Bu çalışma Kanser girişimi, 5U24CA126477-04 Klinik Proteomik Technologies tarafından desteklenmiştir.

1) lektin-konjuge Poros sütun hazırlayın <ol><li> Poros-AL boncuk adımları 1.1 - 1.2 sırasında inhalasyon karşı korumak için bir maske koyun. Poros boncuk istenilen miktarda (100 mg beads/300 ul son hacim) tartılır ve temiz bir Eppendorf tüp içine aktarmak. </li><li> 1 ml fosfat tamponlu salin (PBS) eklenmesi ile boncuk yıkayın. Pelet boncuk, 3 dakika boyunca maksimum hızda bir mikrosantrifüj santrifüj. Süpernatantı ve yıkama tekrarlayın. </li><li> Indirekt lektin istenen miktarda (1 - 4 mg / 200 ul boncuk) tartılır ve temiz bir Eppendorf tüp transfer. 5 formu için PBS - 20 mg / ml. Bu çözüm Rezerv 25 ul. </li><li> Poros boncuk kalan lektin çözüm aktarın. Nihai konsantrasyonu 50 mM sodyum cyanoborohydride ekleyin. Bir rocker tüp yerleştirin ve oda sıcaklığında bir gece tepki. (Not: sodyum cyanoborohydride toksik ve davlumbaz ele alınması gereken Kontamine atıklar uygun şekilde bertaraf edilmelidir.) </li><li> Pelet Poros boncuk adım 1.2 &#39;deki gibi. Süpernatantı ve sonrası konjugasyon çözüm olarak kaydetmek. </li><li> Boncuk 1 ml Quenching Buffer (1 M Tris-Cl, pH 7.4) ile yıkayın. Adım 1.2 ve güvenli bir şekilde Pelet boncuk supernatant atın. </li><li> 1 ml Quenching Tampon Poros boncuk kalan reaktif siteleri engelleme. Nihai konsantrasyonu 50 mM sodyum cyanoborohydride ekleyin. Bir rocker tüp yerleştirin ve oda sıcaklığında 30 dakika inkübe edin. </li><li> Pelet adım 1.2 gibi boncuk ve supernatant atın. </li><li> Boncuk, 1 mL 1 M NaCl ile yıkayın. Pelet boncuk ve supernatant atın. Beş yıkama olmak üzere toplam dört kez tekrarlayın. Boncuk paketi hazır. </li><li> Poros boncuk konjuge protein miktarını belirlemek için, bir protein konsantrasyonu tahlil öncesi konjuge ve post-konjuge lektin çözümleri uygulayın. Konsantrasyonlarda fark boncuklar konjuge protein miktarı. Boncuk hacim başına konjuge protein miktarı boncuklar lektin konsantrasyonu eşittir. Hedef lektin konsantrasyonları 2 -20 mg / ml arasında. </li></ol> 2) bir PEEK sütuna lektin-konjuge Poros boncuk Paketleme <ol><li> (Açıklaması) paketleme sistemi kurma ve bir metal halka standı destek. Paketleme sistemi, alttan üst basınç kısıtlayıcı, sonunda sütun elemanı 1, frit, kolon (2 x 50 mm), son sütun coupler 2, sütun konektörü, son sütun elemanı 3, kolon (4.5 x 50 mm), son sütun coupler 4 ve son montaj. Üst kolon ambalaj materyalleri için bir rezervuar olarak hizmet vermektedir. </li><li> Tampon istenilen hacmi Poros boncuk yeniden süspanse (10 mM Tris tamponu, pH 7.4, 150 mM sodyum klorür, 10 mM kalsiyum klorür, 10 mM magnezyum klorür). Eğer ambalaj 400 ul Tampon A. tekrar süspansiyon bir sütun (~ 200 ul yatak hacmi) </li><li> Üst kolon (rezervuar) konjuge Poros boncuk aktarın. Tampon sütunun üst ulaşıncaya kadar rezervuar Bir Bellek ekleyin. Yavaşça, sütun hava kabarcıklarını önlemek için çalışıyoruz, sütun üstüne son montaj yerleştirin. HPLC sistemi üst sütunun sonuna uydurma bağlayın. </li><li> Tampon sistem üzerinden akan sütun paketleyin. 0.5 ml / dak akış hızı ile başlayın. 4 mL / dak veya maksimum basınç (3000 psi) ya ulaşıncaya kadar akış hızı 0.5 ml / dk her dakika artırın. Mümkün olan en yüksek akış hızında Buffer en az 35 ml A sütunu sayesinde geçene kadar devam edin. </li><li> Pompaların kapatın ve &lt;20 psi düşmesi sütun üzerindeki baskıyı izin. </li><li> Yavaşça, üstten başlayarak, paketleme sistemi sökmeye. Son sütun coupler 2 ulaşıldığında, dikkatli bir şekilde kaldırın. Bazı paketlenmiş malzeme sütun üst ekstrüzyon olmalıdır. Bir jilet veya benzer keskin kenar hafifçe sütun üstüne bile bir boncuk yüzeyinde bırakarak, ekstrüzyon boncuk silerek temizleyin. Bunu yaparken boncuklar basınç uygulama yapmayınız. </li><li> Halka standından dolgulu kolon ayırın. Yeni bir bitiş bağlaştırıcısına yeni bir frit yerleştirin ve açık (eski üst) sütun ucunda frit / bitiş coupler ile bağlamak için dolgulu kolon ters çevirin. </li><li> Sütunu uygun şekilde etiketleyin. Artık kullanıma hazırdır. 