Name: a quantitative glycomics and proteomics combined purification strategy
Uploaded: 2016-03-08T14:00:00
Duration: 11 min 38 s
Description: Watch this Scientific Journal Video about A Quantitative Glycomics and Proteomics Combined Purification Strategy at JoVE.com

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>
	<li>Wisniewski, J. R., Zougman, A., Nagaraj, N., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Universal+sample+preparation+method+for+proteome+analysis.">Universal sample preparation method for proteome analysis.</a> Nat. Meth. 6, 359-362 (2009).</li><li>Preston, R. J. S., Rawley, O., Gleeson, E. M., O'Donnell, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Elucidating+the+role+of+carbohydrate+determinants+in+regulating+hemostasis:+insights+and+opportunities.">Elucidating the role of carbohydrate determinants in regulating hemostasis: insights and opportunities.</a> Blood. 121, 3801-3810 (2013).</li><li>Hasnain, S. Z., Gallagher, A. L., Grencis, R. K., Thornton, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+new+role+for+mucins+in+immunity:+Insights+from+gastrointestinal+nematode+infection.">A new role for mucins in immunity: Insights from gastrointestinal nematode infection.</a> Int. J. Biochem. Cell Biol. 45, 364-374 (2013).</li><li>Roseman, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reflections+on+glycobiology.">Reflections on glycobiology.</a> J. Biol. Chem. 276, 41527-41542 (2001).</li><li>Laine, R. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+calculation+of+all+possible+oligosaccharide+isomers+both+branched+and+linear+yields+1.05+x+10(12)+structures+for+a+reducing+hexasaccharide:+The+isomer+barrier+to+development+of+single-method+saccharide+sequencing+or+synthesis+systems.">A calculation of all possible oligosaccharide isomers both branched and linear yields 1.05 x 10(12) structures for a reducing hexasaccharide: The isomer barrier to development of single-method saccharide sequencing or synthesis systems.</a> Glycobiology. 4, 759-767 (1994).</li><li>Zaia, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mass+spectrometry+and+glycomics.">Mass spectrometry and glycomics.</a> Omics. 14, 401-418 (2010).</li><li>Hirabayashi, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Lectin-based+structural+glycomics:+Glycoproteomics+and+glycan+profiling.">Lectin-based structural glycomics: Glycoproteomics and glycan profiling.</a> Glycoconjugate J. 21, 35-40 (2004).</li><li>Nilsson, J., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Enrichment+of+glycopeptides+for+glycan+structure+and+attachment+site+identification.">Enrichment of glycopeptides for glycan structure and attachment site identification.</a> Nat. Methods. 6, 809-811 (2009).</li><li>Redmond, J. W., Packer, N. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+use+of+solid-phase+extraction+with+graphitised+carbon+for+the+fractionation+and+purification+of+sugars.">The use of solid-phase extraction with graphitised carbon for the fractionation and purification of sugars.</a> Carbohyd. Res. 319, 74-79 (1999).</li><li>Ruhaak, L. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrophilic+interaction+chromatography-based+high-throughput+sample+preparation+method+for+N-glycan+analysis+from+total+human+plasma+glycoproteins.">Hydrophilic interaction chromatography-based high-throughput sample preparation method for N-glycan analysis from total human plasma glycoproteins.</a> Anal. Chem. 80, 6119-6126 (2008).</li><li>Kim, Y. -. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Rapid+and+high-throughput+analysis+of+N-glycans+from+ovarian+cancer+serum+using+a+96-well+plate+platform.">Rapid and high-throughput analysis of N-glycans from ovarian cancer serum using a 96-well plate platform.</a> Anal. Biochem. 391, 151-153 (2009).