This research was generously funded by the NIH NCI IMAT Program Grant R33 (CA147988-02), the NIH NIGMS Graduate Training in Molecular Biotechnology at NC State Grant (T32GM008776), the US Dept. of Education GAANN Fellowship Program in Molecular Biotechnology at NC State Grant (P200A140020), the W.M. Keck Foundation, and North Carolina State University. Hen plasma was obtained with the assistance of Dr. James N. Petitte and Rebecca Wysocky in the NC State University Dept. of Poultry Science.

<ol>
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We have a collaborative relationship with Cambridge Isotope Laboratories (CIL) to disseminate the P2GPN reagent and technology to the broader scientific community in the form of a product (GTK-1000). However, we do not receive any revenue based on the sale of this product.

High-throughput quantitative methods are needed to facilitate routine glycan analysis. For the last thirty years, glycomics analysis has been limited to a subset of research groups, despite its importance in disease, clinical applications, and pharmaceuticals. The FANGS-P2GPN purification and tagging method for glycomics and proteomics performs the same analysis on a single aliquot of sample, reducing the cost of supplies and the amount of material needed (particularly important in human and mouse studies). Furthermore, efforts to minimize variability in preparations are critically important, as every additional step contributes to error, potentially masking important but low-abundant changes in case-control studies. Coupling of FANGS to hydrophobic hydrazide tagging allows protein and glycan samples to be run on the same RPLC column, enhances glycan ionization, provides for relative quantification, and can be quantitatively applied to plasma.
For N-glycan analysis, it is critical to use the suggested level of PNGase F to achieve full de-glycosylation. Though glycans are solvent exposed, denaturation of proteins and excess enzyme help ensure efficient and complete cleavage. For accurate quantitation of the glycans, it is necessary to ensure that they are completely dried after derivatization to quench the reaction and prevent cross-reactions when mixing the NAT and SIL species. Finally, when extending the workflow to glycosite analysis, timing of the steps is critical to minimize non-specific deamidation. The modified protocols provided for combined glycomics and proteomics analysis work consistently when performed accordingly.
The workflow achieves accurate relative quantitation of N-glycans from plasma compared to the gold-standard, SPE method. There is no apparent bias in the types of glycans extracted in terms of molecular weight, hydrophilicity, and compositional structure. Though we have not explored the qualitative analysis of O-linked glycans, we expect that FANGS could accommodate the addition of a &#946;-elimination step post-PNGase F digestion of N-linked glycans. However, procedures would require significant modification for reagent cleanup prior to mass spectrometry, and peptide analysis will be significantly impacted. For proteomics, the same depth of proteome coverage is achieved compared to traditional FASP methods. Importantly, methods achieve a minimal false discovery rate for N-glycan deamidation. While the method is compatible with 18O labeling of Asn during the PNGase digestion step22,23, the low glycosylation site false discovery rate suggests that it may not be necessary, further reducing costs and complexity.
The proteome is not enriched for glycoproteins in this method, which has both advantages and disadvantages. Certain low abundant glycoproteins may not be detected in the analysis. However, the occupancy of glycosylated sites per protein, can be compared between biological samples. Additionally, the error and bias introduced from lectin affinity purification or chemical enrichment is eliminated. In conclusion, coupling of FANGS to the individuality normalization when labeling with glycan hydrazide tags strategy results in a simplified, quantitative, high-throughput method for the tandem analysis of the glycome and proteome with great potential for application in clinical case-control studies.

