리기 튜브 및 불용 성 물질을 제거 20 분 4 ° C에서 14000 × g 에서 원심 슬러리를 전송 합니다.</li>
	<li>새로운 50 mL 원심 분리기 튜브에는 상쾌한을 신중 하 게 전송 하 고 2m trichl...

<ol>
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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. -. 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우리가 여기 제시 하는 프로토콜 무의 다양 한 재배의 N-glycan 프로 파일의 비교를 허용 한다. 기존 프로토콜에 비해이 방법의 중요 한 장점은 Nglycans의 효소 출시와 2 AB와 derivatization 반응 사이의 버퍼 변경할 필요가 없습니다입니다. 이 절차의 가장 중요 한 단계는 N-glycans SPE 열을 사용 하 여 염 분을 제거 하는 실패의 정화 또는 반응 혼합물에 다른 불순물 형광 derivatization 효율?...

식물에서 N-glycans 그린 관심 증가 최근 몇 년 동안, 이전 연구로 알레르기 성 응답1,2 유도할 수 있는 면역 cross-reactions의 N-glycans을 강조 표시 . 그것은 입증 되었습니다 이전 촉매 활동3,4, 내열성 및 접는5,6 또는 subcellular 지 방화 N-glycans 공장 glycoproteins에 영향을 미칠 수 있는 고 분 비7. Glycan 구조 그들의 각각 기능 연관, 위하여 N-glycans 먼저 해제 되어야 합니다 glycoproteins에서 화학적 또는 효소. N-및 O-glycans 방출에 대 한 고전적인 화학 방법은 β-제거, 알칼리 샘플 치료 borohydride alditol8를 가진 감소에 의해 동반입니다. 그러나,이 절차는 fluorophore로 하는 걸로 하 고 glycan 구조의 줄이는 끝에서 단 당 류 단위의 상당한 감퇴의 원인. 수산화 암모늄/탄산 치료에 따라 화학 deglycosylation는 또한 일반적으로 사용 되는 대체 방법9. 이러한 화학 릴리스 방법의 그대로 단백질 같은 대량 범위에 레이블이 없는 glycan 풀 펩 티 드 파편의 간섭 없이 대량 spectrometric 분석을 수 있는 성능이 저하 됩니다. 그러나, 이러한 방법의 한 가지 단점이 일반적인 탄수화물 구조 발견 식물10에서 α1, glycans-3-fucosylated N의 증가 저하 속도입니다. 또는, 펩 티 드를 사용 하 여 효소 분리 방법:N-glycanases (PNGases, EC 3.5.1.52) 또한 광범위 하 게 적용 됩니다. 재조합 PNGase F ( Flavobacterium meningosepticum)에서 가장 일반적인 선택 하 고 N-glycans, 코어 α1, fucose 311,12베어링 구조를 제외 하 고 모든 종류의 릴리스 합니다. 따라서, PNGase A (아몬드 씨에서 분리)으로 식물 N-glycans13의 분석을 위해 사용 됩니다. 그러나,이 효소 deglycosylates만 proteolytically-파생 된 glycopeptides, deglycosylate 기본 glycoproteins14수입니다. 따라서, 다단계 샘플 검사 결과 추가 분석, glycans, 특히 낮은 풍부15의 그의 광범위 한 손실이 발생 하기 전에 필요 합니다. N-glycan 릴리스 및 형광 간단 하 고 강력한 방식으로 라벨에 대 한 최적화 된 워크플로우를 제시 하는 방법의 전반적인 목표가입니다. 기본 근거 PNGase H+는 Terriglobus roseus 에서 최근에 발견 되었다 recombinantly 대장균에 표현 될 수 있다, N-glycans 산 성에서 단백질 비 계에서 직접은 수은 조건16. PNGase H+ 를 사용 하 여 대체 방법의 주요 장점은입니다 형광 라벨 반응 반응 버퍼17,18를 변경 하지 않고 동일한 반응 관에서 수행할 수 있습니다. 간단한 준비 조건 및 낮은 풍부 oligosaccharides의 높은 복구 확인이 방법은 유용한 도구 Nglycans의 분석에. 이 프로토콜은 다양 한 식물 종에서 N-glycans의 분석에 적합 합니다.

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

최근 몇 년 동안, 식물의 탄수화물 moieties 받은 관심, 그들은 cross-reactive, 알레르기 자극 면역 반응의. 또한, 탄수화물 구조 또한 식물 물질 대사에 중요 한 역할을 담당할. 여기, 우리가 준비 하 고 사용 하는 N-glycanase 특정 식물에서 파생 된 탄수화물 구조의 릴리스에 대 한 무 (Raphanus sativus)의 다른 재배 품 정에서 N-glycans를 분석 하는 간단 하 고 빠른 방법을 제시. 이 위해 무 homogenates의 원유 trichloroacetic 산 침전 PNGase H+로 치료 되었고 형광 태그 2-aminobenzamide를 사용 하 여 표시. 레이블이 지정 된 N-glycan 샘플 이후 울트라 성능 액체 크로마토그래피 (UPLC) 분리 및 세부 구조에 대 한 비행 (TOF MALDI) 질량 분석의 매트릭스 보조 레이저 탈 착 이온화 시간으로 분석 되었다 평가 및 무 파생 N-glycan 구조 상대 abundancies 척도를. 이 프로토콜 사용할 수 있습니다 또한 다양 한 다른 식물 종에서 N-glycans의 분석 그리고 함수 추가 조사 및 인간의 건강에 N-glycans의 효과 대 한 유용할 수 있습니다.

그림 1 (glyco-) 무, N-glycans의 준비, UPLC 분석 및 이러한 구성 요소의 MALDI TOF MS 분석에서 단백질의 격리를 포함 하 여 설명된 프로토콜의 구조 개요를 보여 줍니다. 그림 2 분석된 무 재배의 derivatized Nglycans의 대표 UPLC chromatograms를 보여 줍니다. 그림 3 2AB derivatized N-glycan 구조 MALDI TOF 질량 ?...

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-glycans Raphanus sativus Cultivars 사용 PNGase H+ 에서 분석

우리는 준비 및 N-glycans 무 (Raphanus sativus)의 다른 재배에서의 분석을 위한 간단 하 고 빠른 방법을 설명합니다.

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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