</li><li>Zielinska, D. F., Gnad, F., Wi&#347;niewski, J. R., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Precision+mapping+of+an+in+vivo+N-glycoproteome+reveals+rigid+topological+and+sequence+constraints.">Precision mapping of an in vivo N-glycoproteome reveals rigid topological and sequence constraints.</a> Cell. 141, 897-907 (2010).</li><li>Abdul Rahman, S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Filter-aided+N-glycan+separation+(FANGS):+a+convenient+sample+preparation+method+for+mass+spectrometric+N-glycan+profiling.">Filter-aided N-glycan separation (FANGS): a convenient sample preparation method for mass spectrometric N-glycan profiling.</a> J. Proteome Res. 13, 1167-1176 (2014).</li><li>Walker, S. H., Carlisle, B. C., Muddiman, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Systematic+comparison+of+reverse+phase+and+hydrophilic+interaction+liquid+chromatography+platforms+for+the+analysis+of+N-linked+glycans.">Systematic comparison of reverse phase and hydrophilic interaction liquid chromatography platforms for the analysis of N-linked glycans.</a> Anal. Chem. 84, 8198-8206 (2012).</li><li>Walker, S. H., Taylor, A. D., Muddiman, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Individuality+normalization+when+labeling+with+isotopic+glycan+hydrazide+tags+(INLIGHT):+A+novel+glycan-relative+quantification+strategy.">Individuality normalization when labeling with isotopic glycan hydrazide tags (INLIGHT): A novel glycan-relative quantification strategy.</a> J. Am. Soc. Mass Spectr. 24, 1376-1384 (2013).</li><li>Walker, S. H., Lilley, L. M., Enamorado, M. F., Comins, D. L., Muddiman, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hydrophobic+derivatization+of+N-linked+glycans+for+increased+ion+abundance+in+electrospray+ionization+mass+spectrometry.">Hydrophobic derivatization of N-linked glycans for increased ion abundance in electrospray ionization mass spectrometry.</a> J. Am. Soc. Mass. Spectr. 22, 1309-1317 (2011).</li><li>Baldwin, M. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Permethylation+and+tandem+mass+spectrometry+of+oligosaccharides+having+free+hexosamine:+Analysis+of+the+glycoinositol+phospholipid+anchor+glycan+from+the+scrapie+prion+protein.">Permethylation and tandem mass spectrometry of oligosaccharides having free hexosamine: Analysis of the glycoinositol phospholipid anchor glycan from the scrapie prion protein.</a> Anal. Biochem. 191, 174-182 (1990).</li><li>Harvey, D. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Derivatization+of+carbohydrates+for+analysis+by+chromatography;+electrophoresis+and+mass+spectrometry.">Derivatization of carbohydrates for analysis by chromatography; electrophoresis and mass spectrometry.</a> J. Chromatogr. B. 879, 1196-1225 (2011).</li><li>Bradford, M. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+rapid+and+sensitive+method+for+the+quantitation+of+microgram+quantities+of+protein+utilizing+the+principle+of+protein-dye+binding.">A rapid and sensitive method for the quantitation of microgram quantities of protein utilizing the principle of protein-dye binding.</a> Anal. Biochem. 72, 248-254 (1976).</li><li>Smith, P. K., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Measurement+of+protein+using+bicinchoninic+acid.">Measurement of protein using bicinchoninic acid.</a> Anal. Biochem. 150, 76-85 (1985).</li><li>Hecht, E. S., McCord, J. P., Muddiman, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Definitive+screening+design+optimization+of+mass+spectrometry+parameters+for+sensitive+comparison+of+filter+and+solid+phase+extraction+purified,+inlight+plasma+N-glycans.">Definitive screening design optimization of mass spectrometry parameters for sensitive comparison of filter and solid phase extraction purified, inlight plasma N-glycans.</a> Anal. Chem. 87, 7305-7312 (2015).