Name: analysis of n-glycans from raphanus sativus cultivars using pngase h+
Uploaded: 2018-06-25T15:00:00.000+00:00
Duration: 8 min 26 s
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重に転送し、2 M トリクロロ酢酸 (TCA) ソリューションの等しい量を追加します。 注: TCA を追加は、水溶性 (糖) タンパク質を沈殿させます。</li>
	<li>4 ° C、30 分で 14,000 × gで遠心分離機、ペレット (糖) 蛋白質から上澄みを取り外します。</li>
	<li>14,000 × g水溶性オリゴ糖や多糖類を削除する 5 分の 4 ° c で 20 ml の脱イオン水と遠心分離機のペレットを洗浄します。</li>
	<li>手順 2.5 を繰り返します。4 回。</li>
	<li>再 1 mL の脱イオン水と新鮮な 1.5 mL 遠心チューブに転送でペレットを中断します。</li>
</ol>3. N型糖鎖の調製
<ol><li>手順 2.7 からタンパク質溶液のミックス 50 μ L。(新鮮な大根の 5 g に等しい) 組換え PNGase h+ 10 mM 酢酸 0.2 mu。</li>
	<li>12 h の 37 ° C で反応混合物を孵化させなさい。、孵化後余分な蛋白質および酵素を削除する 5 分 14,000 × gで遠心分離機します。</li>
	<li>新鮮な 1.5 mL 遠心チューブに上清を転送します。</li>
</ol>4 N型糖鎖の精製
<ol><li>N型糖鎖を豊かにし、 N型糖鎖の蛍光 2 aminobenzamide (2 AB) ラベル エージェントの選択性を増やす反応混合物から塩および他の不純物を削除する固相抽出 (SPE) の列を準備します。誘導体化。<ol>
<li>3 mL の脱イオン水を洗い流すを追加します。</li>
		<li>アクティブにトリフルオロ酢酸 (TFA, 0.1 %v/v) を含む 80% アセトニ トリル溶液 3 mL SPE カラムを追加します。</li>
		<li>SPE コラムを平衡させ脱イオン水 3 mL を追加します。</li>
	</ol>
</li>
	<li>ステップ 3.3 列上からサンプルを転送し、流れを破棄します。</li>
	<li>1.5 mL の脱イオン水コラムを洗浄し、流れを破棄するを追加します。</li>
	<li>1.5 mL の 0.1% を含む 20% アセトニ トリル水溶液を使用して、列からリリースされたN型糖鎖を溶出 TFA (v/v) 2 mL の遠心管に。</li>
	<li>室温で遠心蒸発溶剤を削除します。このサンプルは、完全に乾燥するまでに蒸発させます。</li>
</ol>5 N型糖鎖の蛍光誘導体化
<ol><li>35 mM 2 AB と 0.1 M ナトリウム シアノ水素化ホウ素ジメチル ジメチルスルホキシド/酢酸酸溶液 (7:3 v/v) から成る、2 AB 溶液 1 mL を準備します。</li>
	<li>六つの異なる大根由来の乾燥サンプル (ステップ 4.5.) 2 AB ソリューションの 5 μ L を追加します。渦各サンプルまで完全に分解します。</li>
	<li>65 ° C で 2 h の混合物を孵化させなさい</li>
	<li>5 分間室温までサンプルの涼しい許可し、5 μ L の脱イオン水とアセトニ トリル 40 μ L を追加します。3 分間 14,000 x g でチューブを遠心します。</li>
	<li>300 μ L 高回復高速液体クロマトグラフィー バイアルに上澄みから 48 μ L を転送します。1 ヶ月の-20 ° C で derivatized のN型糖鎖のサンプルを格納します。</li>
