Bu çalışma, Enerji Enstitüsü Biosciences hibe OO0G01 tarafından finanse edildi.

Olimp yöntemi glycosylhydrolase olarak endoglucanase kullanarak, dikotil bitkiler, xyloglucan hücre duvarlarında bulunan başlıca hemiselüloz örneklediği. Bu yöntem bir bitki doku kaynağı olarak tüm Arabidopsis fideler ile gösterilmiştir. Enzim ve hücre dışı matriks malzemesi, aynı prosedür kullanılarak istenilen analiz bağlı olarak değiştirilebilir. 1. Hücre duvar izolasyonu <ol><li> Hasat beş 5 gün eski tüm Arabidopsis fide veya eşdeğer miktarda istenilen malzemenin ve 1.5mL bir reaksiyon tüpü içine aktarabilirsiniz . Malzeme tüp alt yerleştirildiğinden emin olun. </li><li> Sıvı nitrojen içinde örnek ve ek-freeze üst üste iki 3mm metal topları reaksiyon tüpüne ekleyin. </li><li> Bilyalı değirmen (02:30 dakika, 25Hz) kullanılarak dondurulmuş örnek Grind. </li><li> Örnek 1 ml% 70 sulu etanol ekleyin. Bir mıknatıs kullanarak metal topları çıkarın. Iyice Vortex. </li><li> Pelet için 10dk hücre duvarı malzemesi içeren alkol çözünmeyen kalıntı 14.000 rpm&#39;de örnek santrifüjleyin. </li><li> Aspirasyon ile süpernatantı dikkatlice çıkarın. Pelet rahatsız değil emin olun. </li><li> Kloroform / metanol (1:1 v / v) solüsyonu 1 ml ekleyin. Pelletini tekrar iyice karıştırın. </li><li> Örnek 10 dakika 14.000 rpm&#39;de santrifüj ve dikkatli bir aspirasyon ile süpernatantı kaldırmak. Pelet rahatsız değil emin olun. </li><li> Örnek bir konsantratör kullanarak kurutun. </li></ol> 2. Oligosakkaritlerin çözünürleştirme <ol><li> Oligosakkaritler şeklinde çözünürleştirme için özellikle polisakkarit uygun bir tampon 25μl kuru pelet tekrar süspansiyon haline getirin. Endoglucanase 50mm Amonyum format, pH4.5 için kullanılır. Endoglucanase, 0.2U ekleyin. Vorteks aşağı süspansiyon ve spin sıvı. </li><li> 37 16h için örnek inkübe ° C bir kuluçka makinesi, 300rpm az sallayarak. </li><li> Konsantratör kullanarak sindirmek (su) çözücü çıkarın. </li></ol> 3. MALDI-TOF analizi yayımlanan oligosakkaritler <ol><li> BioRex MSZ 501 katyon değişim reçine boncuk sindirilir örnek tuzları ve enzim kaldırmak için kullanılır. Boş bir sütun yerleştirme ve bir kısım boncuk bol miktarda su ile yıkama Durumu boncuk. </li><li> 5-10 BioRex boncuk sindirilir ve kurutulmuş örnek ekleyin. Sonra küçük miktarlarda su malzeme 5μl olabilir, 10μl su ekleyin. Kısaca tüp iplik çözüm boncuk bırakın. Oda sıcaklığında en az 10 dakika süreyle inkübe edin. </li><li> MALDI hedef plaka üzerine Spot 2μl matris (2,5-dihydroxybenzoic asit (DHB), su 10mg/ml). Homojen matris kimyasal kristalleri giden vakum altında çözücü matris buharlaşır. </li><li> Spot 2μl hedef plakası matris kristallerin üst desalted örnek çözümü. Örnekleri çok sayıda nokta istiyorsanız sadece 3dk maksimum lekelenme devam. Bu zaman çerçevesi ilk benekli örnek henüz kuru olduğunu garanti eder. Son benekli örnek yeniden çözünmüş matris ve örnek oligosakkarit molekülleri tam bir karıştırma sağlamak için ek bir 2min için bekleyin. Sonra kuru bir vakum altında hedef plakası. Örnek / matris karışımı homojen kristalizasyon etkinleştirmek için 1dk daha az kristalize gerekir. </li><li> Hedef plakası MALDI-TOF kütle spektrometresi içine yerleştirin. Makine 20000V hızlanan voltaj ve 350 nm arasında bir gecikme ile olumlu reflectron moduna ayarlanmış olmalıdır seçilen kütle aralığı (olabilir beklenen oligomerler bağımlı değiştirmek) 500-3000 Da olmalıdır. Lazer ateş başlayın ve bir temsilcisi ortalama spektrumu (Şekil 2A) oluşturmak için derlenmiş örnek başına 100-200 spektrumları, toplamak. Belirli oligosakkaritler temsil İyonlar şarj (m / z) oranı üzerinden kitle tarafından tespit edilebilir. Tüm ilgi çekici iyonlarının iyon yoğunlukları (pik yükseklik) örnek her oligosakkarit göreceli bolluk (Şekil 2B)% 100&#39;e kadar ilave edilmelidir. </li></ol> 4. yerinde Olimp analizi Olimp dokusu üzerinde herhangi bir duvar hazırlık adımları atlayarak doğrudan da kullanılabilir. Bitki doku kaynağı olarak bir örnek Arabidopsis, hypocotyls solmuş. Yine, bir endoglucanase hemiselülozlar xyloglucan yapısını belirlemek için kullanılır. Enzim ve doku malzemesi aynı prosedür kullanılarak istenilen analiz bağlı olarak değiştirilebilir. <ol><li> Solmuş fide Hasat ve boş bir MALDI hedef plaka üzerine koyun ve fide kurumaya bırakın. </li><li> Istenilen konuma kuru fide 0.5μl endoglucanase (50mm Amonyum format içinde çözünmüş 0.4U/μl, pH4.5) ekleyin. Enzim damla hedef plakası kısmen dokunur emin olun. </li><li> Doyurarak nem önlemek için kapalı bir kapta MALDI hedef plaka koyun enzim damla kurur. 