We thank SP Gygi and GM Church for help and guidance. This work was supported by a CNRS chaire d'excellence to ACT.

NOT: Bu yöntem daha önce 12 tarif edilmiştir. 1.. Protein İzolasyonu Tercihen, French pres, boncuk dayak veya sonikasyon gibi fiziksel yöntemlerle, yokedici hücreler tarafından hücre protein 1 mg hazırlayın. Enzim kütle spektrometresi ölçümleri bulandırabilir çünkü lizozim-aracılı hücre parçalanmasını önlemek. Proteinlerin 2. TCA Yağış Proteinlerini çöktürmek için 10 dakika süre ile buz üzerinde 4 hacim protein ve soğuk 1 hacim trikloroasetik asit (TCA) ilave edin. Santrifüj, 4 ° C&#39;de 5 dakika boyunca 12,000 x g ve supernatant çıkarın. 4 ° C&#39;de 5 dakika boyunca 12,000 xg&#39;de buz soğukluğunda aseton ve santrifüj 1 ml pelletini Süpernatantı ve 15 dakika boyunca pelet kurumaya bankta tüp ters. -80 ° C&#39;de saklayın protein granül 3.. Doğasını Proteinler ve disülfıt bağlarını azaltır Reproteinler askıya 500 ul denatürasyon ve indirgeme tamponu içinde ~ 2 mg / ml (50 mM HEPES pH 8.5, 5 mM DTT, 4 M üre ya da 3 ya da% SDS) için. İsteğe bağlı olarak, tampon maddesi içinde bir proteaz inhibitörü içerir. Oda sıcaklığında 10 dakika süre ile 56 ° C&#39;de 30 dakika boyunca inkübe edin proteinleri. 4. Aklylate Ücretsiz Sülfhidril Gruplar geri dönüşü olmayan disülfit bağ oluşumu bozacak Taze su içinde 0.3 M iodoasetamid hazırlayın. DİKKAT! Iodoacetamide son derece zehirlidir. 500 ul protein için 25 ul 0.3 M iodoasetamid (15 mM nihai konsantrasyon) ilave edin ve oda sıcaklığında karanlıkta 20 dakika boyunca inkübe edin. 300 mM DTT (5 mM DTT nihai konsantrasyon) ve 10 ul kadar iyodoasetamid eklenerek söndürülür. -80 ° C&#39;de saklayın alkile proteinleri 5.. Protein Sindirim TCA proteinleri çökeltmek (aşama 2&#39;de tarif edildiği gibi) ve 50 mM HEPES (pH 8.2), 1 ml, 1 M üre içinde tekrar süspansiyon. Lisil endoprotein bir stok solüsyonu hazırlayın2 ug / ul &#39;lik bir konsantrasyonda ve protein solüsyonuna 5 ul ekle suda ase (Lys-C). Oda sıcaklığında 16 saat boyunca karışımın inkübe edin. Nihai Lys-C konsantrasyonu, 10 ng / ml ve protein-to-lysC oranı (a / a) 1/200 ile 1/50 olduğu olduğundan emin olun. , 50 mM asetik asit, 40 ul içinde 20 ug tripsin dizileme gradlı yeniden süspanse Lys-C ile sindirilmiş 5 ul (10 ug tripsin) ekleyin ve 37 ° C de 6 saat inkübe Tripsin için kullanıldığı gibi Lys-C için aynı proteaz konsantrasyon ve proteaz-to-protein oranları kullanın. 6.. Ters-faz peptid Ekstraksiyon <ol><li> % 0.5 &#39;lik bir nihai konsantrasyona (pH ≈ 2), trifluoroasetik asit (TFA) eklenmesi ile peptidleri asitlendirilir. Bir ekstre manifolduna Bir C 18 sütunu takın. Peptidlerin yükleme ve elüsyon dışında tüm adımlar için mümkün olan en yüksek akış hızı kullanın. </li><li> 6 ml asetonitril (ACN) ile ıslak kolon. 6 ml% 80 ACN,% 0.1 TFA ile kolondan yıkanır, daha sonra 6 ml% 0.1 TFA ile dengeye getirin. Izin vermeyinadımlar arasında kuru çalıştırmak için sütun. </li><li> Vakum basıncı durdurmak ve yaklaşık olarak 1 ml / dk &#39;lık bir akış hızında kolona 500 ug peptidlerin yükleyin. Peptidler, kolon, yeniden vakum bağlanmıştır ve sonra, (0.09 M sitrik asit, 0.23 M Na 2 HPO 4, pH 5.5), sitrik asit tamponu, 3 ml ile, 6 ml% 0.1 TFA ile yıkayınız sonra. Not: Yüksek miktarda etiketli peptid