Name: analysis of n-glycans from raphanus sativus cultivars using pngase h+
Uploaded: 2018-06-25T15:00:00.000+00:00
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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) çözüm eşit bir hacmi ekleyin. Not: TCA ekleme çözünür (glyco) proteinler çökelti.</li>
	<li>14.000 × g 30 dk için 4 ° C'de, santrifüj kapasitesi ve süpernatant pelleted (glyco) proteinler kaldırın.</li>
	<li>14.000 × g çözünür oligosakkaritler ve polisakkaritler kaldırmak 5 min için 4 ° C'de, Pelet 20 mL deiyonize su ve santrifüj ile yıkayın.</li>
	<li>2.5 arasındaki adımları yineleyin. dört kez.</li>
	<li>Granül 1 ml deiyonize su ve taze 1,5 mL santrifüj tüpü transfer yeniden askıya alma.</li>
</ol>3. N- glukanlardir hazırlanması
<ol><li>Mix 50 µL protein çözüm adım 2.7. (5 g taze turp eşit) 0.2 mU rekombinant PNGase h+ 10 mM asetik asit ile.</li>
	<li>Reaksiyon karışımı için 12 h 37 ° C'de kuluçkaya. Kuluçka sonra santrifüj kapasitesi 14.000 × g ekstra protein ve enzim kaldırmak 5 min için de.</li>
	<li>Süpernatant temiz 1.5 mL santrifüj tüpü aktarın.</li>
</ol>4. N- glukanlardir arınma
<ol><li>N- glukanlardir zenginleştirmek ve N- glycan için fluorogenic 2-aminobenzamide (2-AB) etiketleme Aracısı selectivity artan tepki karışımından tuzları ve diğer kirleri çıkarmak için katı fazlı ayıklama (SPE) sütun hazırlamak derivatization.<ol>
<li>3 mL deiyonize su sütun yıkamak için ekleyin.</li>
		<li>Trifluoroacetic asit (TFA, % 0,1 v/v) etkin içeren % 80 Asetonitril çözüm 3 mL SPE-sütun ekleyin.</li>
		<li>3 mL deiyonize su SPE-sütun equilibrate ekleyin.</li>
	</ol>
</li>
	<li>Örnek adım 3.3 sütuna aktarmak ve akışı aracılığıyla atın.</li>
	<li>1.5 mL deiyonize su sütun yıkayın ve akışı üzerinden atmak için ekleyin.</li>
	<li>Yayımlanan N- glukanlardir 1.5 mL %0,1 içeren sulu % 20 Asetonitril çözüm kullanarak sütundan elute TFA (v/v) 2 mL santrifüj tüpü içine.</li>
	<li>Çözücü, oda sıcaklığında santrifüj buharlaşma çıkarın. Örnek tamamen kuru olana buharlaşır.</li>
</ol>5. floresan Derivatization, N- glukanlardir
<ol><li>1 mL dimetil sülfoksit/asetik asit solüsyonu (7:3, v/v) 35 mM 2-AB ve 0.1 M sodyum cyanoborohydride oluşan 2-AB çözeltisi hazırlamak.</li>
	<li>2-AB çözüm kurutulmuş örnekleri (adım 4.5.) 5 µL altı farklı turp türetilmiş ekleyin. Girdap her örnek kadar tamamen dağıldı.</li>
	<li>65 ° C'de 2 h için karışım kuluçkaya</li>
	<li>5 min için örnek soğumaya aşağı oda sıcaklığına ve 5 µL deiyonize su ve Asetonitril 40 µL ekleyin. 14.000 x g 3 dk de tüpler santrifüj kapasitesi.</li>
	<li>48 µL süpernatant 300 µL yüksek-kurtarma HPLC şişe aktarın. Derivatized N- glycan örnekleri-20 ° c ilâ bir ay için saklayın.</li>
</ol>6. HILIC UPLC N- glukanlardir profil oluşturma
<ol><li>Bir online floresan detectorset 330 nm/420 nm, uyarma/emisyon dalga boylarında, sırasıyla bağlı standart bir UPLC sistem kullanarak örnekleri analiz.</li>
	<li>Hidrofobik etkileşim sıvı kromatografi (HILIC) kullanın-60 ° c analizi için bir sütun ısıda UPLC glycan sütun.</li>
	<li>Çözelti A hisse senedi çözüm (1 M amonyum Formiat çözüm, pH 4.5) 50 mL sıvı kromatografi-kütle spektrometresi (LCMS) 950 mL ile sulandrarak hazırlamak-sınıf su. Hisse senedi çözüm aşağıdaki gibi hazırlayın:<ol>
<li>Formik asit 43 mL LCMS-grade H2o 700 mL için eklemek</li>
		<li>PH 4.5 için dropwise buna ek olarak sulu amonyak çözüm (% 25 w/w) tarafından ayarlayın.</li>
		<li>Çözücü bir ölçüm silindir aktarmak ve 1000 mL 3 aya kadar LCMS-grade H2o mağaza 4-6 ° c ile doldurun.</li>
	</ol>
</li>
	<li>LCMS-grade Asetonitril solvent B. kullanın</li>
	<li>N- glukanlardir ile aşağıdaki degrade elüsyon ayırın:<ol>
<li>Çözücü B % 95'i solvent A. küme akış hızı 0.5 mL/dk 0 ile 44,5 min için ekleyerek başlayın.</li>
		<li>6 dk 0 dk yavaş yavaş çözücü B doğrusal degrade to78% oranını azaltmak.</li>
		<li>44,5 dk 6 dk yavaş yavaş çözücü B %55.9 doğrusal gradyan ile oranını azaltmak.</li>
		<li>44,5 min 46,5 dk dan hızla çözücü B %0 %55.9 doğrusal gradyan ile oranını azaltmak ve %0 2 min için tutun ve çamaşır sütun başlatın. 0.25 mL/dk akış oranını 44.5 üzerinden 55 dk. için azaltmak.</li>
		<li>Çözücü B %95 50,5 dk 48,5 dk dan artırarak daha fazla sütun yıkama ve %95 4,5 dakika tutun.</li>
		<li>Sütun % 95 çözücü B 0.5 mL/dk başlangıç koşulları 50,5 57 dk dan yeniden equilibrate.</li>
	</ol>
</li>
	<li>Örnek 45 µL UPLC sistemi içine enjekte.</li>
	<li>15 ila 40 dk arasında saklama süreleri ile N- glukanlardir UPLC tarafından elute.</li>
	<li>2 mL santrifüj tüpleri kullanarak her gözlenen UPLC tepe kısmını toplamak ve örnekler santrifüj buharlaşma tarafından kuru.</li>
	<li>2-AB dextran standart (2−20 glikoz birimleri) bitki -N- glycan profil oluşturma kalibre ve onların elüsyon kez standart glikoz birimlerine atamak için etiketli yaklaşık 1 pmol enjekte.</li>
</ol>7. MALDI-TOF MS Analizi
<ol><li>Adım 5 6,8 µL LCMS, örneklerinden çözülür-sınıf su. Mix 1 µL örnek çözümü ve 6-aza-2-thiothymine (ATT) çözüm (% %0,3 70 v/v sulu Asetonitril içinde w/v) ve pipet MALDI-TOF örnek taşıyıcı üzerine karışımı 1 µL.</li>
	<li>Başlat düğmesine basarak pozitif iyon modunda bir MALDI-TOF Kütle Spektrometre enstrüman kullanarak örnekleri analiz.</li>
	<li>Kitle spectra eşlik eden MALDI-TOF-Bayan analiz yazılımı kullanarak analiz.</li>
	<li>Bir açık kaynak glycan yorumu yazılım19kullanarak spectra yorumlamak. 2-AB [M + Na]+, etiketleme, arama parametrelerini belirlemek ve 2.0 Da doğruluk parametresini ayarlayın.</li>
</ol>