</li><li>Nepomuceno, A. I., Gibson, R. J., Randall, S. M., Muddiman, D. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Accurate+identification+of+deamidated+peptides+in+global+proteomics+using+a+quadrupole+orbitrap+mass+spectrometer.">Accurate identification of deamidated peptides in global proteomics using a quadrupole orbitrap mass spectrometer.</a> J. Proteome Res. 13, 777-785 (2013).</li><li>Yen, T. -. Y., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Overcoming+challenges+and+opening+new+opportunities+in+glycoproteomics.">Overcoming challenges and opening new opportunities in glycoproteomics.</a> Biomolecules. 3, 270-286 (2013).</li><li>Meiring, H. D., van der Heeft, E., ten Hove, G. J., de Jong, A. P. J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nanoscale+LC-MS(n):+technical+design+and+applications+to+peptide+and+protein.">Nanoscale LC-MS(n): technical design and applications to peptide and protein.</a> J. Sep. Sci. 25, 557-568 (2002).</li><li>Apweiler, R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=UniProt:+the+Universal+Protein+knowledgebase.">UniProt: the Universal Protein knowledgebase.</a> Nucleic Acids Res. 32, 115-119 (2004).</li><li>Perkins, D. N., Pappin, D. J. C., Creasy, D. M., Cottrell, J. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Probability-based+protein+identification+by+searching+sequence+databases+using+mass+spectrometry+data.">Probability-based protein identification by searching sequence databases using mass spectrometry data.</a> Electrophoresis. 20, 3551-3567 (1999).</li><li>Eng, J. K., McCormack, A. L., Yates, J. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+approach+to+correlate+tandem+mass+spectral+data+of+peptides+with+amino+acid+sequences+in+a+protein+database.">An approach to correlate tandem mass spectral data of peptides with amino acid sequences in a protein database.</a> J. Am. Soc. Mass. Spectrom. 5, 976-989 (1994).</li><li>Gonzalez-Galarza, F. F., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+critical+appraisal+of+techniques,+software+packages,+and+standards+for+quantitative+proteomic+analysis.">A critical appraisal of techniques, software packages, and standards for quantitative proteomic analysis.</a> Omics. 16, 431-442 (2012).</li><li>Spivak, M., Weston, J., Bottou, L., K&#228;ll, L., Noble, W. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improvements+to+the+Percolator+Algorithm+for+Peptide+Identification+from+Shotgun+Proteomics+Data+Sets.">Improvements to the Percolator Algorithm for Peptide Identification from Shotgun Proteomics Data Sets.</a> J Proteome Res. 8, 3737-3745 (2009).</li><li>Collier, T. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comparison+of+stable-isotope+labeling+with+amino+acids+in+cell+culture+and+spectral+counting+for+relative+quantification+of+protein+expression.">Comparison of stable-isotope labeling with amino acids in cell culture and spectral counting for relative quantification of protein expression.</a> Rapid Commun Mass Sp. 25, 2524-2532 (2011).</li><li>Kronewitter, S. R., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+development+of+retrosynthetic+glycan+libraries+to+profile+and+classify+the+human+serum+N-linked+glycome.">The development of retrosynthetic glycan libraries to profile and classify the human serum N-linked glycome.</a> Proteomics. 9, 2986-2994 (2009).</li><li>Sheeley, D. M., Reinhold, V. N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Structural+characterization+of+carbohydrate+sequence,+linkage,+and+branching+in+a+quadrupole+ion+trap+mass+spectrometer:+Neutral+oligosaccharides+and+N-linked+glycans.">Structural characterization of carbohydrate sequence, linkage, and branching in a quadrupole ion trap mass spectrometer: Neutral oligosaccharides and N-linked glycans.</a> Anal. Chem. 70, 3053-3059 (1998).</li></ol>