edilebilir, ancak bağlama kapasitesi C18 kolon, en azından örnek kaybını önlemek için peptid miktarına göre iki kat daha yüksek olması gerekir. </li></ol> 7. On-kolonu İndirgemeli dimetilasyonu tarafından Peptit Etiketleme (Redi Etiketleme) Hidrojen siyanür Etiketleme işlemi sırasında düşük bir konsantrasyonda serbest olarak kimyasal bir başlık altında bu adımı gerçekleştirin. <ol><li> Peptid serbest aminler metillenmesi &quot;hafif&quot; ve &quot;ağır&quot; Redi tampon 12 ml hazırlayın. Işık Redi tamponu% 0.8 formaldehit ve inci hidrojeni taşıyan 0.12 M sodyumsiyanoborhidrid oluşursitrik asit tampon maddesi içinde EIR doğal izotopik dağılımları. Ağır Redi tamponu% 0.8 döteryumlu formaldehit ve sitrik asit tampon maddesi içinde 0.12 M döteryumlu sodyum siyanoborohidrid oluşur. </li><li> 1 ml / dakika akış hızında, peptid ihtiva eden sütunlar 10 ml hafif ya da ağır Redi tampon ya da eklenmesi ile peptidleri ihtiva eden bir sütun inkübe edin ve tam etiketleme sağlamak için tekrarlayın. Yıkama 6 ml% 0.1 TFA ile kolon ve daha sonra 1 ml% 0.5 asetik asit ile. </li><li> Vakum durdurmak ve yaklaşık olarak 0.5 ml / dk &#39;lık bir akış oranı kullanılarak, daha sonra 1 ml% 80 ACN ile% 0.5 asetik asit 1 ml% 40 ACN,% 0.5 asetik asit ile ilk işaretli peptidlerin elüsyonu. Eğer istenirse, ağır ve hafif örnekler karıştırma önce kütle spektrometresi ile bireysel örneklerin etiketleme verimliliği ölçmek (&quot;Temsilci Sonuçları&quot; bakın). Kütle spektrometresi ile belirlenebilir 1:1 ağır ve hafif-etiketli peptid örnekleri karıştırın. </li></ol> Temel pH Ters Faz (BPRP) kromatografi &lt;8. Ayrı Peptide Karışım/ P&gt; Temel pH bağımsız bir şekilde, proteom kapsamını arttırmak için LC-MS/MS ile analiz edilir çok sayıda kısmın içine peptid karışımını ayırmak için aşama (BPRP) kromatografisi. <ol><li> 10 mM amonyum bikarbonat (pH 8) içinde artan ACN konsantrasyon gradyanı uygulanarak bir C18-HPLC kolonu üzerinde peptit karışımının fraksiyonlanması. 5 dakika için% 5 (v / v) ACN ile başlayın, 60 dakika içinde,% 35 ACN artması ve daha sonra 1 dakika içinde% 90 ACN. 9 dakika boyunca yeniden dengeye sütun için% 5 ACN indirgeme önce, 4 dakika boyunca% 90 ACN saklayın. 96 oyuklu bir plaka (H12 için A1) eşit hacimde 96 fraksiyonları toplayın. Peptidler kolonu (burada tarif edilen koşullar için 10-70 dakika) kapalı elüsyon ise 220 nm&#39;de bir UV detektör kullanılarak fraksiyonasyon izleyin. </li><li> Kuyu A2, C2, E2, ve G2 (fraksiyon A2) için ve buna bağlı olarak için, kuyular, B1, D1, F1 ve H1 (fraksiyon B1) için, kuyu A1, C1, E1 ve G1 (fraksiyon A1) ikinci kısımları birleştiriniz Geri kalan kısımlar. Solvent çıkarınbir vakum santrifüj kullanılarak t. Fraksiyonlardan peptitler tekrar A1, B2, A3, B4, A5, B6, A7, B8, A9, B10, A11 ve B12 1 M urea/0.5% TFA 130 ul ve aşama 9&#39;da tarif edildiği gibi StageTips kullanılarak arındırmak. Mağazası fraksiyonlar B1, A2, B3, A4, B5, A6, B7, A8, B9, A10, B11, ve -20 ° C de A12 </li></ol> 9. Stop and Go Ekstraksiyon tarafından Arındırmak peptidler (StageTips) 1.07 mm iç çapı (ID) ile iki C18 diskler ile 200 ul pipet uçları ambalaj ile C18-StageTip 7 microcolumns hazırlayın. Eppendorf tüpleri içine Sahne ipuçları koydu. 130 | il metanol, daha sonra 130 ul% 80 ACN,% 0.5 asetik asit ile ipuçları yıkamak için, bir mikro santrifüj kullanın. 