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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><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+

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, ...

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

Video: 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.

Yapısal Mannan hücre duvarı polisakkaritler hızı kullanarak tesislerinde karakterizasyonu

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

Biyokimya

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.

Nicel Glycomics ve Proteomik Kombine Arıtma Stratejisi

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

Kimya

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- glukanlardir dizi geliştirme ve HIV antikor için kemoterapi enzimatik sentezi profil oluşturma

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.

Memeli Hücrelerinde Glikoproteinler N-bağlı Şeker Zincirler Pulse-kovalamaca Analizi

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

Biyoloji

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>Kolorektal Kanser Dokularının Glikomikler Kılavuzluğunda Glikoproteomik Kullanılarak Profillenmesi

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>Glikomik kılavuzluğunda glikoproteomikler, karmaşık tümör mikro ortamlarında glikoproteomun kapsamlı profilini kolaylaştırır

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.

Glikomik kılavuzluğunda glikoproteomikler, karmaşık tümör mikro ortamlarında glikoproteomun kapsamlı profilini kolaylaştırır 

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

Kanser Araştırmaları

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.

Spesifik endo-ve exoglycosidases kullanarak protein glikozilasyon tanımlanması ve karakterizasyonu

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

N- glukanlardir Raphanus sativus çeşitlerin kullanarak PNGase h⁺ Analizi

Özet

Daha Fazla Video Keşfet

Bu videodaki bölümler

Yapısal Mannan hücre duvarı polisakkaritler hızı kullanarak tesislerinde karakterizasyonu

Nicel Glycomics ve Proteomik Kombine Arıtma Stratejisi

N- glukanlardir dizi geliştirme ve HIV antikor için kemoterapi enzimatik sentezi profil oluşturma

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

Memeli Hücrelerinde Glikoproteinler N-bağlı Şeker Zincirler Pulse-kovalamaca Analizi

Nonspecific Proteolysis-Based Glycomics Strategy for Bacterial Glycoproteins

Glikomik kılavuzluğunda glikoproteomikler, karmaşık tümör mikro ortamlarında glikoproteomun kapsamlı profilini kolaylaştırır

Glikomik kılavuzluğunda glikoproteomikler, karmaşık tümör mikro ortamlarında glikoproteomun kapsamlı profilini kolaylaştırır

Glikomik kılavuzluğunda glikoproteomikler, karmaşık tümör mikro ortamlarında glikoproteomun kapsamlı profilini kolaylaştırır

Spesifik endo-ve exoglycosidases kullanarak protein glikozilasyon tanımlanması ve karakterizasyonu