Proteomik alanında, filtre destekli numune hazırlama (FASP), yaygın olarak, başlangıç ​​malzemesinin miktarını en aza indirmek örnek hazırlama eserler azaltmak ve numune sayısını en üst düzeye çıkarmak 1 kabiliyeti için kabul edilmiştir. Bununla birlikte, bu tür bir yöntem henüz ortaya çıkmakta ve Glikomik alanında için çekiş gücü elde etmek için yer alır. Yüksek verimlilik gelişimi, nicel iş akışları nedeniyle ayrılmaz biyolojik savunma glycoslasyonun rolü ve kanser veya hastalıkların 2,3 ederek modülasyon ihtiyaç vardır. Memelilerde, N -glycans bir çekirdek yapısı dekore sakarit birimi (heksozlar (Hex), hekzoaminler (HexNac), sialik asitleri (NeuAc) ve fucoses (Fuc)), (Hex 3 HexNac 2) 4 kovalent olarak bağlı tekrar oluşur asparajin. Izomerleri sayılır zaman glycospace oldukça geniş olmasına rağmen (&gt; 10 ila 12), bu gibi bir bileşim olarak oldukça küçük ve moleküler ağırlıkları tipik olarak 1,000-8,000 da 5 &lt;arasında değişir/ Sup&gt;. Sınıfının kompozisyon homojenliği ve glıkanların hidrofiliklik saflaştırma, ayırma özel bir sorun teşkil ve kütle spektrometrisi (MS) 6 akışları. Geleneksel olarak, N -glycans F (PNGase F) ile Peptid N protein veya peptidlerden glikosidaz sindirilir ve daha sonra hidrazid boncuklar 8 ile lektin afinite kromatografisi 7 ile zenginleştirilmiş yakalanan veya katı faz ekstraksiyonu (SPE), 9,10 ile saflaştırılmıştır. Bu yöntemlerin hepsi son derece etkili olsa da, tuzsuzlaştırma için ekstra adımlar tanıtmak ve örnek sayısı aynı anda işleme sınırlamak. Son on yılda, Glikomik için yüksek verimli platformlar bir dizi önerilmiştir. Kim ve ark., Bir vakum çalışan, SPE 96 oyuklu plaka 11 ile, bir yarı-otomatik bir yöntem yayınlanmıştır. Alternatif olarak, bir afinite filtre yöntemi (N -glyco-FASP) fi ilk türetme gerekli Mann grubu tarafından geliştirilenLektinlerin 12 bir kompozit ile ltresi. Son olarak, Thomas-Oates grup glycospace 13 dar kompozisyon boyutunu sömürülen bir yarı-kantitatif bir yöntem, N -Glycan Ayırma (dişleri) Destekli Filtre, önerdi. Molekül ağırlığı kesme filtreleri dayanarak küçük kirletici maddeler ilk atık için yıkandı ve daha sonra N -glycans sindirilir ve yıkandı. Deglikolize proteinler Bu protokol filtre üzerinde kalır ve hat FASP tabi tutulabilir. Tanımlama ve elektrosprey iyonizasyon ile glıkanların miktar (ESI) MS düşük miktardaki türlerinin tespiti için izomerleri ve türetme (kısmen) çözümlemesi için off-line ayrımları gerektirir. Bireysellik etiketleme glikan hidrazit etiketleri stratejisi ile normalize ters-faz sıvı kromatografisi (RPLC) 14,15 ile uyumluluğu bahşeder. 4-fenetil-benzohidrazid (P2PGN) hidrofobik etiketi enh, glıkanların hidrofilikliğini aracılıkortalama ile iyonizasyon ancing, 16 dört kat. Basit şartlarda stoikiometrik 1: örneğin permethylation 17 ya da amin-reaktif etiketleme kimyaları 18 gibi diğer teknikler, benzer avantajlar sunmaktadır rağmen, hidrazid reaksiyonda, glikanlar 1 tepkimeye sokulur. Rölatif ölçümü yerli (NAT) veya 13 C 6 stabil izotop etiket (SIL) ile derive edilmiş numunelerin ikili analizi ile elde edilmektedir. Aşağıdaki yöntem doğru göreceli ölçümü için P2GPN hidrofobik etikete plazma uygulamaları ve çiftler bunu için dişlerini geliştikçe. Ayrıca, bu analizler bütünlüğünden ödün vermeden, numunenin tek alikot üzerinde atış-silah proteomik, deamidasyon profilleme ve kantitatif Glikomik gerçekleştirmek için tasarlanmıştır.