130 ul% 0.1 TFA ile StageTips dengelenmesi. StageTips peptit karışımı aktarın ve 40 ul,% 0.5 asetik asit, daha sonra 40 ul% 0.1 TFA, daha sonra 130 ul% 0.1 TFA ile yıkayın. 20 ul% 40 ACN,% 0.5 asetik asit, daha sonra 20 ul% 80 ACN,% 0.5 asetik asit ile ilk Peptidlerin elüsyonu. Kombine eluatlar and vakumla süzme ile kuru. 10. Microcapillary LC-MS/MS <ol><li> , Yaklaşık 1 ug / ul &#39;lik bir konsantrasyona 1-5 ul,% 5 formik asit,% 5 ACN peptitler çözülür. On ~ 1 ug peptidler gidermek bir 100 um x C18-ters 0.125% formik asit içinde% 6-22 ACN gradyanı ile faz HPLC kolonu ~ 300 nl / dk &#39;lık bir akış oranında, 75 veya 100 dakikalık bir süre içinde tatbik 20 cm. </li><li> Bir LTQ Orbitrap Velos 12 veya kütle spektrometresi, yüksek çözünürlük ve yüksek kütle doğruluğu sağlayarak benzer sıvı kromatografisi-kütle spektrometresi platformunu kullanarak peptidler tanımlayın. Orbitrap analizörü edinilen tam bir MS tarama (60.000 çözünürlük) ile veri-bağımlı modunda kütle spektrometresi çalıştırın. Tam MS spektrum tespit 20 en bol bulunan iyonlar için lineer iyon kapanı MS / MS spektrumunu üretir. MS / MS için 1 x 10 tam MS için 6 ve 2000 için otomatik kazanç kontrolü (AGC) hedefleri ayarlayın. 1000 msn maksimum iyon birikimi kez ayarlayabilirsinizMS ve MS / MS için 150 msn için. 20-60 sn MS / MS uzak seçimden parçalanmış peptid öncüsü iyonları dışlamak. </li></ol> 11. MS / MS Veri Toplama Bu parametreleri (Tablo 1) kullanılarak bu SEQUEST 8 gibi bir algoritma ile bir teorik veritabanı MS / MS spektrumları RAW dosyaları karşılaştırarak peptidler tanımlamak. Tablo 1. Peptit Veritabanı Arama Parametreleri. <table border="0" cellpadding="0" cellspacing="0" width="600"><tbody><tr height="19"><td height="19" rowspan="3" style="height:19px;" width="180"> Genel Parametreler </td><td> kadar 2 ile tam triptik sindirim bölünmelere kaçırdı </td></tr><tr height="19"><td> 25 ppm öncül iyon tolerans </td></tr><tr height="19"><td> 1.0 Da fragman iyonu tolerans </td></tr><tr height="35"><td height="35" rowspan="2" style="height:36px;"> Statik Değişiklikler </strong&gt; </td><td> Sistein, carboxyamidomethylation üzerinde 57,02146 Da </td></tr><tr height="19"><td> Lisin ve peptid N-terminalinde on 28,03130 Da, hafif dimetilasyonu etiket </td></tr><tr height="19"><td height="19" rowspan="2" style="height:19px;"> Dinamik Değişiklikler </td><td> Metiyonin, oksidasyonu üzerindeki 15,99491 Da </td></tr><tr height="19"><td> Lisin ve peptid N-terminalinde, ağır dimetilasyonu etikette 6,03766 Da </td></tr></tbody></table><ol><li> Fiili ve ters yönlerde açık okuma çerçevelerinin bir veri tabanı kullanılarak bu tür hedef yem 9 strateji olarak bir yöntem ile,% 1 yanlış keşif oranına filtre peptidler. </li></ol> 12. Peptide Kantitasyonu Hesaplayın MS1 ağır ve hafif çiftlerinin alanlar iyon kromatogramları (MS1 pik alanları) ekstre edilmiş ve peptid sinyal-gürültü (S / N) oranları 10. Peptid çiftleri içerir yalnızca onların ortalama sinyal-noise oranı beş üzerindedir. Aynı peptid (MS1 tepe alanı oranı) ağır ve hafif versiyonlarının MS1 ​​pik alanlarının oranı olarak, iki örnekteki nispi bir peptidin bolluk ölçmek. Proteinin tüm peptidler için ortalama MS1 tepe alanı oranı olarak görece protein zengindi hesaplayın.