1 odasında plaka inkübeAz 6 saat 37 ° C </li><li> Bitki doku ve vakum altında enzim damla MALDI hedef plakası kurulayın. </li><li> Her kuru enzim spot üstünde; 0.5μl matrisi (10 mg / l DHB) ekleyin. 2min için bekletin. </li><li> Vakum altında MALDI hedef plakası kurulayın. </li><li> MALDI-TOF örnekleri analiz edin. MALDI-TOF kütle spektrometresi içine doku ve enzim / matris spot (ler) ile hedef plakası yerleştirin. Makine 20000V hızlanan voltaj ve 350 nm arasında bir gecikme ile olumlu reflectron moduna ayarlanmış olmalıdır seçilen kütle aralığı (olabilir beklenen oligomerler bağımlı değiştirmek) 500-3000 Da olmalıdır. </li><li> Doku SONRAKİ matris / örnek kristalleri üzerine lazer ateş başlayın. Zaman moleküller uçuş kapalı olacaktır Böyle bir durumda, doku kendisi basmayın emin olun. Nokta, bir temsili ortalama spektrumu (Şekil 3) oluşturmak için derlenmiş başına yaklaşık 20-50 spektrumları toplayın. Belirli oligosakkaritler temsil İyonlar şarj (m / z) oranı üzerinden kitle tarafından tespit edilebilir. Iyon yoğunlukları (iyon yükseklik) olabilir ve tüm ilgi çekici iyonları örnek her oligosakkaritler göreceli bolluk içinde% 100 ilave edilmelidir. </li></ol> 5. Temsilcisi Sonuçlar Arabidopsis fide hemiselülozlar xyloglucan Olimp analizinin bir örneği Şekil 2&#39;de gösterilmiştir. Iyonlarının kütle farklılıkları ve bilinen iyi karakterize bir enzim sayesinde (endoglucanase) özgüllüğü iyonları özel oligosakkarit yapılar (Şekil 2A) atanabilir. Açıkçası, yapısal izomerleri ayırt edilemez. Belirlenmesi için çeşitli oligosakkaritler göreceli bolluk temel varsayım (Şekil 2B), kitlesel spec tepki faktörü bu oligosakkaritler için çok benzer olmasıdır. Burada görüldüğü gibi, Olimp kantifikasyon yüksek tekrarlanabilir. Bununla birlikte, yöntemin sağlamlık sinyali çeşitli oligosakkarit iyonları gürültü oranları son derece bağımlı olduğunu lütfen unutmayın. Örneğin bu oran düşük miktarlarda tuz veya oligosakkaritler ile kirlenme azaltabilirsiniz. Olimp doku (in situ analizi) analizi, çok küçük ve belirli alanlarda çalışma sağlar ve çok az numune hazırlama içerir. Örneğin aynı kalitatif (iyonlar) (Şekil 3) Burada sunulan ancak kantitatif farklılıklar (iyon şiddetleri değişimi) Arabidopsis fide sürgün ve kök doku arasında gözlendi. Olimp yöntemi Permütasyonlar böylece doku &quot;görüntüleme&quot; yol açabilir. <img src="/files/ftp_upload/2046/2046fig1.jpg" alt="Şekil 1" /> Şekil 1: Akış şeması bir bitki kaynağı olarak tüm Arabidopsis fide kullanarak Olimp prosedürü. İlk olarak, doku yumuşatılır ve hücre duvarı malzemesi (resim, Fujino ve ark modifiye 10) hazırlanır. Sonra belirli bir hidrolaz kullanarak belirli bir duvar polisakkarit oligosakkaritler serbest bırakılır. Son olarak, çözünür oligosakkaritler bağıl bollukları MALDI-TOF kütle spektrometresi ile belirlenir. <img src="/files/ftp_upload/2046/2046fig2.jpg" alt="Şekil 2" /> Şekil 2 Olimp tarafından belirlenir Arabidopsis solmuş fide xyloglucan oligosakkaritler göreceli bolluk. A) Temsilci xyloglucan oligosakkarit kütle spektrumu, her iyon belirli bir oligosakkarit yapısını temsil eder, yapısal izomerleri ayırt edilebilir değildir. B) belirlenmesi için, göreceli oligosakkarit bolluk Sorumlu çubuk diyagramı. <img src="/files/ftp_upload/2046/2046fig3.jpg" alt="Şekil 3" /> Şekil 3 Örnek olarak Arabidopsis solmuş fide kullanarak yerinde Olimp analiz. Her enzim / sindirim damlacık (renkli daireler) bağımsız olarak analiz edilebilir ve buna karşılık gelen bir kütle spektrumları elde edilir ve analiz edilebilir. <img src="/files/ftp_upload/2046/2046fig4.jpg" alt="Şekil 4" /> Şekil 4. Xylanase (Megazyme) Miscanthus yaprak malzeme sindirerek elde hemiselüloz Xylan Olimp spektrumu.