<table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always">
	<tbody>
		<tr >
			<td >Acetic Acid (50%):&#160;</td>
			<td >Sigma Aldrich&#160;</td>
			<td >45754</td>
			<td ></td>
		</tr>
		<tr >
			<td >Acetonitrile, HPLC grade</td>
			<td >Burdick &#38; Jackson&#160;</td>
			<td >AH015-4</td>
			<td ></td>
		</tr>
		<tr >
			<td >Ammonium Bicarbonate</td>
			<td >Sigma Aldrich&#160;</td>
			<td >A6141</td>
			<td ></td>
		</tr>
		<tr >
			<td >Bradford Reagent</td>
			<td >Sigma Aldrich&#160;</td>
			<td >B6916</td>
			<td>Alternative: Bicinchoninic acid kit (Sigma Aldrich BCA1)</td>
		</tr>
		<tr >
			<td >Calcium chloride</td>
			<td >Sigma Aldrich&#160;</td>
			<td >C1016</td>
			<td></td>
		</tr>
		<tr >
			<td >Centrifuge</td>
			<td >Eppendorf</td>
			<td >5804 R</td>
			<td >Alternate centrifuges that reach 14,000 x g are suitable</td>
		</tr>
		<tr >
			<td >DL-Dithiothreitol, 1M in solution</td>
			<td >Sigma Aldrich&#160;</td>
			<td >646563</td>
			<td ></td>
		</tr>
		<tr >
			<td >Easy-nLC 1000</td>
			<td >Thermo Scientific</td>
			<td >LC120</td>
			<td >Alternate nano or ultra high pressure LCs will produce similar data, such as: 1. Dionex UltiMate&#210; 3000 LC (Thermo Scientific) 2. Acquity UPLC (Waters)</td>
		</tr>
		<tr >
			<td >Floating Tube Rack</td>
			<td >TedPella</td>
			<td >20831-20</td>
			<td></td>
		</tr>
		<tr >
			<td >Fetuin</td>
			<td >New England Biolabs&#160;</td>
			<td >P6042S</td>
			<td ></td>
		</tr>
		<tr >
			<td >Fisher Scientific Isotemp Standard Lab Ovens</td>
			<td >Fisher Scientific</td>
			<td >11-690-625F</td>
			<td >Alternate incubators that reach 56 &#176;C are suitable</td>
		</tr>
		<tr >
			<td >Formic Acid</td>
			<td >Sigma Aldrich&#160;</td>
			<td >56302</td>
			<td ></td>
		</tr>
		<tr >
			<td >GE Microwave Oven</td>
			<td >General Electric</td>
			<td >57B5 E82904</td>
			<td >Any microwave with adjustable power settings is suitable</td>
		</tr>
		<tr >
			<td >INLIGHT Glycan Tagging Kit&#160;</td>
			<td >Cambridge Isotope Laboratories</td>
			<td >GTK-1000</td>
			<td >The INLIGHT kit provides NAT and SIL versions of the P2GPN reagent.</td>
		</tr>
		<tr >
			<td >Iodoacetamide</td>
			<td >Sigma Aldrich&#160;</td>
			<td >A3221</td>
			<td></td>
		</tr>
		<tr >
			<td >Kinetix 2.6 mM, 100 &#197;, C18 bulk stationary phase</td>
			<td >Phenomenex&#160;</td>
			<td>Bulk Media</td>
			<td >Alternative: Any C18 stationary phase &#163; 5 mM&#160;</td>
		</tr>
		<tr >
			<td >Mascot Daemon Software and Server</td>
			<td >Matrix Science</td>
			<td ></td>
			<td >Alternative: Proteome Discoverer Software (Thermo Scientific)</td>
		</tr>
		<tr >
			<td >Methanol, HPLC grade</td>
			<td >Burdick &#38; Jackson&#160;</td>
			<td >AH230-4</td>
			<td ></td>
		</tr>
		<tr >
			<td >PicoFrit Self-Pack Column: 360 um, OD 75um ID, 15 um tip, non-coated, 5 per box, 50 cm</td>
			<td >New Objective</td>
			<td >1 5 PF360-75-15-N-5</td>
			<td></td>
		</tr>
		<tr >
			<td >PNGase F (glycerol-free), 75,000 units/ml</td>
			<td >New England BioLabs&#160;</td>
			<td >P0705L</td>
			<td ></td>
		</tr>
		<tr >
			<td >Q Exactive HF Hybrid Quadrupole-Orbitrap Mass Spectrometer</td>
			<td >Thermo Scientific</td>
			<td ></td>
			<td >Alternate high mass accuracy (&#163; 5 ppm) mass spectrometers will provide similar data</td>
		</tr>
		<tr >
			<td >RNase B</td>
			<td >New England Biolabs&#160;</td>
			<td >P7817S</td>
			<td ></td>
		</tr>
		<tr >
			<td >Trypsin from Porcine Pancreas</td>
			<td >Sigma Aldrich&#160;</td>
			<td >T6567-5X</td>
			<td></td>
		</tr>
		<tr >
			<td >Urea</td>
			<td >Sigma Aldrich&#160;</td>
			<td >51456</td>
			<td></td>
		</tr>
		<tr >
			<td >Vacuum ConcentratorSavant SPD131DDA SpeedVac Concentrator</td>
			<td >Thermo Scientific</td>
			<td >SPD131DDA</td>
			<td >Alternate vacuum concentrators are suitable</td>
		</tr>
		<tr >
			<td >Vivacon 500 30 kDa Filters&#160;</td>
			<td >Sartorius Stedim Biotech</td>
			<td >VN01H22</td>
			<td >Alternative: Amicon Ultra 0.5 Centrifugal Filter Units with Ultracel-10 kDa Membrane (Millipore UFC501096)</td>
		</tr>
		<tr >
			<td >Water, HPLC grade</td>
			<td >Burdick &#38; Jackson&#160;</td>
			<td >AH365-4</td>
			<td ></td>
		</tr>
		<tr >
			<td >Water, 18O</td>
			<td >Cambridge Isotope Laboratories</td>
			<td >OLM-240-97-1</td>
			<td >The addition of 18O in the PNGase F digest step is optional and may not be necessary for deamidation studies completed with 95% confidence</td>
		</tr>
		<tr >
			<td >Xcalibur 2.0</td>
			<td >Thermo Scientific</td>
			<td>XCALIBUR20</td>
			<td></td>
		</tr>
		<tr >
			<td >Zwittergent Test Kit</td>
			<td >Merck Millipore</td>
			<td >693030</td>
			<td></td>
		</tr>
	</tbody>
</table>

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.