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Yazarlar herhangi bir mali çıkarlarını ya da diğer ilgi çatışmalarını beyan.

Yüksek ucuz etiketleme reaktifleri (reaktif numune başına 1 dolardan daha az maliyeti), hızlı reaksiyon hızı (~ 10 dk), yan ürünlerin yokluğu: Birkaç puan nicel Proteomikte için cazip bir yöntem indirgeyici dimetilasyonu (Redi etiketleme) kullanılarak peptidlerin kararlı izotop etiketleme yapmak tekrarlanabilirliği (Şekil 3, 4), sabit reaksiyon ürünleri, bir proteaz kullanma yeteneği ve işaretli peptidlerin yüksek iyonlaşma verim. Bir sentetik ortam üzerinde spesifik amino asit o...

Karmaşık numuneler arasında birçok proteinin konsantrasyonu farklılıklarını ölçme proteomikteki merkezi bir sorundur. Giderek, bu farklı izotopik etiketler, her numune proteinlerin etiketleme örnekleri birleştirilmesi ve konsantrasyon farkları ölçmek için kütle spektrometresi kullanılarak yapılmaktadır. Çeşitli yöntemler, protein ve peptid kararlı izotopik etiketleme için vardır. ICAT 3, 4 iTRAQ ve indirgeme dimetilasyonu 5 protein ekstraksiyonu ve sindirim sonra izotop etiketleri ekleme ise 15 N etiketleme 1 ve SILAC 2, in vivo olarak metabolik izotopik etiketler tanıtmak. Bu yöntemler arasında, indirgeyici dimetilasyonu (Redi etiketleme) numune hemen hemen her tür protein konsantrasyonu farklılıkları ölçmek için ucuz, tekrarlanabilir bir yöntem olarak popülerlik kazanıyor. Redi etiketleme düşürülür bir Schiff bazı oluşturmak üzere formaldehit ile reaksiyona sokulmasını içerir peptidlerisiyanoborohidrit. Bu reaksiyon, N-termini ve lisin yan zincirleri ve monomethylates N-terminal prolinler on serbest amino grupları dimethylates. Burada açıklanan protokol döteryumlu formaldehit ve siyanoborohidrit (Şekil 1) kullanılarak bir &quot;ağır&quot; etiketli doğal izotopik dağıtım ve örnek 2 hidrojen atomuna sahip reaktifler kullanılarak bir &quot;ışık&quot; etiketi ile örnek 1 &#39;de peptidler metile. Işık ve bir kütle spektrometresi kullanılarak, iki form arasında ayırt etmek için kullanılır ağır biçimleri arasında, 6,0377 Da&#39;lık bir kütle farkı, bir peptit sonuçlarına her dimetile amino grubudur. Özel olarak, nispi bolluk peptid ışığın MS1 ​​ekstre iyon kromatogram alanlarında oranında (MS1 alan doruk olan oranı) ve her bir peptit iyon çifti için ağır bir versiyonu olarak nicelendirilir. Bir proteinin nispi bolluk, proteinin tüm peptidler arasında orta MS1 tepe alanı oranı olarak hesaplanır. Bu yazıda, davranış için sağlam bir protokol açıklarters-faz peptid katı faz ekstraksiyonu, kolon Redi etiketleme, peptid fraksiyonasyon bazik pH fazı (BPRP) kromatografi tersine ve StageTips kullanarak peptid karışımları saflaştırılmasını (Şekil 2) içerir LC-MS/MS ile Redi etiketleme deneyleri ing . Biz kantitatif Proteomikte için Redi etiketleme kullanmanın avantajları ve sınırlamaları tartışmak.

<table border="0" cellpadding="0" cellspacing="0" width="973">
	<tbody>
		<tr height="19">
			<td height="19" style="height:19px;width:377px;">trichloroacetic acid</td>
			<td style="width:141px;">Sigma-Aldrich</td>
			<td style="width:136px;">T9159</td>
			<td style="width:319px;">protein precipitation</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">acetone</td>
			<td>Sigma-Aldrich</td>
			<td style="width:136px;">650501</td>
			<td>protein precipitation</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Sodium dodecyl sulfate</td>
			<td>Sigma-Aldrich</td>
			<td style="width:136px;">71736</td>
			<td>denature, reduce protein</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">sodium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td style="width:136px;">S8045</td>
			<td>denature, reduce protein</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">DL-Dithiothreitol</td>
			<td>Sigma-Aldrich</td>
			<td style="width:136px;">43816</td>
			<td>denature, reduce, alkylate protein</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">protease Inhibitor Complete Mini Cocktail</td>
			<td>Roche</td>
			<td>4693124001</td>
			<td>denature, reduce protein</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">iodoacetamide</td>
			<td>Sigma-Aldrich</td>
			<td>I6125</td>
			<td>alkylate protein</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">HEPES</td>
			<td>Sigma-Aldrich</td>
			<td style="width:136px;">H7523</td>
			<td>resuspend, extract, label protein</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">calcium chloride</td>
			<td>Sigma-Aldrich</td>
			<td style="width:136px;">C5670</td>
			<td>resuspend protein</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:377px;">Lysyl Endoprotease</td>
			<td>Wako Chemicals</td>
			<td style="width:136px;">129-02541</td>
			<td>protein digestion</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">sequencing grade trypsin</td>
			<td>Promega</td>
			<td style="width:136px;">V5111</td>
			<td>protein digestion</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">acetic acid</td>
			<td>Sigma-Aldrich</td>
			<td style="width:136px;">320099</td>
			<td>protein digestion</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">trifluoroacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>299537</td>
			<td>Reversed-phase peptide extraction</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">tC18 Sep-Pak C18 cartridges</td>
			<td>Waters</td>
			<td>WAT054960</td>
			<td>Reversed-phase peptide extraction</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">extraction manifold</td>
			<td>Waters</td>
			<td>WAT200609</td>
			<td>Reversed-phase peptide extraction</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">acetonitrile</td>
			<td>Sigma-Aldrich</td>
			<td style="width:136px;">14261</td>
			<td>various</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">formaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>252549</td>
			<td>&#8220;light&#8221; peptide labeling</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">cyanoborohydride</td>
			<td>Sigma-Aldrich</td>
			<td style="width:136px;">71435</td>
			<td>&#8220;light&#8221; peptide labeling</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">deuterated formaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>492620</td>
			<td>&#8220;heavy&#8221; peptide labeling</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">sodium cyanoborodeuteride</td>
			<td>CDN isotopes</td>
			<td style="width:136px;">D-1797</td>
			<td>&#8220;heavy&#8221; peptide labeling</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">MES</td>
			<td>Sigma-Aldrich</td>
			<td>M3671</td>
			<td>peptide labeling</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">C18-HPLC column (4.6 x 250 mm, 5 &#181;m particle size)</td>
			<td>Agilent</td>
			<td style="width:136px;">770450-902</td>
			<td>basic pH reversed-phase chromatography</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">formic acid</td>
			<td>Sigma-Aldrich</td>
			<td style="width:136px;">399388</td>
			<td>various</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">C18 Empore Disks</td>
			<td>3M</td>
			<td style="width:136px;">14-386-3&#160;</td>
			<td>STAGE tips</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">methanol</td>
			<td>Sigma-Aldrich</td>
			<td style="width:136px;">494437</td>
			<td>STAGE tips</td>
		</tr>
	</tbody>
</table>

quantitative proteomics using reductive dimethylation for stable isotope labeling