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.

Dans le domaine de la protéomique, filtre aidé la préparation de l&#39; échantillon (FASP) a été largement adopté pour sa capacité à réduire la quantité de matériau de départ, diminuer l&#39; échantillon des artefacts de préparation, et de maximiser le débit d&#39; échantillons 1. Cependant, une telle méthode n&#39;a pas encore d&#39;émerger et de gagner du terrain pour le domaine de la glycomique. Développement de haut débit, les flux quantitatifs sont nécessaires en raison du rôle essentiel de la glycosylation dans la défense biologique et sa modulation par le cancer ou les maladies 2,3. Chez les mammifères, N -glycans sont composées de motifs répétitifs saccharides (hexoses (Hex), hexosamines (HexNAc), acides sialique (NeuAc), et fucoses (Fuc)), qui décorent une structure de noyau (Hex 3 HexNAc 2) 4 à liaison covalente asparagine. Bien que le glycospace est considérablement élevée lorsque les isomères sont comptés (&gt; 10 12), il est tout à fait petite sur une base de la composition et les poids moléculaires varient généralement de 1,000-8,000 Da 5 &lt;/ Sup&gt;. L&#39;homogénéité de la composition de la classe et l&#39;hydrophilie de glycanes posent des défis uniques pour la purification, la séparation et la spectrométrie de masse (MS) workflows 6. Traditionnellement, N -glycans sont digérées à partir des protéines ou des peptides par le peptide N -glycosidase F (PNGase F), puis enrichi par chromatographie d&#39;affinité sur lectine 7, capturé par des billes 8 hydrazide ou purifiés par extraction en phase solide (SPE) 9,10. Bien que ces méthodes sont très efficaces, ils introduisent des étapes supplémentaires pour le dessalage et de limiter le nombre d&#39;échantillons traités simultanément. Au cours de la dernière décennie, un certain nombre de plates-formes à haut débit pour glycomique ont été proposées. Kim et al. , Publié un procédé semi-automatisé à l&#39; aide, d&#39; une plaque SPE de 96 puits à commande par aspiration 11. Alternativement, une méthode d&#39; affinité-filtre (N -glyco-FASP) a été développé par le groupe Mann, qui a nécessité la dérivation initiale de la filtre avec un composite de lectines 12. Enfin, le groupe Thomas-Oates a proposé une méthode semi-quantitative, le filtre Aided N Séparation -Glycan (FANGS), qui exploitait la taille étroite de composition du glycospace 13. En se fondant sur ​​les filtres coupe-off poids moléculaire, de petits contaminants ont d&#39; abord été lavés à perdre et ensuite les -glycans N ont été digérés et élues. protéines déglycosylée restent sur le filtre dans ce protocole et peuvent être soumis à FASP en ligne. Identification et quantification des glycanes par ionisation électrospray (ESI) MS exige des séparations hors ligne pour (partielle) la résolution des isomères et dérivatisation pour la détection d&#39;espèces à faible abondance. Marquer l&#39;individualité quand la normalisation avec la stratégie de balises hydrazide glycanes confère une compatibilité avec la chromatographie liquide en phase inverse (RECH) 14,15. Le 4-phénéthyl-benzohydrazide (P2PGN) tag hydrophobe médie l&#39;hydrophilie des glycanes, enhFINANCEZ ionisation par, en moyenne, quatre fois 16. Bien que d&#39; autres techniques, telles que perméthylation ou 17 chimies de marquage amine réactive 18, présentent des avantages similaires hydrazide dans la réaction, on fait réagir glycanes 1: 1 stoechiométriquement dans des conditions faciles. Quantification relative est obtenue par l&#39; analyse en tandem d&#39;échantillons dérivés avec native (NAT) ou 13 C 6 étiquettes stabile isotopiques (SIL). La méthode suivante évolue FANGS pour les applications de plasma et les couples à l&#39;étiquette hydrophobe P2GPN pour la quantification relative précise. En outre, il a été conçu pour effectuer la protéomique shot-gun, désamidation profilage, et glycomique quantitatives sur une seule aliquote de l&#39;échantillon, sans compromettre l&#39;intégrité des analyses.