6 aya kadar, sütun 4% 0.02 sodyum azid ° C ile PBS içinde saklanabilir olabilir. </li></ol> 3) Program HPLC yöntemi <ol><li> HPLC programlama ayrıntıları üretici yazılım özelliklerine göre değişecektir. Biz bir Michrom Paradigma MG4 HPLC kullanın. Bu makine üzerinde, ekranın üst kısmında yöntemleri inşa ve &quot;Setup&quot; sekmesi altında erişilebilir. Programı aşağıdaki yöntemi: <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> Zaman </td><td> Akış hızı (ul / dak) </td><td> % A </td><td> % B </td></tr><tr><td> 00:00 </td><td> 50 </td><td> 100 </td><td> 0 </td></tr><tr><td> 09:00 </td><td> 50 </td><td> 100 </td><td> 0 </td></tr><tr><td> 09:01 </td><td> 500 </td><td> 0 </td><td> 100 </td></tr><tr><td> 13:50 </td><td> 500 </td><td> 0 </td><td> 100 </td></tr><tr><td> 13:51 </td><td> 3000 </td><td> 100 </td><td> 0 </td></tr><tr><td> 19:50 </td><td> 3000 </td><td> 100 </td><td> 0 </td></tr></tbody></table> 10 mM Tris tamponu, pH 7.4, 150 mM sodyum klorür, 10 mM kalsiyum klorür, 10 mM magnezyum klorür: arabelleğe Tampon B: 0,5 M asetik asit 280 nm UV detektör 0:00 ile 19:50 okumak için programlanabilir olmalıdır. - 50 AU y ekseni düşük absorbans seviyeleri, örneğin, 0 algılamak için ayarlanabilir olmalıdır. </li><li> Otomatik örnekleyici, program fraksiyon toplanması için aşağıdaki programa ise. Aksi takdirde, kromatografi performans zaman belirtildiği gibi elle kesirleri toplamak. <table border="1" cellspacing="0" cellpadding="0"><tbody><tr><td> Kesir </td><td> Numune enjeksiyon toplama gecikme </td><td> Toplama süresi (dakika) </td></tr><tr><td> Flow-through </td><td> 2,8 </td><td> 4,1 </td></tr><tr><td> Bağlı </td><td> 9 </td><td> 2,6 </td></tr></tbody></table></li></ol> 4) HPLC için örnek hazırlayın <ol><li> Bu protokolü kullanmak için önce, insan plazma MARS sütun (Santa Clara, CA Agilent) kullanarak 14 en bol proteinler tükenmiş olmalıdır. Tüketilmiş plazma, insan laktoferrin ve maya invertaz ayrı ayrı tripsin sindirilir ve önceden 1&#39;de açıklandığı gibi, desalted olmalıdır . </li><li> Lektin özel laktoferrin standart hazırlamak için, Bölüm 3 yöntemi kullanarak AAL veya SNA sütun üzerinde 50 ug tripsin-sindirilmiş laktoferrin kromatografi gerçekleştirin. Bağlı fraksiyonu lektin özel laktoferrin glikopeptid içerecektir. Bölüm 6 Kısım 4.4 &#39;te gibi örnekleri ve desalt nötralize eder. Çıkan örnek kullanıma hazırdır, 1 ml Tampon A. örnek tekrar </li><li> Üç deneysel yapılacaktır çoğaltır. Üç numune hazırlamak için çoğaltır, 30 ul MARS-tükenmiş, tripsin-sindirilmiş, plazma benzerleri (PE), tripsin sindirilir invertaz 3 pmol ve lektin özel laktoferrin (ya AAL-LAC veya SNA-LAC) 1 ul ekleyin. PE işlenmiş plazma (MARS yetersizliği ve tripsin sindirilir) belirli bir miktar elde edildiği non-processed/intact/original plazma hacmi olarak tanımlanır. 330 ul son bir hacme ulaşmak için örnek bir Tampon ekleyin. </li><li> Eluat toplayacak numune şişeleri, nötralizasyon tampon (1 M Tris pH: 8.0) ekleyin. Sizinle birlikte çalışmaktan şişeleri sayısı ve hacmi, sizin otosampler kısıtlamaları bağlıdır. MG4 Paradigma için, bir flakon flow-through ve iki eluat toplamak toplar. Yaklaşık 1.5 ml Tris tampon eluat 1 mL nötralize gerekecektir. Hedef nötralize pH 7,0-8,0; pH indikatör kağıdı ile test test nihai pH. </li></ol> 5) kromatografisi gerçekleştirin <ol><li> Izleyen tüm boş ve örnek çalışır Bölüm 1&#39;de anlatılan yöntemi kullanın. Sütun ve tamponlar, kullanım sırasında oda sıcaklığında. Tamponlar oda sıcaklığında saklandığında ise sütunlar, 4 ° C&#39;de saklanır. </li><li> HPLC sistemi ya AAL veya SNA lektin sütununda takın ve iki boş yöntemleri (sadece Tampon enjekte