<img alt="Şekil 1" src="/files/ftp_upload/53735/53735fig1.jpg" /> Şekil 1:. Kombine proteomik ve Glikomik analizi için dişleri-P2GPN hidrofobik etiketleme birleştiğinde yöntemin şeması (Yöntem A) verilen glikozilasyon sitesi tanımlama ile Glikomik sadece işleme ve tandem Glikomik ve proteomik analizi arasında farklılık adımlar, vurgulanır. <a href="https://www.jove.com/files/ftp_upload/53735/53735fig1large.jpg" target="_blank">Bu rakamın büyük halini görmek için lütfen buraya tıklayınız.</a> Içinde hat proteomik ve Glikomik, filtre tabanlı P2GPN hidrofobik etiketleme yöntemi (Yöntem A) toplanmış tavuk plazma örnekleri (Şekil 1) kullanılarak belirlenmesi, kantitatif ve molekül ağırlığı önyargı için doğrulandı. Yalnızca Glikomik deneyler için, N bolluk arası karşılaştırmalar Yöntemi ile ekstre -glycansBir SPE (altın standardı, Metot B) yapılmıştır (Şekil 2) carbograph için. İki yöntem arasındaki glukanlardır bolluk açısından anlamlı bir fark yoktu. aynı yöntemle ekstre glıkanların NAT oranı: içi değişkenliği de SIL nicelleştirilmesiyle tayin edilmiştir. Her iki yöntemin günlük 2 dağıtımı: sıfır merkezli, Gauss vardı ve (Şekil 3A-B) anlamlı olarak farklı değildi. Molekül ağırlığı filtresi N molekül ağırlığı aralığı ve hidrofil (Şekil 4) dayalı -glycans ayrımcılık olmadığını düşündüren, tamamen üst üste iki protokol arasında değişmektedir. <img alt="şekil 2" src="/files/ftp_upload/53735/53735fig2.jpg" /> Şekil 2:. NAT ve SIL N eşdeğer moldeki bir karışımı Yöntem A veya Yöntem B ile ekstre -glycans 2.5 tavuk plazma ul (n = 4) Örnekler edildi anal hazırlandıHer NAT / SIL kanalında bölüm 6. Her glikan için bolluğu önerilen önerilen parametrelere göre UPLC-MS ile yzed, moleküler düzeltmek, çıkarılan iyon kromatogram (MMA = 3 ppm) altındaki alanların integrasyonu ile hesaplanmıştır TGNF ağırlık örtüşme ve ayarlama. Bu oranlar standart hata ile gösterilir. Yöntem B bolluğu. Yöntem A N -glycans (p&gt; 0.05) anlamlı bir fark yoktu <a href="https://www.jove.com/files/ftp_upload/53735/53735fig2large.jpg" target="_blank">bu rakamın daha büyük bir versiyonunu görmek için lütfen buraya tıklayınız.</a> <img alt="Şekil 3," src="/files/ftp_upload/53735/53735fig3.jpg" /> Şekil 3, (A) yöntemi, bir içi değişkenliği (N = 133), ya da (b) Yöntem B (n = 123) stratejiler karşılaştırılmıştır. Aynı çıkarma düzeni N -glycans NAT ve SIL eşit molar karışımları,Üç teknik çoğaltır üzerinde analiz edildi. Günlük 2 dağıtımı: sıfır merkezli ve iki iş akışları arasında. Anlamlı fark edildi <a href="https://www.jove.com/files/ftp_upload/53735/53735fig3large.jpg" target="_blank">bu rakamın daha büyük bir versiyonunu görmek için lütfen buraya tıklayınız.</a> <img alt="Şekil 4," src="/files/ftp_upload/53735/53735fig4.jpg" /> Şekil 4: Her stratejiden glıkanların molekül ağırlığı aralıkları karşılaştırıldı. İki iş akışları aynı molekül ağırlığı aralığı kapsayan glıkanları vermiştir. N, diğer sadece algılama (1 × 10 5 bolluk) sınırının üzerinde düştü karşı bir protokolde tespit -glycans ve sistematik önyargı yansıtmıyor. <a href="https://www.jove.com/files/ftp_upload/53735/53735fig4large.jpg" target="_blank">Bir büyük görmek için tıklayınız Bu rakamın sürümü.</a> <p class="jove_content" fo:keep-together.içinde sayfa = &quot;1&quot;&gt; proteom geniş analizi ve glikozilasyon sitesi tanımlama in-line etkin molekül ağırlığı filtresi tarafından deglycosylated proteinlerin Tutma. Glikanların ve proteinlerin bir araya saflaştırılması için birden çok protokol Deglikosilasyondan (Tablo 5) olmadan geleneksel FASP hazırlanması ile karşılaştırılmıştır. FASP tripsin sindirmek (Std protokolü) tarafından takip Bizim tipik 18 saat filtre tabanlı PNGase F sindirim, glycosites tanımlanması engelleyebilir non-spesifik deaminasyondan önemli düzeylerde sonuçlandı. Bu nedenle, yüksek bir sıcaklıkta (50 ° C) ve bir PNGase F başak aşama (SPI protokolü) de kısa bir kuluçkalama süresinde kullanan bir yöntem olup, bir mikrodalga sindirim protokolü (MD protokolü) ile birlikte, bir alternatif olarak araştırılmıştır, sivil en aza indirmek için belirli bir deamidasyon. Yeni sindirim yöntemleri gözlenen glıkanlar, standart 18 saat gelen kompozisyonlar ya da bolluk içinde anlamlı bir farklılık yoktu 37 ° C filtre PNGase F özeti ( <strong&gt; Şekil 5). Kısa bir tripsin inkübasyon ile birlikte, non-spesifik deamidasyon anlamlı glycosites &lt;% 5 (Tablo 5) için bir yalancı pozitiflik oranı ile azalmıştır. <table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td colspan="2"></td><td colspan="4"> proteinler </td><td></td><td colspan="4"> peptitler </td><td></td><td colspan="4"> amitsizleştirilmiş Peptitler </td><td></td><td colspan="4"> Glycosites </td><td></td><td colspan="4"> glikoproteinler </td></tr><tr><td colspan="2"></td><td colspan="2"> + </td><td colspan="2"> - </td><td></td><td colspan="2"> + </td><td colspan="2"> - </td><td></td><td colspan="2"> + </td><td colspan="2"> - </td><td></td><td colspan="2"> + </td><td colspan="2"> - </td><td></td><td colspan="2"> + </td><td colspan="2"> - </td></tr><tr><td colspan="2"> FASP </td><td colspan="2"></td><td colspan=&quot;2&quot;&gt; 249 </td><td></td><td colspan="2"></td><td colspan="2"> 3083 </td><td></td><td colspan="2"></td><td colspan="2"> 795 </td><td></td><td colspan="2"></td><td colspan="2"> 5 </td><td></td><td colspan="2"></td><td colspan="2"> 5 </td></tr><tr><td colspan="2"> 18hr </td><td colspan="2"> 217 </td><td colspan="2"> 304 </td><td></td><td colspan="2"> 6013 </td><td colspan="2"> 5086 </td><td></td><td colspan="2"> 1029 </td><td colspan="2"> 733 </td><td></td><td colspan="2"> 257 </td><td colspan="2"> 24 </td><td></td><td colspan="2"> 112 </td><td colspan="2"> 18 </td></tr><tr><td colspan="2"> MD </td><td colspan="2"> 270 </td><td colspan="2"> 266 </td><td></td><td colspan="2"> 5190 </td><td colspan="2"> 5125 </td><td></td><td colspan="2"> 465 </td><td colspan="2"> 455 </td><td></td><td colspan="2"> 254 </td><td colspan="2"> 8 </td><td></td><td colspan="2"> 102 </td><td colspan="2"> 7 </td></tr><tr><td colspan="2"> SPI </td><td colspan="2"> 281 </td><td colspan="2"> 288 </td><td></td><td colspan="2"> 4729 </td><td colspan="2"> 4482 </td><td></td><td colspan="2"> 602 </td><td colspan="2"> 573 </td><td></td><td colspan="2"> 232 </td><td colspan="2"> 10 </td><td></td><td colspan="2"> 145 </td><td colspan="2"> 8 </td></tr></tbody></table> . Tablo 5: Standart bir tripsin ile proteomik verilerin karşılaştırılması in-line dişleri-P2GPN etiketleme yöntemi yapılmıştır karşı sindirmek hazırlıklar ile gerçekleştirildi (+) ve olmayan (-) PNGase F non-spesifik deaminasyondan ve tahmin arka plan oranlarını belirlemek için yalancı pozitif oranları glycosite. Proteinler% 1 yanlış keşif oranı (FDR) ile tespit edilmiştir; peptidler Proteome Discoverer 1.4 &quot;yüksek&quot; peptid güvene dayalı filtre edildi (q &lt;0.01); glycosites saptanmasına edildied korunmuş glikosilasyon motifi (NXS / T) deamidasyonu içeren benzersiz dizilerinin dayalı; ve glikoproteinler, en az bir tespit glycosite ihtiva tanımlanan proteinler olarak tanımlandı. <img alt="Şekil 5," src="/files/ftp_upload/53735/53735fig5.jpg" /> Şekil 5: glikanlara kısaltılmış protokoller örneklerde deaminasyonu en aza indirmek için geliştirilmiş ve 18hr dişleri PNGase F Digest karşılaştırılmıştır. bollukları ortalama farklılıklar% 10 ve sırasıyla MD (2.2.3) ve SPI (2.2.2) protokolleri,% 5 idi. Tespit 48 glukanlardır,% 90 daha az 1,5 kat varyasyon daha. Görüntülenen <a href="https://www.jove.com/files/ftp_upload/53735/53735fig5large.jpg" target="_blank">bu rakamın daha büyük bir versiyonunu görmek için lütfen buraya tıklayınız.</a>