<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="Figure 1" src="/files/ftp_upload/53735/53735fig1.jpg" /> Figure 1:. Le schéma de la méthode FANGS-P2GPN marquage hydrophobe couplé (méthode A) pour la protéomique et glycomique combinés analyse est donnée étapes qui diffèrent entre glycomique-seulement le traitement et tandem glycomique et analyse protéomique, avec l&#39; identification des sites de glycosylation, sont mis en évidence. <a href="https://www.jove.com/files/ftp_upload/53735/53735fig1large.jpg" target="_blank">S&#39;il vous plaît cliquer ici pour voir une version plus grande de cette figure.</a> L&#39;en-ligne protéomique et glycomique, P2GPN méthode de marquage hydrophobe à base de filtre (méthode A) a été validée pour l&#39; identification, la quantification, et le biais de poids moléculaire en utilisant des échantillons de plasma de poule groupée (figure 1). Pour uniquement glycomique expériences, inter-comparaisons dans les abondances de N -glycans extraites par la méthodeA à Carbograph SPE (étalon-or, méthode B) ont été faites (Figure 2). Il n&#39;y avait aucune différence significative dans les abondances de glycanes entre les deux méthodes. La variabilité intra a également été évaluée par la quantification de la SIL: NAT rapport de glycanes obtenus par la même méthode. Le log 2 -distributions des deux méthodes étaient gaussienne, centrée sur zéro, et non significativement différents (figure 3A-B). Le poids moléculaire se situe entre les deux protocoles complètement superposées, ce qui suggère que le filtre ne discriminait pas N -glycans basée sur la gamme de poids moléculaire et hydrophilie (Figure 4). <img alt="Figure 2" src="/files/ftp_upload/53735/53735fig2.jpg" /> Figure 2: Un mélange équimolaire de NAT et SIL N -glycans extrait par la méthode A ou B a été préparé à partir de 2,5 pi de poule plasma (N = 4) Les échantillons ont été anal.yzed par UPLC-MS selon les paramètres recommandés suggérés dans la section 6. Les abondances pour chaque glycane, dans chaque canal NAT / SIL, ont été calculés en intégrant la zone sous le chromatogramme ionique extrait (MMA = 3 ppm), la correction de la molécule poids de chevauchement, et l&#39;ajustement par le TGNF. Ces ratios sont présentés avec leurs erreurs standard. Les abondances de la Méthode B: Méthode A N -glycans sont pas significativement différentes (p&gt; 0,05). <a href="https://www.jove.com/files/ftp_upload/53735/53735fig2large.jpg" target="_blank">S&#39;il vous plaît cliquer ici pour voir une version plus grande de cette figure.</a> <img alt="Figure 3" src="/files/ftp_upload/53735/53735fig3.jpg" /> Figure 3: La variabilité intra-en (A) Méthode A (N = 133) ou (B) Méthode B (n = 123) des stratégies ont été comparées. NAT et SIL mélanges équimolaires de N -glycans, du même schéma d&#39;extraction, Ont été analysés sur trois répétitions techniques. Le journal 2 -distributions étaient centrés sur zéro et ne sont pas significativement différents entre les deux flux de travail. <a href="https://www.jove.com/files/ftp_upload/53735/53735fig3large.jpg" target="_blank">S&#39;il vous plaît cliquer ici pour voir une version plus grande de cette figure.</a> <img alt="Figure 4" src="/files/ftp_upload/53735/53735fig4.jpg" /> Figure 4: Les gammes de poids moléculaires de glycanes à partir de chacune de ces stratégies ont été comparées. Les deux flux de travail ont donné glycanes couvrant la même gamme de poids moléculaire. N -glycans détecté dans un protocole par rapport à l&#39;autre est tombé juste au- dessus de la limite de détection (1 × 10 5 abondance) et ne reflètent pas de biais systématique. <a href="https://www.jove.com/files/ftp_upload/53735/53735fig4large.jpg" target="_blank">S&#39;il vous plaît cliquer ici pour voir une plus grande version de ce chiffre.</a> <p class="jove_content" fo:keep-together.dans les pages = &quot;1&quot;&gt; Rétention des protéines déglycosylées par le filtre de poids moléculaire activé en ligne protéome analyse et site de glycosylation large identification. De nombreux protocoles pour la purification combinée des glycanes et des protéines ont été comparées à une préparation traditionnelle , sans FASP déglycosylation (tableau 5). Notre typique filtre 18 h base PNGase F de digestion, suivie par FASP trypsine digest (protocole Std) a donné lieu à des niveaux significatifs de désamidation non spécifique, ce qui peut interférer avec l&#39;identification des glycosites. Par conséquent, un procédé utilisant un temps d&#39;incubation plus courte à une température élevée (50 ° C) et une étape de pointe dans la PNGase F (protocole SPI) a été étudiée comme une alternative, ainsi qu&#39;un protocole de micro-ondes de digestion (protocole MD), pour réduire au minimum la non- désamidation spécifique. Les glycanes observées dans les nouvelles méthodes de digestion ne sont pas significativement différents dans les compositions ou les abondances de 18 h standard, 37 ° C filtre PNGase F digest ( <strong&gt; La figure 5). Combiné avec une incubation de trypsine courte, désamidation non spécifique a été réduite de manière significative, avec un taux de faux positifs glycosites &lt;5% (tableau 5). <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"> Protéines </td><td></td><td colspan="4"> peptides </td><td></td><td colspan="4"> peptides Désaminée </td><td></td><td colspan="4"> Glycosites </td><td></td><td colspan="4"> glycoprotéines </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"> MARYLAND </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"> dix </td><td></td><td colspan="2"> 145 </td><td colspan="2"> 8 </td></tr></tbody></table> . Tableau 5: Comparaison des données protéomiques d&#39;une trypsine norme digérer par rapport à la méthode de marquage en ligne FANGS-P2GPN ont fait des préparatifs ont été effectués avec (+) et sans (-) PNGase F pour déterminer les taux de désamidation et une estimation non spécifique fond glycosite taux de faux positifs. Les protéines ont été identifiées avec 1% le taux de fausses découvertes (FDR); peptides ont été filtrés en fonction de &quot;haut&quot; la confiance des peptides dans Proteome Discoverer 1.4 (q &lt;0,01); glycosites ont été identified sur la base de séquences uniques contenant désamidation du motif de glycosylation conservés (NXS / T); et glycoprotéines ont été définies comme des protéines identifiées contenant au moins une glycosite identifiée. <img alt="Figure 5" src="/files/ftp_upload/53735/53735fig5.jpg" /> Figure 5: Raccourcissement des protocoles de glycanes ont été mis au point pour minimiser la désamination dans les échantillons et on les compare à la PNGase F 18hr FANGS digestion. Les différences moyennes en abondances étaient de 10% et 5% pour le rapport de gestion (2.2.3) et SPI (2.2.2) protocoles, respectivement. Sur les 48 glycanes identifiés, 90% affiché moins de variation de 1,5 fois. <a href="https://www.jove.com/files/ftp_upload/53735/53735fig5large.jpg" target="_blank">S&#39;il vous plaît cliquer ici pour voir une version plus grande de cette figure.</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.