A) çalıştırın. Lektin sütun lektin özel laktoferrin (Bölüm 2) uygun olduğundan emin olun. </li><li> Toplam 7 HPLC çalışır, her enjekte örnek arasında boş bir enjekte üç plazma numuneleri kromatograflanır. Boş çalışır Kesirler toplanmasına gerek yoktur. </li></ol> 6) Desalt toplanan fraksiyonları Her bir kısmı aşağıdaki gibi bir 1 ml Waters Oasis HLB SPE kartuş kullanmak için: <ol><li> Vakum manifoldunun için gerekli kartuşları takın. </li><li> Her bir kartuş% 1 formik asit 1 ml% 80 ACN (20 lnHg manifoldu vakum göstergesi 5 okumalısınız) ile ıslatın. </li><li> 1.5 ml 0.1% Formik asit (20 lnHg manifoldu vakum göstergesi 5 okumalısınız) ile kartuşları dengelenmesi </li><li> Bir kartuş (- 2,5 lnHg ve akış hızı 1 ml / dk geçmemelidir manifoldu üzerinde Vakum göstergesi 2 okumalısınız) üzerine bir örnek tüm hacim yükleyin </li><li> 3 x 1 ml% 0.1 formik asid (20 lnHg manifoldu vakum göstergesi 5 okumalısınız) ile kartuşları yıkayın </li><li> 1 x 1 ml% 0.1 formik asit% 80 asetonitril içine Zehir peptidler / glikopeptid Eppendorf tüpleri etiketli. Tek bir toplama tüpü her kartuş için kullanılmalıdır (2.5 lnHg, ve akış hızı 1 ml / dk geçmemelidir manifoldunun üzerine Vakum göstergesi 2 okumalısınız.) </li><li> Her bir toplama tüpüne 60 ul 0.5 M amonyum bikarbonat ekleyerek eluat nötralize. Hedef nötralized pH 7,0-8,0; pH indikatör kağıdı ile test nihai pH. </li><li> ~ 2 saat 35 ° C (örneğin, Thermo Savant SpeedVac) bir vakum santrifüj çalışan tarafından yıkandı, peptidler / ~ 50 mcL glikopeptid Konsantre </li></ol> 7) PNGase F-sindirim. <ol><li> Emin olmak için test örneği pH 7,0 - 8,0 arasındadır. Gerekirse, 50 mM pH artırmak için amonyum bikarbonat ekleyin. Ul 50-100 arasında olduğu kadar son örnek hacmi&gt; 100 ul, speedvac örneğidir. </li><li> 37 ° C gecede her bir numune tüpü ve inkübe ul 0.5 (250 U) gliserol-PNGase F ekleyin. </li></ol> 8) Zip İpucu örnekleri <ol><li> PNGase F-sindirilmiş örnekleri ile başlayarak, aşağıdaki gibi her biri ziptip. </li><li> 10 ul 100% asetonitril, 10 ul% 80 asetonitril,% 0.1 formik asit takip Islak ziptip. </li><li> 20 ul% 0.1 formik asite ziptip dengeye. </li><li> Ziptip matris üzerinden 10 kat yukarı ve aşağı pipetleme ziptip üzerine örnek yükleyin. </li><li> 10 ul% 0.1 formik asit, 5 kez pipetleme ziptip yıkayın. </li><li> 10 ul% 80 asetonitril,% 0.1 formik asit ile temiz bir Eppendorf tüp içine örnek Zehir. </li><li> Elüsyon tekrarlayın ve aynı tüpe ikinci 10 ul eklemek. </li><li> &lt;2 ul bir ses örnekleri Speedvac. </li><li> 20 ul% 0.1 formik asite süspanse edin örnekleri. Örnekleri LC-MS/MS analiz için hazır. </li></ol> 9) Temsilcisi Sonuçlar: 20 mg / ml - Poros konjugasyon genellikle 2 aralığında on-boncuk lektin konsantrasyonları elde edilir. Bu afinite kromatografisi için en uygunudur. Tripsin sindirilir MARS-tükenmiş insan plazma lektin kromatografi genellikle bağlı bir fraksiyon olarak glikopeptid zenginleştirir. Glikopeptid PNGase önce LC-MS/MS F-tedavi olarak, N-bağlantılı bir konsensüs sırası ile peptitler olarak tanımlanır asparajin dönüştürülür edildiği NXS / T (burada X herhangi bir kalıntı ama prolin), PNGase F. Peptitler eylem yoluyla bu özelliklere sahip bir aspartik asit deglycosylated peptidler (veya &quot;deglycopeptides&quot;) olarak kabul edilir. Bağlı bir fraksiyon olarak ele peptidlerin% 30-50 deglycopeptides ise ortalama akış-through (FT) AAL veya SNA kromatografisi fraksiyonu,% 2-4 oranında deglycopeptides bulunur. Tipik olarak, bir QStar Elite kullanarak, 1000-1300 peptidler FT fraksiyonu tespit olacak ve 200-400 peptidler bağlı bir fraksiyon olarak tespit edilecektir. İki veya daha fazla farklı Invertase deglycopeptides FT fraksiyonu ve bağlı fraksiyonu (Tablo 1) iki veya daha fazla farklı laktoferrin deglycopeptides dikkat edilmelidir. <img src="/files/ftp_upload/1398/1398table.jpg" alt="Tablo 1" /> Tablo 1.