</ol>6. 構築高精度のN型糖鎖のプロファイリング
<ol><li>それぞれ 330 nm/420 nm の励起/蛍光波長オンライン蛍光 detectorset に接続されている標準の高精度システムを使用してサンプルを分析します。</li>
	<li>疎水性相互作用クロマトグラフィー (構築) を使用して-分析のための 60 ° C のカラム温度の高精度糖鎖列。</li>
	<li>50 mL の原液 (1 M, ギ酸アンモニウム溶液、pH 4.5) の液体クロマトグラフィー質量分析計 (LCMS) 950 mL で希釈することによって溶剤 A を準備-水のグレードします。とおり、在庫ソリューションを準備します。<ol>
<li>LCMS グレード H2O を 700 mL にギ酸の 43 mL を追加します。</li>
		<li>(奥行 25% アンモニア水を滴下して 4.5 に pH を調整します。</li>
		<li>溶媒をメスシリンダーに転送し、3 ヵ月の LCMS グレード H2o. ストア 4-6 ° c と 1000 mL を入力します。</li>
	</ol>
</li>
	<li>溶剤 B として LCMS グレード アセトニ トリルを使用します。</li>
	<li>N型糖鎖を次のグラジエントで区切ります。<ol>
<li>まず 0 から 44.5 分溶媒 A. セット 0.5 mL/min の流量に溶剤 B の 95% を追加します。</li>
		<li>0 分から 6 分、線形のグラデーションの to78% の溶媒 B の割合を徐々 に減少します。</li>
		<li>6 分から 44.5 分、55.9% に線形グラデーションの溶媒 B の割合を徐々 に減少します。</li>
		<li>46.5 分 44.5 分からには、急速に 55.9% から 0% への線形グラデーションで溶剤 B の割合を減らすと 2 分 0% を保持し、列の洗濯を開始します。55 分 44.5 から 0.25 mL/分への流量を減らします。</li>
		<li>50.5 分 48.5 分から 95% に溶剤 B を増やすことによってさらに列を洗って、4.5 分の 95% を保持します。</li>
		<li>再度 0.5 mL/min で 95% 溶剤 B の開始条件にコラムを平衡させ、57 分 50.5 から。</li>
	</ol>
</li>
	<li>高精度システムに 45 μ L のサンプルを注入します。</li>
	<li>15 と 40 分間保持時間と、高精度での糖鎖を溶出します。</li>
	<li>2 mL 遠心管を使用して各観測の高精度ピーク分数を収集し、遠心蒸発によってサンプルを乾燥します。</li>
	<li>2 AB デキストラン標準 (2−20 グルコース単位) 工場 -N型糖鎖プロファイリング、調整し、標準化されたグルコース単位への溶出時間を割り当てるをラベルの約 1 pmol を注入します。</li>
</ol>7. MALDI-TOF 質量分析
<ol><li>LCMS のステップ 5 で 6.8 μ L のサンプルを溶解-水のグレードします。ミックス 1 μ L の試料溶液及び 1 μ L 6 字-2 thiothymine (ATT) ソリューション (0.3 %w/v 70 %v/v アセトニ トリル水溶液中で)、およびピペット MALDI-TOF サンプル キャリアの上に混合物の。</li>
	<li>[スタート] ボタンを押すことによって肯定的なイオン モードで MALDI-TOF 質量分析計の計測器を使用してサンプルを分析します。</li>
	<li>付属の MALDI TOF MS 解析ソフトウェアを使用して質量スペクトルを分析します。</li>
	<li>オープン ソース糖解釈ソフトウェア19を使用してスペクトルを解釈します。2 AB のラベル、[M+na]+の検索パラメーターを設定し、2.0 da 精度パラメーターを設定します。</li>
</ol>