A Quantitative Glycomics et protéomique Stratégie Purification Combined

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

Research

JoVE Journal

Chemistry

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

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

À grande échelle non ciblée profilage métabolomique du sérum par 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 ...

Recherche

Chimie

Quantitative Proteomics Using Reductive Dimethylation for Stable Isotope Labeling

Protéomique quantitative à l&#39;aide réductrice diméthylation pour les isotopes stables étiquetage

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

Synthèse et purification de Iodoaziridines implication quantitative de sélection de la phase stationnaire optimale pour chromatographie

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

Improved In-gel Reductive ÃƒÅ½Ã‚Â²-Elimination for Comprehensive O-linked and Sulfo-glycomics by Mass Spectrometry

Amélioration dans le gel sulfo-réductrices glycomique β-élimination complète pour O-liés et par spectrométrie de masse

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

CN-GELFrEE - Clear natif Gel-élué Fraction liquide Entrapment Electrophoresis

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

Glycan Analyse Node: une approche bottom-up à Glycomics

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)

Peptide and Protein Quantification Using Automated Immuno-MALDI iMALDI

Peptide et protéine Quantification moyen automatisé 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 ...

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

Protéomique de haut vers le bas à grande échelle à l’aide de l’électrophorèse de Zone capillaire 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 ...

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

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

Phase solide 11C-Méthylation, purification et formulation pour la production de traceurs de PET

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

Versatile CO2 Transformations into Complex Products: A One-pot Two-step Strategy

Versatile CO2 Transformations into Complex Products: A One-pot Two-step Strategy

Transformations polyvalentes de CO2 en produits complexes : une stratégie en deux étapes en un seul pot

CO2 transformations using a one-pot two-step method are presented herein. The purpose of the method is to give access to a variety of value-added products ...