<ol>
	<li>Keshishian, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=multiplexed+assays+for+low+abundance+proteins+in+plasma+by+targeted+mass+spectrometry+and+stable+isotope+dilution.">multiplexed assays for low abundance proteins in plasma by targeted mass spectrometry and stable isotope dilution.</a> Mol Cell Proteomics. 6, 2212-2229 (2007).</li></ol>

Bu protokol, glikopeptid glukanıdır özel bir şekilde izole etmek ve tanımlamak için hızlı bir yöntem sağlar. Glikozilasyon geliştirme ve patogenezi sırasında bir organizmanın içinde yaygın olarak değişir, bu teknik sorular geniş bir yelpazede adresi için geçerli olabilir. Özellikle, kanser ilişkili epitoplar biyomarker keşfi yolunda bir ilk adım olarak modifiye edilmiş glikopeptid seçici zenginleştirmek için yöntem kullanıyor.

<!DOCTYPE html PUBLIC "-//W3C//DTD XHTML 1.0 Transitional//EN" "http://www.w3.org/TR/xhtml1/DTD/xhtml1-transitional.dtd">
<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>Human plasma</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>&nbsp;</td>
<td>We make this in house</td>
</tr>
<tr>
<td>Human lactoferrin</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>L0520</td>
<td>Trypsin-digest before use</td>
</tr>
<tr>
<td>Yeast invertase</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>I0408</td>
<td>Trypsin-digest before use</td>
</tr>
<tr>
<td>Oass HLB SPE cartridges</td>
<td>&nbsp;</td>
<td>Waters</td>
<td>WAT094225</td>
<td>1 cc volume</td>
</tr>
<tr>
<td>ZipTip pipet tips</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>ZTC1 8M0 96</td>
<td>Millipore, C18 for 10 ml pipette</td>
</tr>
<tr>
<td>Tris</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>BP154-1</td>
<td>99%</td>
</tr>
<tr>
<td>Ammonium bicarbonate</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>A6141</td>
<td>99%</td>
</tr>
<tr>
<td>Calcium chloride</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>C4901</td>
<td>96%</td>
</tr>
<tr>
<td>Magnesium chloride</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>M8266</td>
<td>98%</td>
</tr>
<tr>
<td>Acetic acid</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>242853</td>
<td>99.7%</td>
</tr>
<tr>
<td>pH indicator paper</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>M95903</td>
<td>Range 0-14</td>
</tr>
<tr>
<td>Acetonitrile</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>A998</td>
<td>99.9%</td>
</tr>
<tr>
<td>Formic acid</td>
<td>&nbsp;</td>
<td>Pierce</td>
<td>28905</td>
<td>99%</td>
</tr>
<tr>
<td>Phosphate-buffered saline</td>
<td>&nbsp;</td>
<td>Invitrogen</td>
<td>14190-136</td>
<td>Without calcium and magnesium</td>
</tr>
<tr>
<td>Sodium azide</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>71289</td>
<td>99.5%</td>
</tr>
<tr>
<td>PNGase F</td>
<td>&nbsp;</td>
<td>New England Biolabs</td>
<td>P0705L</td>
<td>Glycerol-free</td>
</tr>
<tr>
<td>Vacuum-filter flasks</td>
<td>&nbsp;</td>
<td>Fisher</td>
<td>SCGVU05RE</td>
<td>0.2 mm pores</td>
</tr>
<tr>
<td>POROS-AL beads</td>
<td>&nbsp;</td>
<td>Applied Biosystems</td>
<td>1-6028-02</td>
<td>&nbsp;</td>
<tr>
<td>Aleuria aurantia lectin</td>
<td>&nbsp;</td>
<td>Vector Laboratories</td>
<td>L-1390</td>
<td>&nbsp;</td>
<tr>
<td>Sambucus nigra agglutinin</td>
<td>&nbsp;</td>
<td>Vector Laboratories</td>
<td>L-1300</td>
<td>&nbsp;</td>
<tr>
<td>Sodium cyanoborohydride</td>
<td>&nbsp;</td>
<td>Sigma</td>
<td>296945</td>
<td>5.0 M solution</td>
</tr>		</tbody>
		</table>
		</div>

a lectin hplc method to enrich selectively-glycosylated peptides from complex biological samples

Glukanlardır post-translasyonel modifikasyonlar önemli bir sınıf vardır. Genellikle salgılanır ve hücre dışı moleküller üzerinde bulunan, glukanıdır yapıları, hücrenin iç durum sinyali. Tümör hücreleri üzerinde glukanlardır bol miktarda sialik asit ve fucose moieties sahip olma eğilimindedir. Biz, bu kanser ilişkili glukanıdır varyantları erken evre hastalık teşhis amacıyla biyomarker geliştirme için istismar edilmesi öneriyoruz. Buna göre, biz bir kitle lektinleri belirli glukanıdır yapıları bağlayan proteinleri oluşan yakınlık matrisleri kromatografi içermektedir spektrometresi tabanlı iş akışı geliştirdi. Sırasıyla, sialik asit ve fucose bağlayan lektin Sambucus nigra (SNA) ve Aleuria aurantia (AAL), kovalent Poros boncuk (Uygulamalı Biyosistem) birleştiğinde ve yüksek basınçlı sıvı kromatografi (HPLC) PEEK sütunlar halinde dolu. Kısaca, bir çok ilgisi temizleme sistemi (Agilent MARS-14) kullanarak ondört en bol bulunan proteinlerin plazma tükenmiş oldu. Tüketilmiş plazma tripsin-sindirilir ve akış yoluyla ve SNA veya AAL HPLC bağlı fraksiyonları ayrılır. N-bağlı glukanlardır kaldırmak için kesirler PNGaseF ile tedavi ve QStar Elite LC-MS/MS tarafından incelendi. Veriler Maskot yazılımı kullanılarak analiz edildi. Deneysel tasarım kontrolleri fucosylated ve sialylated olumlu dahil insan laktoferrin glikopeptid ve Saccharomyces negatif kontroller yüksek mannoz glikopeptid cerevisiae lektin yakalama özgüllüğü izlemek için kullanılır. Bu iş akışının temel özellikleri glikoprotein standartlar kullanılarak HPLC biçimi, kendi deamidated Asn-Xxx-Ser/Thr motifleri yakalanan ve tedavi edilen PNGaseF glikopeptid olumlu kimlik ve kalite değerlendirmesi türetilen tekrarlanabilirlik. Protokolü optimizasyonu da uygun sütun kapasitesinin malzeme başlayan, en verimli yakalama ve yıkama tamponları tespit ve tam deglycosylation emin olmak için PNGaseF tedavi izleme oranının tespitinde dahildir. Gelecek yönde meme kanseri hastalarında ve kontrol bireylerin plazma kütle spektrometresi tabanlı keşif deneyler gerçekleştirmek için bu iş akışını kullanarak.