<ol>
	<li>Lerouxel, O. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+structural+phenotyping+of+plant+cell+wall+mutants+by+enzymatic+oligosaccharide+fingerprinting.">Rapid structural phenotyping of plant cell wall mutants by enzymatic oligosaccharide fingerprinting.</a> Plant Physiology. 130 (4), 1754-1754 (2002).</li><li>Obel, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Microanalysis+of+plant+cell+wall+polysaccharides.">Microanalysis of plant cell wall polysaccharides.</a> Molecular Plant. 2 (5), 922-922 (2009).</li><li>Mouille, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+trait+loci+analysis+of+primary+cell+wall+composition+in+Arabidopsis.">Quantitative trait loci analysis of primary cell wall composition in Arabidopsis.</a> Plant Physiology. 141 (3), 1035-1035 (2006).</li><li>Bauer, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Development+and+application+of+a+suite+of+polysaccharide-degrading+enzymes+for+analyzing+plant+cell+walls.">Development and application of a suite of polysaccharide-degrading enzymes for analyzing plant cell walls.</a> PNAS. 103 (30), 11417-11417 (2006).</li><li>Pauly, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+xyloglucan-specific+endo-beta-1,4-glucanase+from+Aspergillus+aculeatus:+expression+cloning+in+yeast,+purification+and+characterization+of+the+recombinant+enzyme.">A xyloglucan-specific endo-beta-1,4-glucanase from Aspergillus aculeatus: expression cloning in yeast, purification and characterization of the recombinant enzyme.</a> Glycobiology. 9 (1), 93-93 (1999).</li><li>Aboughe-Angone, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+wall+carbohydrates+from+fruit+pulp+of+Argania+spinosa:+structural+analysis+of+pectin+and+xyloglucan+polysaccharides.">Cell wall carbohydrates from fruit pulp of Argania spinosa: structural analysis of pectin and xyloglucan polysaccharides.</a> Carbohydr Res. 343 (1), 67-67 (2008).</li><li>Brown, D. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+five+xylan+synthesis+mutants+reveals+new+insight+into+the+mechanisms+of+xylan+synthesis.">Comparison of five xylan synthesis mutants reveals new insight into the mechanisms of xylan synthesis.</a> Plant Journal. 52 (6), 1154-1154 (2007).</li><li>Lee, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+PARVUS+gene+is+expressed+in+cells+undergoing+secondary+wall+thickening+and+is+essential+for+glucuronoxylan+biosynthesis.">The PARVUS gene is expressed in cells undergoing secondary wall thickening and is essential for glucuronoxylan biosynthesis.</a> Plant Cell Physiol. 48 (12), 1659-1659 (2007).</li><li>Obel, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Pectin+may+hinder+the+unfolding+of+xyloglucan+chains+during+cell+deformation:+implications+of+the+mechanical+performance+of+Arabidopsis+hypocotyls+with+pectin+alterations.">Pectin may hinder the unfolding of xyloglucan chains during cell deformation: implications of the mechanical performance of Arabidopsis hypocotyls with pectin alterations.</a> Molecular Plant . , (2009).</li><li>Cavalier, D. 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H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+irregular+xylem9+mutant+is+deficient+in+xylan+xylosyltransferase+activity.">The irregular xylem9 mutant is deficient in xylan xylosyltransferase activity.</a> Plant and Cell Physiology. 48 (11), 1624-1624 (2007).</li><li>Iglesias, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Apoplastic+glycosidases+active+against+xyloglucan+oligosaccharides+of+Arabidopsis+thaliana.">Apoplastic glycosidases active against xyloglucan oligosaccharides of Arabidopsis thaliana.</a> Plant and Cell Physiology. 47 (1), 55-55 (2006).</li><li>Fujino, T., Sone, Y., Mitsuishi, Y., Itoh, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Characterization+of+cross-links+between+cellulose+microfibrils,+and+their+occurrence+during+elongation+growth+in+pea+epicotyl.">Characterization of cross-links between cellulose microfibrils, and their occurrence during elongation growth in pea epicotyl.</a> Plant Cell Physiol. 41 (4), 486-486 (2000).</li><li>Vanzin, G. 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M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Disrupting+two+Arabidopsis+thaliana+xylosyltransferase+genes+results+in+plants+deficient+in+xyloglucan,+a+major+primary+cell+wall+component.">Disrupting two Arabidopsis thaliana xylosyltransferase genes results in plants deficient in xyloglucan, a major primary cell wall component.</a> Plant Cell. 20 (6), 1519-1519 (2008).</li><li>Hilz, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+comparison+of+liquid+chromatography,+capillary+electrophoresis,+and+mass+spectrometry+methods+to+determine+xyloglucan+structures+in+black+currants.">A comparison of liquid chromatography, capillary electrophoresis, and mass spectrometry methods to determine xyloglucan structures in black currants.</a> Journal of Chromatography A. 1133 (1-2), 275-275 (2006).</li><li>Pauly, M. <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+the+mur1+mutation+on+xyloglucans+produced+by+suspension-cultured+Arabidopsis+thaliana+cells.">Effects of the mur1 mutation on xyloglucans produced by suspension-cultured Arabidopsis thaliana cells.</a> Planta. 214 (1), 67-67 (2001).</li></ol>