Indirgeyici dimetilasyonu (Redi etiketleme) tarafından peptidlerin kararlı izotop etiketleme doğru kütle spektrometresi kullanılarak örnekler arasında protein ekspresyon farklılıkları ölçmek için bir yöntemdir. Redi etiketleme her bir serbest amin için iki metil grubu eklemek için düzenli olarak (ışık) veya formaldehid ve sodyum siyanoborohidrid döteryumlanmış (ağır) formları kullanılarak gerçekleştirilir. Burada Redi etiketleme ve kompleks protein karışımları kantitatif karşılaştırılması için sağlam bir protokol göstermektedir. Karşılaştırma için Protein örnekleri hafif ya da ağır metil etiketler, karıştırılmış ve LC-MS/MS ile ko-analiz ya da taşımak için etiketli peptidler içine sindirilir. Bağıl protein bolluğu tam MS spektrumları elde oluşturucu peptidin ağır ve hafif olarak işaretlenmiş versiyonlarının iyon kromatogram tepe alanlarının karşılaştırılması ile nicelendirilir. Burada tarif edilen yöntem örnek ters-fazlı, katı faz ekstraksiyonu ile hazırlama, peptidlerin üzerinde kolon Redi etiketleme, bazik pH rev tarafından peptid içerir fraksiyonasyonersed faz (BPRP) kromatografi ve StageTip peptid saflaştırma. Biz kararlı izotop birleşme için diğer yöntemlere göre Redi etiketleme avantajları ve sınırlamaları tartışmak. Biz örnek hemen hemen her tür protein zengindi karşılaştırmak için, hızlı, ucuz ve doğru yöntem olarak Redi etiketleme kullanarak yeni uygulamalarını vurgulayın.

Biz Saccharomyces cerevisiae kullanılarak doğruluk, kesinlik ve Redi etiketleme tekrarlanabilirliği değerlendirildi ve Clostridium tüm hücre lizatlarını phytofermentans. Önce C bir karışımı Redi etiketleme verimliliği sayılabilir selüloz (ağır etiketli, H) ve glukoz (açık etiketli, L) kültürler protein lizatları phytofermentans. Bir% 1 peptit yanlış keşif orana süzüldü, bu örnek, bir% 98 verimle Redi etiketleme 11194 benzersiz peptid dizilerini i...

Watch this Scientific Journal Video about Quantitative Proteomics Using Reductive Dimethylation for Stable Isotope Labeling at JoVE.com

Quantitative Proteomics Using Reductive Dimethylation for Stable Isotope Labeling

Indirgeyici dimetilasyonu (Redi etiketleme) tarafından peptidlerin kararlı izotop etiketleme doğru kütle spektrometresi tabanlı nicel Proteomikte için hızlı, ucuz bir stratejidir. Burada, hemen hemen her tür örnek uygulanabilir Redi yaklaşım kullanılarak protein karışımlarının hazırlanması ve analizi için güçlü bir yöntem ortaya koymaktadır.

Kararlı İzotop Etiketleme için indirgeyici dimetilasyonu kullanma Sayısal Proteomiks

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a method to accurately quantify protein expression differences between ...

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Research

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Chemistry

16.0K Görüntüleme.  Genoscope.  Indirgeyici dimetilasyonu (Redi etiketleme) tarafından peptidlerin kararlı izotop etiketleme doğru kütle spektrometresi tabanlı nicel Proteomikte için hızlı, ucuz bir stratejidir. Burada, hemen hemen her tür örnek uygulanabilir Redi yaklaşım kullanılarak protein karışımlarının hazırlanması ve analizi için güçlü bir yöntem ortaya koymaktadır. 

Video: Quantitative Proteomics Using Reductive Dimethylation for Stable Isotope Labeling

Large Scale Non-targeted Metabolomic Profiling of Serum by Ultra Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS)

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to investigate metabolism. The approach offers an unbiased and in-depth analysis that can enable the development of diagnostic tests, novel therapies, and further our understanding of disease processes. The inherent chemical diversity of the metabolome creates significant analytical challenges and there is no single experimental approach that can detect all metabolites. Additionally, the biological variation in individual metabolism and the dependence of metabolism on environmental factors necessitates large sample numbers to achieve the appropriate statistical power required for meaningful biological interpretation. To address these challenges, this tutorial outlines an analytical workflow for large scale non-targeted metabolite profiling of serum by UPLC-MS. The procedure includes guidelines for sample organization and preparation, data acquisition, quality control, and metabolite identification and will enable reliable acquisition of data for large experiments and provide a starting point for laboratories new to non-targeted metabolite profiling by UPLC-MS.