Watch this Scientific Journal Video about A Lectin HPLC Method to Enrich Selectively-glycosylated Peptides from Complex Biological Samples at JoVE.com

A Lectin HPLC Method to Enrich Selectively-glycosylated Peptides from Complex Biological Samples

Lektin-konjuge Poros boncuk HPLC için istihdam edildi. Glikopeptid standartları, pozitif ve negatif kontroller olarak görev yaptı. MARS-14 tükenmiş, tripsin-sindirilmiş insan plazma Kromatografi ve flow-through (FT) ve bağlı kesirler ESI-LC-MS/MS analizler için toplanmıştır. FT&#39;ye göre glikopeptid bağlı fraksiyon zenginleştirilmiş.

Kompleks Biyolojik örnekler seçerek glikozile Peptitler zenginleştirin lektin HPLC Yöntemi

Glycans are an important class of post-translational modifications. Typically found on secreted and extracellular molecules, glycan structures signal the ...

a-lectin-hplc-method-to-enrich-selectively-glycosylated-peptides-from

Research

JoVE Journal

Biology

15.9K Görüntüleme.  University of California, San Francisco - UCSF. Lektin-konjuge Poros boncuk HPLC için istihdam edildi. Glikopeptid standartları, pozitif ve negatif kontroller olarak görev yaptı. MARS-14 tükenmiş, tripsin-sindirilmiş insan plazma Kromatografi ve flow-through (FT) ve bağlı kesirler ESI-LC-MS/MS analizler için toplanmıştır. FT'ye göre glikopeptid bağlı fraksiyon zenginleştirilmiş.

Video: A Lectin HPLC Method to Enrich Selectively-glycosylated Peptides from Complex Biological Samples

T-wave Ion Mobility-mass Spectrometry: Basic Experimental Procedures for Protein Complex Analysis

Ion mobility (IM) is a method that measures the time taken for an ion to travel through a pressurized cell under the influence of a weak electric field. The speed by which the ions traverse the drift region depends on their size: large ions will experience a greater number of collisions with the background inert gas (usually N2) and thus travel more slowly through the IM device than those ions that comprise a smaller cross-section. In general, the time it takes for the ions to migrate though the dense gas phase separates them, according to their collision cross-section (&Omega;). 
Recently, IM spectrometry was coupled with mass spectrometry and a traveling-wave (T-wave) Synapt ion mobility mass spectrometer (IM-MS) was released. Integrating mass spectrometry with ion mobility enables an extra dimension of sample separation and definition, yielding a three-dimensional spectrum (mass to charge, intensity, and drift time). This separation technique allows the spectral overlap to decrease, and enables resolution of heterogeneous complexes with very similar mass, or mass-to-charge ratios, but different drift times. Moreover, the drift time measurements provide an important layer of structural information, as &Omega; is related to the overall shape and topology of the ion. The correlation between the measured drift time values and &Omega; is calculated using a calibration curve generated from calibrant proteins with defined cross-sections1.
The power of the IM-MS approach lies in its ability to define the subunit packing and overall shape of protein assemblies at micromolar concentrations, and near-physiological conditions1. Several recent IM studies of both individual proteins2,3 and non-covalent protein complexes4-9, successfully demonstrated that protein quaternary structure is maintained in the gas phase, and highlighted the potential of this approach in the study of protein assemblies of unknown geometry. Here, we provide a detailed description of IMS-MS analysis of protein complexes using the Synapt (Quadrupole-Ion Mobility-Time-of-Flight) HDMS instrument (Waters Ltd; the only commercial IM-MS instrument currently available)10. We describe the basic optimization steps, the calibration of collision cross-sections, and methods for data processing and interpretation. The final step of the protocol discusses methods for calculating theoretical &Omega; values. Overall, the protocol does not attempt to cover every aspect of IM-MS characterization of protein assemblies; rather, its goal is to introduce the practical aspects of the method to new researchers in the field.

Ion mobility-mass spectrometry is an emerging gas-phase technology that separates ions, based on their collision cross-section and mass. The method provides three-dimensional information on the overall topology and shape of protein complexes. Here, we outline a basic procedure for instrument setting and optimization, calibration of drift times, and data interpretation.

T-dalgası İyon Hareketlilik-Kütle Spektrometresi: Protein Karmaşık Analiz Temel Deneysel Prosedürler

Ion mobility (IM) is a method that measures the time taken for an ion to travel through a pressurized cell under the influence of a weak electric field. ...

Biyoloji

Peptides from Phage Display Library Modulate Gene Expression in Mesenchymal Cells and Potentiate Osteogenesis in Unicortical Bone Defects

Two novel synthetic peptides accelerate bone formation and can be delivered using a collagen matrix. The aim of this study was to investigate the effects on bone repair in a unicortical defect model. Treatment of mesenchymal cells produced an increase in alkaline phosphatase activity, showed nodule formation by the cells, and increased the expression of genes for runx2, osterix, bone sialoprotein, and osteocalcin. A collagen sponge soaked with peptide promoted repair of bone defects, whereas the control was less effective. The results from this study demonstrated that mesenchymal cells treated with peptide in vitro differentiate towards osteogenesis, and, that peptides delivered in vivo using a collagen sponge promote the repair of unicortical defects.