Burada sunulan Olimp yöntemi ekstrasellüler matrisler bulunan polimerlerin çok hassas ve hızlı analiz sağlar. Olimp enzimatik sonraki MALDI-TOF analizi ile oligomerler sürümü birleştirir. MALDI-TOF yelpazenin nesil bir dakikadan az sürer, dolayısıyla Olimp mutant ekranlar gibi yüksek verimli çalışmaları da dahil olmak üzere geniş bir uygulama yelpazesi için uygundur. Olimp bitki polisakkaritler ile sınırlı değildir ancak potansiyel olarak sadece özel hidrolitik enzimlerin durumu ile sınırlı polimerler, geniş bir yelpazede uygulanabilir. Ancak, Olimp bir sınırlama polimer mutlak bir bereket elde edilemez. Olimp çeşitli türlerin çeşitliliği ekstraselüler matriks polisakkaritlerin yapısını incelemek için, kullanılmadan önce de belirtildiği gibi. Bir örnek olarak, Şekil 4, ot türleri başlıca hemiselüloz, Xylan bir Olimp spektrum temsil eder. Burada, hücre duvar malzemesi ılıman çim Miscanthus türetilmiş bir xylanase kullanılarak sindirilir.

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		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
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<tr>
<td>2,5-dihydroxybenzoic acid</td>
<td>&nbsp;</td>
<td>Sigma-Aldrich</td>
<td>37550</td>
<td>10mg/mL in water</td>
</tr>
<tr>
<td>BioRex MSZ 501(D) Resin</td>
<td>&nbsp;</td>
<td>BioRad</td>
<td>142-7425</td>
<td>&nbsp;</td>
<tr>
<td>Endoglucanase</td>
<td>&nbsp;</td>
<td>Megazyme</td>
<td>E-CELTR</td>
<td>&nbsp;</td>
<tr>
<td>Xylanase M6</td>
<td>&nbsp;</td>
<td>Megazyme</td>
<td>E-XYRU6</td>
<td>&nbsp;</td>
<tr>
<td>3mm metal balls</td>
<td>&nbsp;</td>
<td>Retsch</td>
<td>22.455.0011</td>
<td>&nbsp;</td>
<tr>
<td>Beat mill</td>
<td>&nbsp;</td>
<td>Retsch</td>
<td>Mixer Mill MM400</td>
<td>&nbsp;</td>
<tr>
<td>MALDI-TOF</td>
<td>&nbsp;</td>
<td>Shimadzu BioTech</td>
<td>Axima Performance</td>
<td>&nbsp;</td>
<tr>
<td>MALDI target plate</td>
<td>&nbsp;</td>
<td>Kratos Analytical</td>
<td>DE4555TA</td>
<td>&nbsp;</td>
<tr>
<td>SpeedVac</td>
<td>&nbsp;</td>
<td>Eppendorf</td>
<td>Vacufuge 5301</td>
<td>&nbsp;</td>
<tr>
<td>Vacuum manifold</td>
<td>&nbsp;</td>
<td>Millipore</td>
<td>MSVMHTS00</td>
<td>&nbsp;</td>
<tr>
<td>Vacuum pump</td>
<td>&nbsp;</td>
<td>Welch</td>
<td>DryFast Ultra 2032</td>
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oligo mass profiling (olimp) of extracellular polysaccharides