Watch this Scientific Journal Video about A Quantitative Glycomics and Proteomics Combined Purification Strategy at JoVE.com

A Quantitative Glycomics and Proteomics Combined Purification Strategy

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 ...

a-quantitative-glycomics-and-proteomics-combined-purification-strategy

Research

JoVE Journal

Chemistry

14.8K Görüntüleme.  North Carolina State University. 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.

Video: A Quantitative Glycomics and Proteomics Combined Purification Strategy

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

Quantitative Proteomics Using Reductive Dimethylation for Stable Isotope Labeling

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a method to accurately quantify protein expression differences between samples using mass spectrometry. ReDi labeling is performed using either regular (light) or deuterated (heavy) forms of formaldehyde and sodium cyanoborohydride to add two methyl groups to each free amine. Here we demonstrate a robust protocol for ReDi labeling and quantitative comparison of complex protein mixtures. Protein samples for comparison are digested into peptides, labeled to carry either light or heavy methyl tags, mixed, and co-analyzed by LC-MS/MS. Relative protein abundances are quantified by comparing the ion chromatogram peak areas of heavy and light labeled versions of the constituent peptide extracted from the full MS spectra. The method described here includes sample preparation by reversed-phase solid phase extraction, on-column ReDi labeling of peptides, peptide fractionation by basic pH reversed-phase (BPRP) chromatography, and StageTip peptide purification. We discuss advantages and limitations of ReDi labeling with respect to other methods for stable isotope incorporation. We highlight novel applications using ReDi labeling as a fast, inexpensive, and accurate method to compare protein abundances in nearly any type of sample.

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a rapid, inexpensive strategy for accurate mass spectrometry-based quantitative proteomics. Here we demonstrate a robust method for preparation and analysis of protein mixtures using the ReDi approach that can be applied to nearly any sample type.

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 ...

Synthesis and Purification of Iodoaziridines Involving Quantitative Selection of the Optimal Stationary Phase for Chromatography

The highly diastereoselective preparation of cis-N-Ts-iodoaziridines through reaction of diiodomethyllithium with N-Ts aldimines is described. Diiodomethyllithium is prepared by the deprotonation of diiodomethane with LiHMDS, in a THF/diethyl ether mixture, at -78 &#176;C in the dark. These conditions are essential for the stability of the LiCHI2 reagent generated. The subsequent dropwise addition of N-Ts aldimines to the preformed diiodomethyllithium solution affords an amino-diiodide intermediate, which is not isolated. Rapid warming of the reaction mixture to 0 &#176;C promotes cyclization to afford iodoaziridines with exclusive cis-diastereoselectivity. The addition and cyclization stages of the reaction are mediated in one reaction flask by careful temperature control.
Due to the sensitivity of the iodoaziridines to purification, assessment of suitable methods of purification is required. A protocol to assess the stability of sensitive compounds to stationary phases for column chromatography is described. This method is suitable to apply to new iodoaziridines, or other potentially sensitive novel compounds. Consequently this method may find application in range of synthetic projects. The procedure involves firstly the assessment of the reaction yield, prior to purification, by 1H NMR spectroscopy with comparison to an internal standard. Portions of impure product mixture are then exposed to slurries of various stationary phases appropriate for chromatography, in a solvent system suitable as the eluent in flash chromatography. After stirring for 30 min to mimic chromatography, followed by filtering, the samples are analyzed by 1H NMR spectroscopy. Calculated yields for each stationary phase are then compared to that initially obtained from the crude reaction mixture. The results obtained provide a quantitative assessment of the stability of the compound to the different stationary phases; hence the optimal can be selected. The choice of basic alumina, modified to activity IV, as a suitable stationary phase has allowed isolation of certain iodoaziridines in excellent yield and purity.