A phage display library was used to identify peptide sequences that target bone. The objective was to investigate the effect of these peptides on mesenchymal cell differentiation and to determine their effect on bone regeneration.

Bakteriyofaj Görüntüleme Kütüphane Peptitler Mezenkimal Hücreler Gen ekspresyonunu modüle ve Unicortical Kemik Hastalıkları Osteogenezi potansiyalize

Two novel synthetic peptides accelerate bone formation and can be delivered using a collagen matrix. The aim of this study was to investigate the effects ...

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in 73 different countries throughout Africa, Asia, Europe, New Zealand, Oceania and the Americas1-2. Cogongrass forms rapidly-spreading, monodominant stands that displace a large variety of native plant species and in turn threaten the native animals that depend on the displaced native plant species for forage and shelter. To add to the problem, an ornamental variety [I. cylindrica var. koenigii (Retzius)] is widely marketed under the names of Imperata cylindrica 'Rubra', Red Baron, and Japanese blood grass (JBG). This variety is putatively sterile and noninvasive and is considered a desirable ornamental for its red-colored leaves. However, under the correct conditions, JBG can produce viable seed (Carol Holko, 2009 personal communication) and can revert to a green invasive form that is often indistinguishable from cogongrass as it takes on the distinguishing characteristics of the wild-type invasive variety4 (Figure 1). This makes identification using morphology a difficult task even for well-trained plant taxonomists. Reversion of JBG to an aggressive green phenotype is also not a rare occurrence. Using sequence comparisons of coding and variable regions in both nuclear and chloroplast DNA, we have confirmed that JBG has reverted to the green invasive within the states of Maryland, South Carolina, and Missouri. JBG has been sold and planted in just about every state in the continental U.S. where there is not an active cogongrass infestation. The extent of the revert problem in not well understood because reverted plants are undocumented and often destroyed.
Application of this molecular protocol provides a method to identify JBG reverts and can help keep these varieties from co-occurring and possibly hybridizing. Cogongrass is an obligate outcrosser and, when crossed with a different genotype, can produce viable wind-dispersed seeds that spread cogongrass over wide distances5-7. JBG has a slightly different genotype than cogongrass and may be able to form viable hybrids with cogongrass. To add to the problem, JBG is more cold and shade tolerant than cogongrass8-10, and gene flow between these two varieties is likely to generate hybrids that are more aggressive, shade tolerant, and cold hardy than wild-type cogongrass. While wild-type cogongrass currently infests over 490 million hectares worldwide, in the Southeast U.S. it infests over 500,000 hectares and is capable of occupying most of the U.S. as it rapidly spreads northward due to its broad niche and geographic potential3,7,11. The potential of a genetic crossing is a serious concern for the USDA-APHIS Federal Noxious Week Program. Currently, the USDA-APHIS prohibits JBG in states where there are major cogongrass infestations (e.g., Florida, Alabama, Mississippi). However, preventing the two varieties from combining can prove more difficult as cogongrass and JBG expand their distributions. Furthermore, the distribution of the JBG revert is currently unknown and without the ability to identify these varieties through morphology, some cogongrass infestations may be the result of JBG reverts. Unfortunately, current molecular methods of identification typically rely on AFLP (Amplified Fragment Length Polymorphisms) and DNA sequencing, both of which are time consuming and costly. Here, we present the first cost-effective and reliable PCR-based molecular genotyping method to accurately distinguish between cogongrass and JBG revert.

A PCR-based Genotyping Method to Distinguish Between Wild-type and Ornamental Varieties of Imperata cylindrica

We provide a cost-effective and rapid molecular genotyping protocol that employs variety-specific PCR primers that target DNA sequence differences within the chloroplast trnL-F spacer region to differentiate between varieties of Imperata cylindrica (cogongrass) that cannot be distinguished by morphology alone. These varieties include the federally listed noxious weed, cogongrass and closely-related, wide-spread ornamental variety, I. cylindrica var. koenigii (Japanese blood grass).

Wild-tip ve Süs Çeşitler arasında ayırt A PCR tabanlı Genotiplendirme Yöntemi Imperata cylindrica</em

Wild-type I. cylindrica (cogongrass) is one of the top ten worst invasive plants in the world, negatively impacting agricultural and natural resources in ...

The Logic, Experimental Steps, and Potential of Heterologous Natural Product Biosynthesis Featuring the Complex Antibiotic Erythromycin A Produced Through E. coli

The heterologous production of complex natural products is an approach designed to address current limitations and future possibilities. It is particularly useful for those compounds which possess therapeutic value but cannot be sufficiently produced or would benefit from an improved form of production. The experimental procedures involved can be subdivided into three components: 1) genetic transfer; 2) heterologous reconstitution; and 3) product analysis. Each experimental component is under continual optimization to meet the challenges and anticipate the opportunities associated with this emerging approach.
Heterologous biosynthesis begins with the identification of a genetic sequence responsible for a valuable natural product. Transferring this sequence to a heterologous host is complicated by the biosynthetic pathway complexity responsible for product formation. The antibiotic erythromycin A is a good example. Twenty genes (totaling &gt;50 kb) are required for eventual biosynthesis. In addition, three of these genes encode megasynthases, multi-domain enzymes each ~300 kDa in size. This genetic material must be designed and transferred to E. coli for reconstituted biosynthesis. The use of PCR isolation, operon construction, multi-cystronic plasmids, and electro-transformation will be described in transferring the erythromycin A genetic cluster to E. coli.
Once transferred, the E. coli cell must support eventual biosynthesis. This process is also challenging given the substantial differences between E. coli and most original hosts responsible for complex natural product formation. The cell must provide necessary substrates to support biosynthesis and coordinately express the transferred genetic cluster to produce active enzymes. In the case of erythromycin A, the E. coli cell had to be engineered to provide the two precursors (propionyl-CoA and (2S)-methylmalonyl-CoA) required for biosynthesis. In addition, gene sequence modifications, plasmid copy number, chaperonin co-expression, post-translational enzymatic modification, and process temperature were also required to allow final erythromycin A formation.
Finally, successful production must be assessed. For the erythromycin A case, we will present two methods. The first is liquid chromatography-mass spectrometry (LC-MS) to confirm and quantify production. The bioactivity of erythromycin A will also be confirmed through use of a bioassay in which the antibiotic activity is tested against Bacillus subtilis. The assessment assays establish erythromycin A biosynthesis from E. coli and set the stage for future engineering efforts to improve or diversify production and for the production of new complex natural compounds using this approach.