Çevre hücreleri doğrudan temas ekstraselüler matriks tarafından birçok organizmalarda aracılık eder. Ekstrasellüler matrisler bir ortak yönü glikoproteinlerin şeklinde kompleks şeker moieties proteoglikanlar ve / veya polisakkaritler içerir. Örnekler, fibriler protein ve proteoglikanlar veya bitki ve mantar hücre duvarları polisakkarit başlıca oluşan insan ve hayvan hücreleri ekstrasellüler matriks ve proteoglikan / bakteri hücre duvarlarının glikolipid içerir. Tüm bu glycostructures hücre-hücre ve hücre-çevre haberleşme ve sinyalizasyon hayati rol oynamaktadır. Ekstraselüler matriks bir olağanüstü karmaşık bir örnek yüksek bitki hücrelerinin duvarları mevcut. Onların duvar şekerlerin kuru ağırlığının% 75 kadar, neredeyse tamamen, ve bu gezegen üzerindeki en bol biyopolimerlerin oluşur. Bu nedenle, araştırma, fosil yakıt elde edilen petrokimya yerine karbon-nötr bir yenilenebilir kaynak olarak en iyi bu malzemelerin nasıl kullanılacağını yapılır. Yakıt dönüşümü için temel zorluk, bu eşsiz biokompozit benzersiz glycostructures nedeniyle enzimatik veya kimyasal bozunmaya duvarların dinlemezlik kalır. Burada, bitki hücre duvarının glycostructures hızlı ve hassas bir analiz için bir yöntem mevcut. Bu yöntem, oligo Kitle Profil (Olimp) (MALDI-TOF/MS matriks yardımlı lazer desorpsiyon / iyonizasyon zaman-uçuş-kütle spektrometresi kullanarak özel glycosylhydrolases ve çözündüğünü oligosakkarit karışımları sonraki analizi kolaylaştırmak duvar malzemeleri oligosakkaritler enzimatik serbest dayanır 1) (Şekil 1). Olimp, tam bir analiz için sadece 5000 hücrelerinin duvarları, doku kendisini 2 yapılabilir gerektirir ve yüksek verimlilik analizleri 3 için uygun . Mutlak miktarda çözünür oligosakkaritler Olimp tarafından belirlenir mümkün olmasa bile, çeşitli oligosakkarit iyonların göreceli bolluk, duvarda bulunan yerli bir polisakkarit ikame model hakkında anlayışlar veren kitle spektrumu tarif edilebilir. Olimp sadece belirli enzimler 4 durumu ile sınırlı duvar polimerlerin geniş bir yelpazede, analiz için kullanılabilir. Örneğin, bitki hücre duvarı enzimler bulunan polimerlerin analizi için bir endo-β-(1-4)-xylanase 6,7 kullanarak xyloglucanase 5, 11, 12, 13, Xylan kullanarak hemiselülozlar xyloglucan analiz etmek için kullanılabilir. veya polygalacturonase ve methylesterase 8 bir arada kullanarak pektik polisakkaritler. Ayrıca, aynı ilkeler Olimp glycosylhydrolase ve hatta glycosyltransferase faaliyetleri izlenir ve 9 tespit edilebilir.

Watch this Scientific Journal Video about OLIgo Mass Profiling OLIMP of Extracellular Polysaccharides at JoVE.com

OLIgo Mass Profiling OLIMP of Extracellular Polysaccharides

Bir ekstrasellüler matriks polisakkaritlerin yapısına anlayışlar kazanmak için hızlı bir şekilde açıklanmıştır. Yöntemi dakika miktarda malzeme analiz edilmesi için izin kütle spektrometresi glycosylhydrolases özgüllüğü ve duyarlılığı yararlanır. Bu teknikte doku kendisini doğrudan kullanılmak üzere uyarlanabilir.

Ekstrasellüler polisakkaritler Oligo Kitle Profil (Olimp)

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is ...

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Research

JoVE Journal

Biology

OLIgo Mass Profiling (OLIMP) of Extracellular Polysaccharides

13.6K Görüntüleme.  University of California, Berkeley. Bir ekstrasellüler matriks polisakkaritlerin yapısına anlayışlar kazanmak için hızlı bir şekilde açıklanmıştır. Yöntemi dakika miktarda malzeme analiz edilmesi için izin kütle spektrometresi glycosylhydrolases özgüllüğü ve duyarlılığı yararlanır. Bu teknikte doku kendisini doğrudan kullanılmak üzere uyarlanabilir.

Video: OLIgo Mass Profiling OLIMP of Extracellular Polysaccharides

MALDI Sample Preparation: the Ultra Thin Layer Method

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) 1,2. The ultra-thin layer method involves the production of a substrate layer of matrix crystals (alpha-cyano-4-hydroxycinnamic acid) on the sample plate, which serves as a seeding ground for subsequent crystallization of a matrix/analyte mixture. Advantages of the ultra-thin layer method over other sample deposition approaches (e.g. dried droplet) are that it provides (i) greater tolerance to impurities such as salts and detergents, (ii) better resolution, and (iii) higher spatial uniformity. This method is especially useful for the accurate mass determination of proteins. The protocol was initially developed and optimized for the analysis of membrane proteins and used to successfully analyze ion channels, metabolite transporters, and receptors, containing between 2 and 12 transmembrane domains 2. Since the original publication, it has also shown to be equally useful for the analysis of soluble proteins. Indeed, we have used it for a large number of proteins having a wide range of properties, including those with molecular masses as high as 380 kDa 3. It is currently our method of choice for the molecular mass analysis of all proteins. The described procedure consistently produces high-quality spectra, and it is sensitive, robust, and easy to implement.

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS).

MALDI Numune Hazırlama: Ultra İnce Tabaka Yöntemi

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption ...

Biyoloji

Antibody Profiling by Luciferase Immunoprecipitation Systems (LIPS)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease mechanisms and may elucidate new markers to substratify disease with different clinical features and better understand pathogenesis. We have developed a highly quantitative method called Luciferase Immunoprecipitation Systems (LIPS) for profiling patient sera antibody responses to autoantigens and pathogen antigens associated with infection. Unlike ELISAs, the highly sensitive LIPS is easily implemented to survey humoral serological response profiles to different antigens in a universal format and produces dynamic antibody titer ranges up to 6 log10 for some antigens. In these studies, quantitative profiling by LIPS of patient humoral responses against panels of antigens or even the entire proteome of some pathogens (i.e. HIV), is typically more informative than testing a single antigen by ELISA. In addition, LIPS also eliminates time and effort needed to produce highly purified antigens as well as the labor-intensive assay optimization steps needed for standard ELISAs. Here we provide a detailed protocol describing the technical aspects of performing LIPS assays for readily profiling antibody responses to single or multiple antigens.