A protocol for the diastereoselective one-pot preparation of cis-N-Ts-iodoaziridines is described. The generation of diiodomethyllithium, addition to N-Ts aldimines and cyclization of the amino gem-diiodide intermediate to iodoaziridines is demonstrated. Also included is a protocol to rapidly and quantitatively assess the most appropriate stationary phase for purification by chromatography.

Sentez ve Kromatografi için optimal sabit faz kantitatif seçimi dahil edilmesi Iodoaziridines saflaştırılması

The highly diastereoselective preparation of cis-N-Ts-iodoaziridines through reaction of diiodomethyllithium with N-Ts aldimines is described. ...

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.

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 ...

CN-GELFrEE - Clear Native Gel-eluted Liquid Fraction Entrapment Electrophoresis

Protein complexes perform an array of crucial cellular functions. Elucidating their non-covalent interactions and dynamics is paramount for understanding the role of complexes in biological systems. While the direct characterization of biomolecular assemblies has become increasingly important in recent years, native fractionation techniques that are compatible with downstream analysis techniques, including mass spectrometry, are necessary to further expand these studies. Nevertheless, the field lacks a high-throughput, wide-range, high-recovery separation method for native protein assemblies. Here, we present clear native gel-eluted liquid fraction entrapment electrophoresis (CN-GELFrEE), which is a novel separation modality for non-covalent protein assemblies. CN-GELFrEE separation performance was demonstrated by fractionating complexes extracted from mouse heart. Fractions were collected over 2 hr and displayed discrete bands ranging from ~30 to 500 kDa. A consistent pattern of increasing molecular weight bandwidths was observed, each ranging ~100 kDa. Further, subsequent reanalysis of native fractions via SDS-PAGE showed molecular-weight shifts consistent with the denaturation of protein complexes. Therefore, CN-GELFrEE was proved to offer the ability to perform high-resolution and high-recovery native separations on protein complexes from a large molecular weight range, providing fractions that are compatible with downstream protein analyses.

This protocol describes how to prepare and perform clear native gel-eluted liquid fraction entrapment electrophoresis (CN-GELFrEE), a native separation technique for non-covalent biomolecular assemblies and proteins from heterogeneous samples that is compatible with various downstream protein analysis techniques.

CN-GELFrEE - net yerli jel-elüt sıvı kısmı Tuzak Elektroforez

Protein complexes perform an array of crucial cellular functions. Elucidating their non-covalent interactions and dynamics is paramount for understanding ...

Glycan Node Analysis: A Bottom-up Approach to Glycomics

Synthesized in a non-template-driven process by enzymes called glycosyltransferases, glycans are key players in various significant intra- and extracellular events. Many pathological conditions, notably cancer, affect gene expression, which can in turn deregulate the relative abundance and activity levels of glycoside hydrolase and glycosyltransferase enzymes. Unique aberrant whole glycans resulting from deregulated glycosyltransferase(s) are often present in trace quantities within complex biofluids, making their detection difficult and sometimes stochastic. However, with proper sample preparation, one of the oldest forms of mass spectrometry (gas chromatography-mass spectrometry, GC-MS) can routinely detect the collection of branch-point and linkage-specific monosaccharides ("glycan nodes") present in complex biofluids. Complementary to traditional top-down glycomics techniques, the approach discussed herein involves the collection and condensation of each constituent glycan node in a sample into a single independent analytical signal, which provides detailed structural and quantitative information about changes to the glycome as a whole and reveals potentially deregulated glycosyltransferases. Improvements to the permethylation and subsequent liquid/liquid extraction stages provided herein enhance reproducibility and overall yield by facilitating minimal exposure of permethylated glycans to alkaline aqueous conditions. Modifications to the acetylation stage further increase the extent of reaction and overall yield. Despite their reproducibility, the overall yields of N-acetylhexosamine (HexNAc) partially permethylated alditol acetates (PMAAs) are shown to be inherently lower than their expected theoretical value relative to hexose PMAAs. Calculating the ratio of the area under the extracted ion chromatogram (XIC) for each individual hexose PMAA (or HexNAc PMAA) to the sum of such XIC areas for all hexoses (or HexNAcs) provides a new normalization method that facilitates relative quantification of individual glycan nodes in a sample. Although presently constrained in terms of its absolute limits of detection, this method expedites the analysis of clinical biofluids and shows considerable promise as a complementary approach to traditional top-down glycomics.