The Logic, Experimental Steps, and Potential of Heterologous Natural Product Biosynthesis Featuring the Complex Antibiotic Erythromycin A Produced Through E. coli

The heterologous biosynthesis of erythromycin A through E. coli includes the following experimental steps: 1) genetic transfer; 2) heterologous reconstitution; and 3) product analysis. Each step will be explained in the context of the motivation, potential, and challenges in producing therapeutic natural products using E. coli as a surrogate host.

A ile Üretilen Kompleksi Antibiyotik Eritromisin Featuring Mantık, Deneysel Merdivenleri ve Heterolog Doğal Ürün biyosentezi Potansiyeli<em&gt; E. coli</em

The heterologous production of complex natural products is an approach designed to address current limitations and future possibilities.  It is ...

A Method for Microinjection of Patiria minata Zygotes

Echinoderms have long been a favorite model system for studies of reproduction and development, and more recently for the study of gene regulation and evolution of developmental processes. The sea star, Patiria miniata, is gaining prevalence as a model system for these types of studies which were previously performed almost exclusively in the sea urchins, Strongylocentrotus purpuratus and Lytechinus variegatus. An advantage of these model systems is the ease of producing modified embryos in which a particular gene is up or downregulated, labeling a group of cells, or introducing a reporter gene. A single microinjection method is capable of creating a wide variety of such modified embryos. Here, we present a method for obtaining gametes from P. miniata, producing zygotes, and introducing perturbing reagents via microinjection. Healthy morphant embryos are subsequently isolated for quantitative and qualitative studies of gene function. The availability of genome and transcriptome data for this organism has increased the types of studies that are performed and the ease of executing them.

A Method for Microinjection of Patiria minata Zygotes

Methods that produce morphant embryos are essential to study developmental mechanisms and gene regulatory networks. The sea star Patiria miniata is an emerging model system for these studies. Here we present a protocol for obtaining gametes, producing cultures of embryos, and rapid microinjection of zygotes from this species.

Mikroenjeksiyonu Bir Yöntem<em&gt; Patiria Minata</em&gt; Zigotların

Echinoderms have long been a favorite model system for studies of reproduction and development, and more recently for the study of gene regulation and ...

Purifying the Impure: Sequencing Metagenomes and Metatranscriptomes from Complex Animal-associated Samples

The accessibility of high-throughput sequencing has revolutionized many fields of biology. In order to better understand host-associated viral and microbial communities, a comprehensive workflow for DNA and RNA extraction was developed. The workflow concurrently generates viral and microbial metagenomes, as well as metatranscriptomes, from a single sample for next-generation sequencing. The coupling of these approaches provides an overview of both the taxonomical characteristics and the community encoded functions. The presented methods use Cystic Fibrosis (CF) sputum, a problematic sample type, because it is exceptionally viscous and contains high amount of mucins, free neutrophil DNA, and other unknown contaminants. The protocols described here target these problems and successfully recover viral and microbial DNA with minimal human DNA contamination. To complement the metagenomics studies, a metatranscriptomics protocol was optimized to recover both microbial and host mRNA that contains relatively few ribosomal RNA (rRNA) sequences. An overview of the data characteristics is presented to serve as a reference for assessing the success of the methods. Additional CF sputum samples were also collected to (i) evaluate the consistency of the microbiome profiles across seven consecutive days within a single patient, and (ii) compare the consistency of metagenomic approach to a 16S ribosomal RNA gene-based sequencing. The results showed that daily fluctuation of microbial profiles without antibiotic perturbation was minimal and the taxonomy profiles of the common CF-associated bacteria were highly similar between the 16S rDNA libraries and metagenomes generated from the hypotonic lysis (HL)-derived DNA. However, the differences between 16S rDNA taxonomical profiles generated from total DNA and HL-derived DNA suggest that hypotonic lysis and the washing steps benefit in not only removing the human-derived DNA, but also microbial-derived extracellular DNA that may misrepresent the actual microbial profiles.

Using the cystic fibrosis airway as an example, the manuscript presents a comprehensive workflow comprising a combination of metagenomic and metatranscriptomic approaches to characterize the microbial and viral communities in animal-associated samples.

Saf olmayan Arındırıcı: Sıralama Metagenomes ve Metatranscriptomes Kompleksi Hayvan ilişkili Örneklerinden

The accessibility of high-throughput sequencing has revolutionized many fields of biology. In order to better understand host-associated viral and ...