Antibody Profiling by Luciferase Immunoprecipitation Systems LIPS

The technical aspects of performing LIPS (Luciferase Immunoprecipitation Systems) are described. The overall approach involves expressing chimeric genes encoding antigens fused to Renilla luciferase (Ruc) in mammalian cells. Crude Ruc-antigen extracts are then prepared and, without purification, employed in immunoprecipitation assays to quantify antibodies.

Antikor Profil Lusiferaz Immunoprecipitation Sistemleri (LIPS)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease ...

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (CCMS)

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity chromatography 1 or Activity Based Protein Profiling 2. Trifunctional Capture Compounds (CCs, Figure 1A) 3 are the basis for a generic approach, in which the initial equilibrium-driven interaction between a small molecule probe (the selectivity function, here S-adenosyl-L-homocysteine, SAH, Figure 1A) and target proteins is irreversibly fixed upon photo-crosslinking between an independent photo-activable reactivity function (here a phenylazide) of the CC and the surface of the target proteins. The sorting function (here biotin) serves to isolate the CC - protein conjugates from complex biological mixtures with the help of a solid phase (here streptavidin magnetic beads). Two configurations of the experiments are possible: "off-bead" 4 or the presently described "on-bead" configuration (Figure 1B). The selectivity function may be virtually any small molecule of interest (substrates, inhibitors, drug molecules).
S-Adenosyl-L-methionine (SAM, Figure 1A) is probably, second to ATP, the most widely used cofactor in nature 5, 6. It is used as the major methyl group donor in all living organisms with the chemical reaction being catalyzed by SAM-dependent methyltransferases (MTases), which methylate DNA 7, RNA 8, proteins 9, or small molecules 10. Given the crucial role of methylation reactions in diverse physiological scenarios (gene regulation, epigenetics, metabolism), the profiling of MTases can be expected to become of similar importance in functional proteomics as the profiling of kinases. Analytical tools for their profiling, however, have not been available. We recently introduced a CC with SAH as selectivity group to fill this technological gap (Figure 1A).
SAH, the product of SAM after methyl transfer, is a known general MTase product inhibitor 11. For this reason and because the natural cofactor SAM is used by further enzymes transferring other parts of the cofactor or initiating radical reactions as well as because of its chemical instability 12, SAH is an ideal selectivity function for a CC to target MTases. Here, we report the utility of the SAH-CC and CCMS by profiling MTases and other SAH-binding proteins from the strain DH5&alpha; of Escherichia coli (E. coli), one of the best-characterized prokaryotes, which has served as the preferred model organism in countless biochemical, biological, and biotechnological studies. Photo-activated crosslinking enhances yield and sensitivity of the experiment, and the specificity can be readily tested for in competition experiments using an excess of free SAH.

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry CCMS

Capture Compounds are trifunctional small molecules to reduce the complexity of the proteome by functional reversible small molecule-protein interaction followed by photo-crosslinking and purification. Here we use a Capture Compound with S-adenosyl-L-homocysteine-binding as selectivity function to isolate methyltransferases from an Escherichia coli whole cell lysate and identify them by MS.

Methyltransferases, profilleme ve Diğer S Adenosil- L</subYakalama Bileşik Kütle Spektrometresi (CCMS)> homosistein bağlayıcı Proteinler

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity ...

Single Cell Transcriptional Profiling of Adult Mouse Cardiomyocytes

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation between cells within a tissue. Isolation of pure cardiomyocyte populations through a collagenase perfusion of mouse hearts facilitates the generation of single cell microarrays for whole transcriptome gene expression, or qPCR of specific targets using nanofluidic arrays. We describe here a procedure to examine single cell gene expression profiles of cardiomyocytes isolated from the heart. This paradigm allows for the evaluation of metrics of interest which are not reliant on the mean (for example variance between cells within a tissue) which is not possible when using conventional whole tissue workflows for the evaluation of gene expression (Figure 1). We have achieved robust amplification of the single cell transcriptome yielding micrograms of double stranded cDNA that facilitates the use of microarrays on individual cells. In the procedure we describe the use of NimbleGen arrays which were selected for their ease of use and ability to customize their design. Alternatively, a reverse transcriptase - specific target amplification (RT-STA) reaction, allows for qPCR of hundreds of targets by nanofluidic PCR. Using either of these approaches, it is possible to examine the variability of expression between cells, as well as examining expression profiles of rare cell types from within a tissue. Overall, the single cell gene expression approach allows for the generation of data that can potentially identify idiosyncratic expression profiles that are typically averaged out when examining expression of millions of cells from typical homogenates generated from whole tissues.

Single cell expression profiling allows the detailed gene expression analysis of individual cells. We describe methods for the isolation of cardiomyocytes, and preparing the resulting lysates for either whole transcriptome microarray or qPCR of specific targets.

Yetişkin Fare kardiyomiyositlerde Tek Hücre transkripsiyonel Profilleme

While numerous studies have examined gene expression changes from homogenates of heart tissue, this prevents studying the inherent stochastic variation ...

Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on Arabidopsis thaliana, a genetically tractable host plant1,2. The accumulation of reactive oxygen species (ROS) in cotyledons inoculated with DC3000 indicates a role of ROS in modulating necrotic cell death during bacterial speck disease of tomato3. Hydrogen peroxide, a component of ROS, is produced after inoculation of tomato plants with Pseudomonas3. Hydrogen peroxide can be detected using a histochemical stain 3'-3' diaminobenzidine (DAB)4. DAB staining reacts with hydrogen peroxide to produce a brown stain on the leaf tissue4. ROS has a regulatory role of the cellular redox environment, which can change the redox status of certain proteins5. Cysteine is an important amino acid sensitive to redox changes. Under mild oxidation, reversible oxidation of cysteine sulfhydryl groups serves as redox sensors and signal transducers that regulate a variety of physiological processes6,7. Tandem mass tag (TMT) reagents enable concurrent identification and multiplexed quantitation of proteins in different samples using tandem mass spectrometry8,9. The cysteine-reactive TMT (cysTMT) reagents enable selective labeling and relative quantitation of cysteine-containing peptides from up to six biological samples. Each isobaric cysTMT tag has the same nominal parent mass and is composed of a sulfhydryl-reactive group, a MS-neutral spacer arm and an MS/MS reporter10. After labeling, the samples were subject to protease digestion. The cysteine-labeled peptides were enriched using a resin containing anti-TMT antibody. During MS/MS analysis, a series of reporter ions (i.e., 126-131 Da) emerge in the low mass region, providing information on relative quantitation. The workflow is effective for reducing sample complexity, improving dynamic range and studying cysteine modifications. Here we present redox proteomic analysis of the Pst DC3000 treated tomato (Rio Grande) leaves using cysTMT technology. This high-throughput method has the potential to be applied to studying other redox-regulated physiological processes.

Reactive oxygen species level is elevated when cells encounter stress conditions. Here we show the example of 3'-3' diaminobenzidine staining as well as cysTMT labeling and mass spectrometry to profile the redox proteome in Pseudomonas syringae treated tomato leaves.

İzotop Etiketleme kütle spektrometresi kullanarak Tiyol Redoks Proteom profil

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on ...

Glycan Profiling of Plant Cell Wall Polymers using Microarrays

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the raw materials for human societies (e.g. wood, paper, textile and biofuel industries)1,2. However, understanding the biosynthesis and function of these components remains challenging.
Cell wall glycans are chemically and conformationally diverse due to the complexity of their building blocks, the glycosyl residues. These form linkages at multiple positions and differ in ring structure, isomeric or anomeric configuration, and in addition, are substituted with an array of non-sugar residues. Glycan composition varies in different cell and/or tissue types or even sub-domains of a single cell wall3. Furthermore, their composition is also modified during development1, or in response to environmental cues4.
In excess of 2,000 genes have Plant cell walls are complex matrixes of heterogeneous glycans been predicted to be involved in cell wall glycan biosynthesis and modification in Arabidopsis5. However, relatively few of the biosynthetic genes have been functionally characterized 4,5. Reverse genetics approaches are difficult because the genes are often differentially expressed, often at low levels, between cell types6. Also, mutant studies are often hindered by gene redundancy or compensatory mechanisms to ensure appropriate cell wall function is maintained7. Thus novel approaches are needed to rapidly characterise the diverse range of glycan structures and to facilitate functional genomics approaches to understanding cell wall biosynthesis and modification.
Monoclonal antibodies (mAbs)8,9 have emerged as an important tool for determining glycan structure and distribution in plants. These recognise distinct epitopes present within major classes of plant cell wall glycans, including pectins, xyloglucans, xylans, mannans, glucans and arabinogalactans. Recently their use has been extended to large-scale screening experiments to determine the relative abundance of glycans in a broad range of plant and tissue types simultaneously9,10,11.
Here we present a microarray-based glycan screening method called Comprehensive Microarray Polymer Profiling (CoMPP) (Figures 1 &amp; 2)10,11 that enables multiple samples (100 sec) to be screened using a miniaturised microarray platform with reduced reagent and sample volumes. The spot signals on the microarray can be formally quantified to give semi-quantitative data about glycan epitope occurrence. This approach is well suited to tracking glycan changes in complex biological systems12 and providing a global overview of cell wall composition particularly when prior knowledge of this is unavailable.

A technique called Comprehensive Microarray Polymer Profiling (CoMPP) for the characterisation of plant cell wall glycans is described. This method combines the specificity of monoclonal antibodies directed to defined glycan-epitopes with a miniature microarray analytical platform allowing screening of glycan occurrence in a broad range of biological contexts.

Mikroarray&#39;ler kullanarak Bitki Hücre Duvarı Polimerlerin glukanıdır Profilleme

Plant cell walls are complex matrixes of heterogeneous glycans which play an important role in the physiology and development of plants and provide the ...