Large Scale Non-targeted Metabolomic Profiling of Serum by Ultra Performance Liquid Chromatography-Mass Spectrometry UPLC-MS

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to investigate metabolism. This article outlines a typical workflow utilized for non-targeted metabolite profiling of serum including sample organization and preparation, data acquisition, data analysis, quality control, and metabolite identification.

Ultra Performans Sıvı Kromatografisi-Kütle Spektrometresi (UPLC-MS) ile Serum Büyük Ölçekli Sigara hedefli metabolomik Profilleme

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to ...

Kimya

Improved In-gel Reductive &#946;-Elimination for Comprehensive O-linked and Sulfo-glycomics by Mass Spectrometry

Separation of proteins by SDS-PAGE followed by in-gel proteolytic digestion of resolved protein bands has produced high-resolution proteomic analysis of biological samples. Similar approaches, that would allow in-depth analysis of the glycans carried by glycoproteins resolved by SDS-PAGE, require special considerations in order to maximize recovery and sensitivity when using mass spectrometry (MS) as the detection method. A major hurdle to be overcome in achieving high-quality data is the removal of gel-derived contaminants that interfere with MS analysis. The sample workflow presented here is robust, efficient, and eliminates the need for in-line HPLC clean-up prior to MS. Gel pieces containing target proteins are washed in acetonitrile, water, and ethyl acetate to remove contaminants, including polymeric acrylamide fragments. O-linked glycans are released from target proteins by in-gel reductive &#946;-elimination and recovered through robust, simple clean-up procedures. An advantage of this workflow is that it improves sensitivity for detecting and characterizing sulfated glycans. These procedures produce an efficient separation of sulfated permethylated glycans from non-sulfated (sialylated and neutral) permethylated glycans by a rapid phase-partition prior to MS analysis, and thereby enhance glycomic and sulfoglycomic analyses of glycoproteins resolved by SDS-PAGE.

Improved In-gel Reductive &amp;#946;-Elimination for Comprehensive O-linked and Sulfo-glycomics by Mass Spectrometry

In order to comprehensively explore the diversity of O-linked glycans, a new procedure for in-gel reductive &#946;-elimination, combined with permethylation and a rapid phase-partition method, is applied to the analysis of O-linked glycans directly released from glycoproteins resolved by SDS-PAGE and amenable to subsequent glycomic analysis by mass spectrometry.

Kütle spektrometresi ile In-jel Geliştirilmiş İndirgemeli β-Eleme Kapsamlı O-bağlı ve için Sulfo-Glikomik

Separation of proteins by SDS-PAGE followed by in-gel proteolytic digestion of resolved protein bands has produced high-resolution proteomic analysis of ...

Quantitative and Qualitative Examination of Particle-particle Interactions Using Colloidal Probe Nanoscopy

Colloidal Probe Nanoscopy (CPN), the study of the nano-scale interactive forces between a specifically prepared colloidal probe and any chosen substrate using the Atomic Force Microscope (AFM), can provide key insights into physical interactions present within colloidal systems. Colloidal systems are widely existent in several applications including, pharmaceuticals, foods, paints, paper, soil and minerals, detergents, printing and much more.1-3 Furthermore, colloids can exist in many states such as emulsions, foams and suspensions. Using colloidal probe nanoscopy one can obtain key information on the adhesive properties, binding energies and even gain insight into the physical stability and coagulation kinetics of the colloids present within. Additionally, colloidal probe nanoscopy can be used with biological cells to aid in drug discovery and formulation development. In this paper we describe a method for conducting colloidal probe nanoscopy, discuss key factors that are important to consider during the measurement, and show that both quantitative and qualitative data that can be obtained from such measurements.

Colloidal probe nanoscopy can be used within a variety of fields to gain insight into the physical stability and coagulation kinetics of colloidal systems and aid in drug discovery and formulation sciences using biological systems. The method described within provides a quantitative and qualitative means to study such systems.

Kolloidal Probe Nanoscopy kullanarak parçacık-parçacık Etkileşimleri Nicel ve Nitel Sınav

Colloidal Probe Nanoscopy (CPN), the study of the nano-scale interactive forces between a specifically prepared colloidal probe and any chosen substrate ...

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 ...

Standards for Quantitative Metalloproteomic Analysis Using Size Exclusion ICP-MS

Metals are essential for protein function as cofactors to catalyze chemical reactions. Disruption of metal homeostasis is implicated in a number of diseases including Alzheimer's and Parkinson's disease, but the exact role these metals play is yet to be fully elucidated. Identification of metalloproteins encounters many challenges and difficulties. Here we report an approach that allows metalloproteins in complex samples to be quantified. This is achieved using size exclusion chromatography coupled with inductively coupled plasma - mass spectrometry (SEC-ICP-MS). Using six known metalloproteins, the size exclusion column can be calibrated and the respective trace elements (iron, copper, zinc, cobalt, iodine) can be used for quantification. SEC-ICP-MS traces of human brain and plasma are presented. The use of these metalloprotein standards provides the means to quantitatively compare metalloprotein abundances between biological samples. This technique is poised to help shed light on the role of metalloproteins in neurodegenerative disease as well as other diseases where imbalances in trace elements are implicated.