A Hybrid DNA Extraction Method for the Qualitative and Quantitative Assessment of Bacterial Communities from Poultry Production Samples

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses performed. Considering that the use of new bacterial community analysis techniques (e.g., microbiomics, metagenomics) is becoming more prevalent in the agricultural and environmental sciences and many environmental samples within these disciplines can be physiochemically and microbiologically unique (e.g., fecal and litter/bedding samples from the poultry production spectrum), appropriate and effective DNA extraction methods need to be carefully chosen. Therefore, a novel semi-automated hybrid DNA extraction method was developed specifically for use with environmental poultry production samples. This method is a combination of the two major types of DNA extraction: mechanical and enzymatic. A two-step intense mechanical homogenization step (using bead-beating specifically formulated for environmental samples) was added to the beginning of the &#8220;gold standard&#8221; enzymatic DNA extraction method for fecal samples to enhance the removal of bacteria and DNA from the sample matrix and improve the recovery of Gram-positive bacterial community members. Once the enzymatic extraction portion of the hybrid method was initiated, the remaining purification process was automated using a robotic workstation to increase sample throughput and decrease sample processing error. In comparison to the strict mechanical and enzymatic DNA extraction methods, this novel hybrid method provided the best overall combined performance when considering quantitative (using 16S rRNA qPCR) and qualitative (using microbiomics) estimates of the total bacterial communities when processing poultry feces and litter samples.

A novel semi-automated hybrid DNA extraction method for use with environmental poultry production samples was developed and demonstrated improvements over a common mechanical and enzymatic extraction method in terms of the quantitative and qualitative estimates of the total bacterial communities.

Kanatlı Üretim Örneklerden Bakteriyel Topluluklar Nitel ve Nicel Değerlendirme Karma DNA Ekstraksiyon Yöntemi

The efficacy of DNA extraction protocols can be highly dependent upon both the type of sample being investigated and the types of downstream analyses ...

Removal of Exogenous Materials from the Outer Portion of Frozen Cores to Investigate the Ancient Biological Communities Harbored Inside

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. In fact, these frozen materials could reflect conditions over vast time periods and investigation of biological materials harbored inside could provide insight of ancient environments. To appropriately analyze these ecosystems and extract meaningful biological information from frozen soils and ice, proper collection and processing of the frozen samples is necessary. This is especially critical for microbial and DNA analyses since the communities present may be so uniquely different from modern ones. Here, a protocol is presented to successfully collect and decontaminate frozen cores. Both the absence of the colonies used to dope the outer surface and exogenous DNA suggest that we successfully decontaminated the frozen cores and that the microorganisms detected were from the material, rather than contamination from drilling or processing the cores.

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. A protocol is presented to collect and decontaminate permafrost cores of soils and ice. The absence of exogenous colonies and DNA suggest that microorganisms detected represent the material, rather than contamination from drilling or processing.

Antik Biyolojik Topluluklar barındırmıştır İç Araştırma Dondurulmuş Çekirdek dış bölümünden Eksojen Malzeme Kaldırma

The cryosphere offers access to preserved organisms that persisted under past environmental conditions. In fact, these frozen materials could reflect ...

Preparation and Observation of Thick Biological Samples by Scanning Transmission Electron Tomography

This report describes a protocol for preparing thick biological specimens for further observation using a scanning transmission electron microscope. It also describes an imaging method for studying the 3D structure of thick biological specimens by scanning transmission electron tomography. The sample preparation protocol is based on conventional methods in which the sample is fixed using chemical agents, treated with a heavy atom salt contrasting agent, dehydrated in a series of ethanol baths, and embedded in resin. The specific imaging conditions for observing thick samples by scanning transmission electron microscopy are then described. Sections of the sample are observed using a through-focus method involving the collection of several images at various focal planes. This enables the recovery of in-focus information at various heights throughout the sample. This particular collection pattern is performed at each tilt angle during tomography data collection. A single image is then generated, merging the in-focus information from all the different focal planes. A classic tilt-series dataset is then generated. The advantage of the method is that the tilt-series alignment and reconstruction can be performed using standard tools. The collection of through-focal images allows the reconstruction of a 3D volume that contains all of the structural details of the sample in focus.

This report describes a sample preparation protocol and specific imaging conditions for performing scanning transmission electron tomography of thick biological specimens.

Tarama Transmisyon Elektron Tomografi ile hazırlanması ve Kalın Biyolojik Örneklerinin Gözlem

This report describes a protocol for preparing thick biological specimens for further observation using a scanning transmission electron microscope. It ...

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

A method is described for labeling individual messenger RNA (mRNA) transcripts in fixed bacteria for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH allows the measurement of cell-to-cell variability in mRNA copy number of genes of interest, as well as the subcellular location of the transcripts. The main steps involved are fixation of the bacterial cell culture, permeabilization of cell membranes, and hybridization of the target transcripts with sets of commercially available short fluorescently-labeled oligonucleotide probes. smFISH can allow the imaging of the transcripts of multiple genes in the same cell, with limitations imposed by the spectral overlap between different fluorescent markers. Following completion of the protocol illustrated below, cells can be readily imaged using a microscope coupled with a camera suitable for low-intensity fluorescence. These images, together with cell contours obtained from segmentation of phase contrast frames, or from cell membrane staining, allow the calculation of the mRNA copy number distribution of a sample of cells using open-source or custom-written software. The labeling method described here can also be applied to image transcripts with stochastic optical reconstruction microscopy (STORM).

Method for Labeling Transcripts in Individual Escherichia coli Cells for Single-molecule Fluorescence In Situ Hybridization Experiments

This manuscript describes a method for labeling individual messenger RNA (mRNA) transcripts with fluorescently-labeled DNA probes, for use in single-molecule fluorescence in situ hybridization (smFISH) experiments in E. coli. smFISH is a visualization method that allows the simultaneous detection, localization, and quantification of single mRNA molecules in fixed individual cells.