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

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

Universidad de Cartagena UDEC

Research

JoVE Journal

Biochemistry

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

6.6K ビュー.  Nanjing Agricultural University. 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。

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

Structural Characterization of Mannan Cell Wall Polysaccharides in Plants Using PACE

Plant cell wall polysaccharides are notoriously difficult to analyze, and most methods require expensive equipment, skilled operators, and large amounts of purified material. Here, we describe a simple method for gaining detailed polysaccharide structural information, including resolution of structural isomers. For polysaccharide analysis by gel electrophoresis (PACE), plant cell wall material is hydrolyzed with glycosyl hydrolases specific to the polysaccharide of interest (e.g., mannanases for mannan). Large format polyacrylamide gels are then used to separate the released oligosaccharides, which have been fluorescently labeled. Gels can be visualized with a modified gel imaging system (see Table of Materials). The resulting oligosaccharide fingerprint can either be compared qualitatively or, with replication, quantitatively. Linkage and branching information can be established using additional glycosyl hydrolases (e.g., mannosidases and galactosidases). Whilst this protocol describes a method for analyzing glucomannan structure, it can be applied to any polysaccharide for which characterized glycosyl hydrolases exist. Alternatively, it can be used to characterize novel glycosyl hydrolases using defined polysaccharide substrates.

A protocol for the structural analysis of polysaccharides by gel electrophoresis (PACE), using mannan as an example, is described.

マンナン ペースを用いた植物細胞壁多糖の構造解析

Plant cell wall polysaccharides are notoriously difficult to analyze, and most methods require expensive equipment, skilled operators, and large amounts ...

生化学

A Quantitative Glycomics and Proteomics Combined Purification Strategy

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

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

定量的グライコミクスおよびプロテオミクス複合精製戦略

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

化学

Chemo-enzymatic Synthesis of N-glycans for Array Development and HIV Antibody Profiling

We present a highly efficient way for the rapid preparation of a wide range of N-linked oligosaccharides (estimated to exceed 20,000 structures) that are commonly found on human glycoproteins. To achieve the desired structural diversity, the strategy began with the chemo-enzymatic synthesis of three kinds of oligosaccharyl fluoride modules, followed by their stepwise &#945;-selective glycosylations at the 3-O and 6-O positions of the mannose residue of the common core trisaccharide having a crucial &#946;-mannoside linkage. We further attached the N-glycans to the surface of an aluminum oxide-coated glass (ACG) slide to create a covalent mixed array for the analysis of hetero-ligand interaction with an HIV antibody. In particular, the binding behavior of a newly isolated HIV-1 broadly neutralizing antibody (bNAb), PG9, to the mixture of closely spaced Man5GlcNAc2 (Man5) and 2,6-di-sialylated bi-antennary complex type N-glycan (SCT) on an ACG array, opens a new avenue to guide the effective immunogen design for HIV vaccine development. In addition, our ACG array embodies a powerful tool to study other HIV antibodies for hetero-ligand binding behavior.

Chemo-enzymatic Synthesis of N-glycans for Array Development and HIV Antibody Profiling

A modular approach to the synthesis of N-glycans for attachment to an aluminum oxide-coated glass slide (ACG slide) as a glycan microarray has been developed and its use for the profiling of an HIV broadly neutralizing antibody has been demonstrated.

N型糖鎖アレイ開発および HIV 抗体のための化学酵素合成プロファイリング

We present a highly efficient way for the rapid preparation of a wide range of N-linked oligosaccharides (estimated to exceed 20,000 structures) that are ...

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

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

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

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

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

Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although this modification was implicated in several biological processes, additional aspects of its function are emerging, with recent evidence of its role in the production of signals for glycoprotein quality control and trafficking. Thus, phenomena related to N-linked glycans and their processing are being intensively investigated. Methods that have been recently developed for proteomic analysis have greatly improved the characterization of glycoprotein N-linked glycans. Nevertheless, they do not provide insight into the dynamics of the sugar chain processing involved. For this, labeling and pulse-chase analysis protocols are used that are usually complex and give very low yields. We describe here a simple method for the isolation and analysis of metabolically labeled N-linked oligosaccharides. The protocol is based on labeling of cells with [2-3H] mannose, denaturing lysis and enzymatic release of the oligosaccharides from either a specifically immunoprecipitated protein of interest or from the general glycoprotein pool by sequential treatments with endo H and N-glycosidase F, followed by molecular filtration (Amicon). In this method the isolated oligosaccharides serve as an input for HPLC analysis, which allows discrimination between various glycan structures according to the number of monosaccharide units comprising them, with a resolution of a single monosaccharide. Using this method we were able to study high mannose N-linked oligosaccharide profiles of total cell glycoproteins after pulse-chase in normal conditions and under proteasome inhibition. These profiles were compared to those obtained from an immunoprecipitated ER-associated degradation (ERAD) substrate. Our results suggest that most NIH 3T3 cellular glycoproteins are relatively stable and that most of their oligosaccharides are trimmed to Man9-8GlcNAc2. In contrast, unstable ERAD substrates are trimmed to Man6-5GlcNAc2 and glycoproteins bearing these species accumulate upon inhibition of proteasomal degradation.