Analysis of Translation Initiation During Stress Conditions by Polysome Profiling

Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. Alterations of this program can lead to the growth of damaged cells, a hallmark of cancer development, or to premature cell death such as seen in neurodegenerative diseases. Much of what is known concerning the molecular basis for translational control has been obtained from polysome analysis using a density gradient fractionation system. This technique relies on ultracentrifugation of cytoplasmic extracts on a linear sucrose gradient. Once the spin is completed, the system allows fractionation and quantification of centrifuged zones corresponding to different translating ribosomes populations, thus resulting in a polysome profile. Changes in the polysome profile are indicative of changes or defects in translation initiation that occur in response to various types of stress. This technique also allows to assess the role of specific proteins on translation initiation, and to measure translational activity of specific mRNAs. Here we describe our protocol to perform polysome profiles in order to assess translation initiation of eukaryotic cells and tissues under either normal or stress growth conditions.

Here, we describe a method to analyze changes in the initiation of mRNA translation of eukaryotic cells in response to stress conditions. This method is based on the velocity separation on sucrose gradients of translating ribosomes from non-translating ribosomes.

Polizom profilleme Stres Durumlardaki Çeviri başlatılması Analizi

Precise control of mRNA translation is fundamental for eukaryotic cell homeostasis, particularly in response to physiological and pathological stress. ...

Profiling Individual Human Embryonic Stem Cells by Quantitative RT-PCR

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different lineages. A single cell transcriptome assay can be a new approach for dissecting individual variation. We have developed the single cell qRT-PCR method, and confirmed that this method works well in several gene expression profiles. In single cell level, each human embryonic stem cell, sorted by OCT4::EGFP positive cells, has high expression in OCT4, but a different level of NANOG expression. Our single cell gene expression assay should be useful to interrogate population heterogeneities.

Single cell gene expression assay is needed for understanding stem cell heterogeneities.

Kantitatif RT-PCR ile Bireysel İnsan Embriyonik Kök Hücreleri profilleme

Heterogeneity of stem cell population hampers detailed understanding of stem cell biology, such as their differentiation propensity toward different ...

Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of cell proliferation, survival, migration, etc. The ECM plays important roles in development and in diverse pathologies including cardio-vascular and musculo-skeletal diseases, fibrosis, and cancer. Thus, characterizing the composition of ECMs of normal and diseased tissues could lead to the identification of novel prognostic and diagnostic biomarkers and potential novel therapeutic targets. However, the very nature of ECM proteins (large in size, cross-linked and covalently bound, heavily glycosylated) has rendered biochemical analyses of ECMs challenging. To overcome this challenge, we developed a method to enrich ECMs from fresh or frozen tissues and tumors that takes advantage of the insolubility of ECM proteins. We describe here in detail the decellularization procedure that consists of sequential incubations in buffers of different pH and salt and detergent concentrations and that results in 1) the extraction of intracellular (cytosolic, nuclear, membrane and cytoskeletal) proteins and 2) the enrichment of ECM proteins. We then describe how to deglycosylate and digest ECM-enriched protein preparations into peptides for subsequent analysis by mass spectrometry.

This protocol describes a procedure for enriching ECM proteins from tissues or tumors and deglycosylating and digesting the ECM-enriched preparations into peptides to analyze their protein composition by mass spectrometry.

Kütle Spektrometresi Analizi için Peptitler içine Dokular ve Sindirim gelen Ekstrasellüler Matriks Proteinleri zenginleştirilmesi

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of ...

Cellular Redox Profiling Using High-content Microscopy

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, excessive ROS levels induce a state of oxidative stress, which is accompanied by irreversible oxidative damage to DNA, lipids and proteins. Thus, quantification of ROS provides a direct proxy for cellular health condition. Since mitochondria are among the major cellular sources and targets of ROS, joint analysis of mitochondrial function and ROS production in the same cells is crucial for better understanding the interconnection in pathophysiological conditions. Therefore, a high-content microscopy-based strategy was developed for simultaneous quantification of intracellular ROS levels, mitochondrial membrane potential (&#916;&#936;m) and mitochondrial morphology. It is based on automated widefield fluorescence microscopy and image analysis of living adherent cells, grown in multi-well plates, and stained with the cell-permeable fluorescent reporter molecules CM-H2DCFDA (ROS) and TMRM (&#916;&#936;m and mitochondrial morphology). In contrast with fluorimetry or flow-cytometry, this strategy allows quantification of subcellular parameters at the level of the individual cell with high spatiotemporal resolution, both before and after experimental stimulation. Importantly, the image-based nature of the method allows extracting morphological parameters in addition to signal intensities. The combined feature set is used for explorative and statistical multivariate data analysis to detect differences between subpopulations, cell types and/or treatments. Here, a detailed description of the assay is provided, along with an example experiment that proves its potential for unambiguous discrimination between cellular states after chemical perturbation.

This paper presents a high-content microscopy workflow for simultaneous quantification of intracellular ROS levels, as well as mitochondrial membrane potential and morphology &#8211; jointly referred to as mitochondrial morphofunction &#8211; in living adherent cells using the cell-permeant fluorescent reporter molecules 5-(and-6)-chloromethyl-2&#39;,7&#39;-dichlorodihydrofluorescein diacetate, acetyl ester (CM-H2DCFDA) and tetramethylrhodamine methylester (TMRM).

Yüksek İçerikli Mikroskopi Kullanılarak Hücresel Redoks Profillemesi

Reactive oxygen species (ROS) regulate essential cellular processes including gene expression, migration, differentiation and proliferation. However, ...