This article presents an enhanced form of a novel bottom-up glycomics technique designed to analyze the pooled compositional profile of glycans in unfractionated biofluids through the chemical breakdown of glycans into their constituent linkage-specific monosaccharides for detection by GC-MS. Potential applications include early detection of cancer and other glycan-affective disorders.

Glycan Düğüm Analizi: Glikomik A Aşağıdan yukarı Yaklaşım

Synthesized in a non-template-driven process by enzymes called glycosyltransferases, glycans are key players in various significant intra- and ...

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 ...

Rapid Nanoprobe Signal Enhancement by In Situ Gold Nanoparticle Synthesis

The use of nanoprobes such as gold, silver, silica or iron-oxide nanoparticles as detection reagents in bioanalytical assays can enable high sensitivity and convenient colorimetric readout. However, high densities of nanoparticles are typically needed for detection. The available synthesis-based enhancement protocols are either limited to gold and silver nanoparticles or rely on precise enzymatic control and optimization. Here, we present a protocol to enhance the colorimetric readout of gold, silver, silica, and iron oxide nanoprobes. It was observed that the colorimetric signal can be improved by up to a 10000-fold factor. The basis for such signal enhancement strategies is the chemical reduction of Au3+ to Au0. There are several chemical reactions that enable the reduction of Au3+ to Au0. In the protocol, Good&#39;s buffers and H2O2 are used and it is possible to favor the deposition of Au0 onto the surface of existing nanoprobes, in detriment of the formation of new gold nanoparticles. The protocol consists of the incubation of the microarray with a solution consisting of chloroauric acid and H2O2 in 2-(N-morpholino)ethanesulfonic acid pH 6 buffer following the nanoprobe-based detection assay. The enhancement solution can be applied to paper and glass-based sensors. Moreover, it can be used in commercially available immunoassays as demonstrated by the application of the method to a commercial allergen microarray. The signal development requires less than 5 min of incubation with the enhancement solution and the readout can be assessed by naked eye or low-end image acquisition devices such as a table-top scanner or a digital camera.

Rapid Nanoprobe Signal Enhancement by In Situ Gold Nanoparticle Synthesis

In this work, a protocol for signal enhancement of nanoprobe-based biosensing is presented. The protocol is based on the reduction of chloroauric acid onto the surface of existing nanoprobes that consist of gold, silver, silica or iron-oxide nanoparticles.

Hızlı Nanoprobe sinyal geliştirme içinde in Situ altın nanoparçacık sentezi tarafından

The use of nanoprobes such as gold, silver, silica or iron-oxide nanoparticles as detection reagents in bioanalytical assays can enable high sensitivity ...

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 ...

Solid Phase 11C-Methylation, Purification and Formulation for the Production of PET Tracers

Routine production of radiotracers used in positron emission tomography (PET) mostly relies on wet chemistry where the radioactive synthon reacts with a non-radioactive precursor in solution. This approach necessitates purification of the tracer by high performance liquid chromatography (HPLC) followed by reformulation in a biocompatible solvent for human administration. We recently developed a novel 11C-methylation approach for the highly efficient synthesis of carbon-11 labeled PET radiopharmaceuticals, taking advantage of solid phase cartridges as disposable &#34;3-in-1&#34; units for the synthesis, purification and reformulation of the tracers. This approach obviates the use of preparative HPLC and reduces the losses of the tracer in transfer lines and due to radioactive decay. Furthermore, the cartridge-based technique improves synthesis reliability, simplifies the automation process and facilitates compliance with the Good Manufacturing Practice (GMP) requirements. Here, we demonstrate this technique on the example of production of a PET tracer Pittsburgh compound B ([11C]PiB), a gold standard in vivo imaging agent for amyloid plaques in the human brains.

Solid Phase 11C-Methylation, Purification and Formulation for the Production of PET Tracers

We report an efficient carbon-11 radiolabeling technique to produce clinically relevant tracers for Positron Emission Tomography (PET) using solid phase extraction cartridges. 11C-methylating agent is passed through a cartridge preloaded with precursor and successive elution with aqueous ethanol provides chemically and radiochemically pure PET tracers in high radiochemical yields.

Pet İzleyicilerin Üretimi için Katı Faz 11C-Metilasyon, Arıtma ve Formülasyon

Routine production of radiotracers used in positron emission tomography (PET) mostly relies on wet chemistry where the radioactive synthon reacts with a ...