We describe a method for analysis of the alteration of N-linked glycans through the early life of glycoproteins after their biosynthesis in mammalian cells. This is achieved by pulse-chase analysis of metabolically labeled glycans, enzymatic release from glycoproteins and examination by HPLC.

哺乳動物細胞での糖タンパク質からN -結合型糖鎖のパルス - チェイス分析

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although ...

生物学

Nonspecific Proteolysis-Based Glycomics Strategy for Bacterial Glycoproteins

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

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

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

<meta charset="utf8"></head><body>グライコミクスガイド下グライコプロテオミクスを用いた大腸がん組織のプロファイリング

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

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

<meta charset="utf8"></head><body>グライコミクスガイド下グライコプロテオミクスは、複雑な腫瘍微小環境におけるグリコプロテオームの包括的なプロファイリングを容易にします

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

グライコミクスガイド下グライコプロテオミクスは、複雑な腫瘍微小環境におけるグリコプロテオームの包括的なプロファイリングを容易にします 

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

がん研究

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

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

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

特定のエンド - およびエキソグリコシダーゼを用いたタンパク質のグリコシル化の同定と特性評価

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

ダイコン品種使用 PNGase H⁺からN型糖鎖の解析

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マンナンペースを用いた植物細胞壁多糖の構造解析

定量的グライコミクスおよびプロテオミクス複合精製戦略

N型糖鎖アレイ開発および HIV 抗体のための化学酵素合成プロファイリング

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

哺乳動物細胞での糖タンパク質からN -結合型糖鎖のパルス - チェイス分析

Nonspecific Proteolysis-Based Glycomics Strategy for Bacterial Glycoproteins

グライコミクスガイド下グライコプロテオミクスは、複雑な腫瘍微小環境におけるグリコプロテオームの包括的なプロファイリングを容易にします

グライコミクスガイド下グライコプロテオミクスは、複雑な腫瘍微小環境におけるグリコプロテオームの包括的なプロファイリングを容易にします

グライコミクスガイド下グライコプロテオミクスは、複雑な腫瘍微小環境におけるグリコプロテオームの包括的なプロファイリングを容易にします

特定のエンド - およびエキソグリコシダーゼを用いたタンパク質のグリコシル化の同定と特性評価

ダイコン品種使用 PNGase H+からN型糖鎖の解析

要約

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マンナン ペースを用いた植物細胞壁多糖の構造解析

定量的グライコミクスおよびプロテオミクス複合精製戦略

N型糖鎖アレイ開発および HIV 抗体のための化学酵素合成プロファイリング

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

哺乳動物細胞での糖タンパク質からN -結合型糖鎖のパルス - チェイス分析

Nonspecific Proteolysis-Based Glycomics Strategy for Bacterial Glycoproteins

グライコミクスガイド下グライコプロテオミクスは、複雑な腫瘍微小環境におけるグリコプロテオームの包括的なプロファイリングを容易にします

グライコミクスガイド下グライコプロテオミクスは、複雑な腫瘍微小環境におけるグリコプロテオームの包括的なプロファイリングを容易にします

グライコミクスガイド下グライコプロテオミクスは、複雑な腫瘍微小環境におけるグリコプロテオームの包括的なプロファイリングを容易にします

特定のエンド - およびエキソグリコシダーゼを用いたタンパク質のグリコシル化の同定と特性評価

ダイコン品種使用 PNGase H⁺からN型糖鎖の解析

マンナンペースを用いた植物細胞壁多糖の構造解析