Size exclusion chromatography hyphenated with inductively coupled plasma - mass spectrometry (ICP-MS) is a powerful tool to measure changes in the abundance of metalloproteins directly from biological samples. Here we describe a set of metalloprotein standards used to estimate molecular mass and the amount of metal associated with unknown proteins.

Boyut Dışlama ICP-MS kullanarak Nicel Metalloproteomic Analiz Standartları

Metals are essential for protein function as cofactors to catalyze chemical reactions. Disruption of metal homeostasis is implicated in a number of ...

Peptide and Protein Quantification Using Automated Immuno-MALDI (iMALDI)

Mass spectrometry (MS) is one of the most commonly used technologies for quantifying proteins in complex samples, with excellent assay specificity as a result of the direct detection of the mass-to-charge ratio of each target molecule. However, MS-based proteomics, like most other analytical techniques, has a bias towards measuring high-abundance analytes, so it is challenging to achieve detection limits of low ng/mL or pg/mL in complex samples, and this is the concentration range for many disease-relevant proteins in biofluids such as human plasma. To assist in the detection of low-abundance analytes, immuno-enrichment has been integrated into the assay to concentrate and purify the analyte before MS measurement, significantly improving assay sensitivity. In this work, the immuno- Matrix-Assisted Laser Desorption/Ionization (iMALDI) technology is presented for the quantification of proteins and peptides in biofluids, based on immuno-enrichment on beads, followed by MALDI-MS measurement without prior elution. The anti-peptide antibodies are functionalized on magnetic beads, and incubated with samples. After washing, the beads are directly transferred onto a MALDI target plate, and the signals are measured by a MALDI-Time of Flight (MALDI-TOF) instrument after the matrix solution has been applied to the beads. The sample preparation procedure is simplified compared to other immuno-MS assays, and the MALDI measurement is fast. The whole sample preparation is automated with a liquid handling system, with improved assay reproducibility and higher throughput. In this article, the iMALDI assay is used for determining the peptide angiotensin I (Ang I) concentration in plasma, which is used clinically as readout of plasma renin activity for the screening of primary aldosteronism (PA).

Peptide and Protein Quantification Using Automated Immuno-MALDI iMALDI

A protocol for the protein quantification in complex biological fluids using automated immuno-MALDI (iMALDI) technology is presented.

Peptid ve Protein miktar kullanarak otomatik IMMUNO-maldı (iMALDI)

Mass spectrometry (MS) is one of the most commonly used technologies for quantifying proteins in complex samples, with excellent assay specificity as a ...

Large-scale Top-down Proteomics Using Capillary Zone Electrophoresis Tandem Mass Spectrometry

Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) has been recognized as a useful tool for top-down proteomics that aims to characterize proteoforms in complex proteomes. However, the application of CZE-MS/MS for large-scale top-down proteomics has been impeded by the low sample-loading capacity and narrow separation window of CZE. Here, a protocol is described using CZE-MS/MS with a microliter-scale sample-loading volume and a 90-min separation window for large-scale top-down proteomics. The CZE-MS/MS platform is based on a linear polyacrylamide (LPA)-coated separation capillary with extremely low electroosmotic flow, a dynamic pH-junction-based online sample concentration method with a high efficiency for protein stacking, an electro-kinetically pumped sheath flow CE-MS interface with extremely high sensitivity, and an ion trap mass spectrometer with high mass resolution and scan speed. The platform can be used for the high-resolution characterization of simple intact protein samples and the large-scale characterization of proteoforms in various complex proteomes. As an example, a highly efficient separation of a standard protein mixture and a highly sensitive detection of many impurities using the platform is demonstrated. As another example, this platform can produce over 500 proteoform and 190 protein identifications from an Escherichia coli proteome in a single CZE-MS/MS run.

A detailed protocol is described for the separation, identification, and characterization of proteoforms in protein samples using capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS). The protocol can be used for the high-resolution characterization of proteoforms in simple protein samples and the large-scale identification of proteoforms in complex proteome samples.

Büyük ölçekli tepeden proteomik kılcal bölge Elektroforez Tandem kütle spektrometresi kullanılarak

Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) has been recognized as a useful tool for top-down ...

Automated Sample Multiplexing by using Combined Precursor Isotopic Labeling and Isobaric Tagging (cPILOT)

We have introduced a high throughput quantitative proteomics workflow, combined precursor isotopic labeling and isobaric tagging (cPILOT) capable of multiplexing up to 22 or 24 samples with tandem mass tags or isobaric N,N-dimethyl leucine isobaric tags, respectively, in a single experiment. This enhanced sample multiplexing considerably reduces mass spectrometry acquisition times and increases the utility of the expensive commercial isobaric reagents. However, the manual process of sample handling and pipetting steps in the strategy can be labor intensive, time consuming, and introduce sample loss and quantitative error. These limitations can be overcome through the incorporation of automation. Here we transferred the manual cPILOT protocol to an automated liquid handling device that can prepare large sample numbers (i.e., 96 samples) in parallel. Overall, automation increases feasibility and reproducibility of cPILOT and allows for broad usage by other researchers with comparable automation devices.

Automated Sample Multiplexing by using Combined Precursor Isotopic Labeling and Isobaric Tagging cPILOT

Combined precursor isotopic labeling and isobaric tagging (cPILOT) is an enhanced sample multiplexing strategy that is capable of increasing the number of samples that can be analyzed simultaneously with available isobaric tags. Incorporation of a robotic platform has greatly increased experimental throughput, reproducibility, and quantitative accuracy.

Kombine Öncül İzotopik Etiketleme ve İzobarik Etiketleme (cPILOT) kullanılarak Otomatik Numune Çoklama

We have introduced a high throughput quantitative proteomics workflow, combined precursor isotopic labeling and isobaric tagging (cPILOT) capable of ...

Stable Aqueous Suspensions of Manganese Ferrite Clusters with Tunable Nanoscale Dimension and Composition

Manganese ferrite clusters (MFCs) are spherical assemblies of tens to hundreds of primary nanocrystals whose magnetic properties are valuable in diverse applications. Here we describe how to form these materials in a hydrothermal process that permits the independent control of product cluster size (from 30 to 120 nm) and manganese content of the resulting material. Parameters such as the total amount of water added to the alcoholic reaction media and the ratio of manganese to iron precursor are important factors in achieving multiple types of MFC nanoscale products. A fast purification method uses magnetic separation to recover the materials making production of grams of magnetic nanomaterials quite efficient. We overcome the challenge of magnetic nanomaterial aggregation by applying highly charged sulfonate polymers to the surface of these nanomaterials yielding colloidally stable MFCs that remain non-aggregating even in highly saline environments. These non-aggregating, uniform, and tunable materials are excellent prospective materials for biomedical and environmental applications.

We report a one-pot hydrothermal synthesis of manganese ferrite clusters (MFCs) that offers independent control over material dimension and composition. Magnetic separation allows rapid purification while surface functionalization using sulfonated polymers ensures the materials are non-aggregating in biologically relevant medium. The resulting products are well positioned for biomedical applications.

Ayarlanabilir Nano ölçekli Boyut ve Bileşime Sahip Manganez Ferrit Kümelerinin Kararlı Sulu Süspansiyonları

Manganese ferrite clusters (MFCs) are spherical assemblies of tens to hundreds of primary nanocrystals whose magnetic properties are valuable in diverse ...

Tıbbi Bitkilerde 13C6-Glikoz Etiketlemesi Kullanılarak Birincil Organ Tanımlaması

13C6&#45;Glucose Labeling Associated with LC&#45;MS&#58; Identification of Plant Primary Organs in Secondary Metabolite Synthesis

This paper presents a novel and efficient method for certifying primary organs involved in secondary metabolite synthesis. As the most important secondary metabolite in Parispolyphylla var. yunnanensis (Franch.) Hand. -Mzt. (PPY), Paris saponin (PS) has a variety of pharmacological activities and PPY is in increasing demand. This study established leaf, rhizome, and stem-vascular-bundle 13C6-Glucose feeding and non-feeding four treatments to precisely certify the primary organs involved in Paris saponins VII (PS VII) synthesis. By combining liquid chromatography-mass spectrometry (LC-MS), the 13C/12C ratios of leaf, rhizome, stem, and root in different treatments were quickly and accurately calculated, and four types of PS isotopic ion peak(M&#8722;) ratios were found: (M+1) &#8722;/M&#8722;, (M+2) &#8722;/M&#8722;, (M+3) &#8722;/M&#8722; and (M+4) &#8722;/M&#8722;. The results showed that the ratio of 13C/12C in the rhizomes of the stem-vascular-bundle and rhizome feeding treatments was significantly higher than that in the non-feeding treatment. Compared to the non-feeding treatment, the ratio of PS VII molecules (M+2) &#8722;/M&#8722; in the leaves increased significantly under leaf and stem-vascular-bundle feeding treatments. Simultaneously, compared to the non-feeding treatment, the ratio of PS VII molecules (M+2) &#8722;/M&#8722; in the leaves under rhizome treatment showed no significant difference. Furthermore, the ratio of PS VII molecules (M+2) &#8722;/M&#8722; in the stem, root, and rhizome showed no differences among the four treatments. Compared to the non-feeding treatment, the ratio of the Paris saponin II (PS II) molecule (M+2) &#8722;/M&#8722; in leaves under leaf feeding treatment showed no significant difference, and the (M+3) &#8722;/M&#8722; ratio of PS II molecules in leaves under leaf feeding treatment were lower. The data confirmed that the primary organ for the synthesizing of PS VII is the leaves. It lays the foundation for future identification of the primary organs and pathways involved in the synthesis of secondary metabolites in medicinal plants.

Yazar Spotlight: 13C6-Glikoz Etiketleme ve LC-MS Kullanarak Bitki Birincil Organları Profili Oluşturma

The developed method of 13C6-Glucose labeling combined with liquid chromatography high-resolution mass spectrometry is versatile and lays the foundation for future studies on the primary organs and pathways involved in the synthesis of secondary metabolites in medicinal plants, as well as the comprehensive utilization of these secondary metabolites.

13C 6-LC-MS ile İlişkili Glikoz Etiketlemesi: İkincil Metabolit Sentezinde Bitki Birincil Organlarının Tanımlanması

This paper presents a novel and efficient method for certifying primary organs involved in secondary metabolite synthesis. As the most important secondary ...