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>
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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. 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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.

In the field of proteomics, filter aided sample preparation (FASP) has been widely adopted for its ability to minimize the amount of starting material, decrease sample preparation artifacts, and maximize sample throughput1. However, such a method has yet to emerge and gain traction for the field of glycomics. Development of high-throughput, quantitative workflows are needed because of the integral role of glycosylation in biological defense and its modulation by cancer or diseases2,3. In mammals, N-glycans are composed of repeating saccharide units (hexoses (Hex), hexosamines (HexNac), sialic acids (NeuAc), and fucoses (Fuc)), which decorate a core structure (Hex3HexNac2)4 covalently bound to asparagine. Though the glycospace is considerably large when isomers are counted (&#62;1012), it is quite small on a composition basis and molecular weights typically range from 1,000-8,000 Da5. The compositional homogeneity of the class and the hydrophilicity of glycans pose unique challenges to purification, separation, and mass spectrometry (MS) workflows6.
Traditionally, N-glycans are digested from proteins or peptides by peptide-N-glycosidase F (PNGase F) and then enriched by lectin affinity chromatography7, captured by hydrazide beads8, or purified via solid phase extraction (SPE)9,10. While these methods are all highly effective, they introduce extra steps for desalting and limit the number of samples simultaneously processed. Over the last decade, a number of high-throughput platforms for glycomics have been proposed. Kim et al. published a semi-automated method using a vacuum-operated, SPE 96-well plate11. Alternatively, an affinity-filter method (N-glyco-FASP) was developed by the Mann group, which required the initial derivatization of the filter with a composite of lectins12. Lastly, the Thomas-Oates group proposed a semi-quantitative method, the Filter Aided N-Glycan Separation (FANGS), which exploited the narrow composition size of the glycospace13. Relying on molecular weight cut-off filters, small contaminants were first washed to waste and then the N-glycans were digested and eluted. Deglycosylated proteins remain on the filter in this protocol and can be subjected to in-line FASP.
Identification and quantification of glycans by electrospray ionization (ESI) MS requires off-line separations for (partial) resolution of isomers and derivatization for detection of low-abundant species. Labeling in the individuality when normalizing with glycan hydrazide tags strategy confers compatibility with reverse-phase liquid chromatography (RPLC)14,15. The 4-phenethyl-benzohydrazide (P2PGN) hydrophobic tag mediates the hydrophilicity of the glycans, enhancing ionization by, on average, four-fold16. Though other techniques, such as permethylation17 or amine-reactive tagging chemistries18, offer similar advantages, in the hydrazide reaction, glycans are reacted 1:1 stoichiometrically in facile conditions. Relative quantification is achieved by tandem analysis of samples derivatized with native (NAT) or 13C6 stabile isotope labels (SIL).
The following method evolves FANGS for plasma applications and couples it to the P2GPN hydrophobic tag for accurate relative quantification. Furthermore, it was designed to perform shot-gun proteomics, deamidation profiling, and quantitative glycomics on a single aliquot of sample, without compromising the integrity of the 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: The scheme of the FANGS-P2GPN hydrophobic tagging coupled method (Method A) for combined proteomics and glycomics analysis is given. Steps that differ between glycomics-only processing and tandem glycomics and proteomics analysis, with glycosylation site identification, are highlighted. <a href="https://www.jove.com/files/ftp_upload/53735/53735fig1large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
The in-line proteomics and glycomics, filter-based P2GPN hydrophobic tagging method (Method A) was validated for identification, quantitation, and molecular weight bias using pooled hen plasma samples (Figure 1). For solely glycomics experiments, inter-comparisons in the abundances of N-glycans extracted by Method A to carbograph SPE (gold-standard, Method B) were made (Figure 2). There were no significant differences in the abundances of glycans between the two methods. The intra-variability was also assessed by quantifying the SIL: NAT ratio of glycans extracted by the same method. The log2-distributions of both methods were Gaussian, centered on zero, and not significantly different (Figure 3A-B). The molecular weight ranges between the two protocols completely overlapped, suggesting that the filter did not discriminate N-glycans based on molecular weight range and hydrophilicity (Figure 4).
<img alt="Figure 2" src="/files/ftp_upload/53735/53735fig2.jpg" /> 
Figure 2: An equimolar mixture of NAT and SIL N-glycans extracted by Method A or Method B was prepared from 2.5 &#956;l of hen plasma (N = 4). Samples were analyzed by UPLC-MS according to the recommended parameters suggested in section 6. The abundances for each glycan, in each NAT/SIL channel, were calculated by integrating the area under the extracted ion chromatogram (MMA = 3 ppm), correcting for the molecular weight overlap, and adjusting by the TGNF. These ratios are shown with their standard errors. The abundances of Method B: Method A N-glycans were not significantly different (p &#62;0.05). <a href="https://www.jove.com/files/ftp_upload/53735/53735fig2large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 3" src="/files/ftp_upload/53735/53735fig3.jpg" /> 
Figure 3: The intra-variability in the (A) Method A (N = 133) or (B) Method B (N = 123) strategies was compared. NAT and SIL equimolar mixtures of N-glycans, from the same extraction scheme, were analyzed over three technical replicates. The log2-distributions were centered on zero and were not significantly different between the two workflows. <a href="https://www.jove.com/files/ftp_upload/53735/53735fig3large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
<img alt="Figure 4" src="/files/ftp_upload/53735/53735fig4.jpg" /> 
Figure 4: The molecular weight ranges of glycans from each strategy were compared. The two workflows yielded glycans spanning the same molecular weight range. N-glycans detected in one protocol versus the other fell just above the limit of detection (1 &#215; 105 abundance) and do not reflect systematic bias. <a href="https://www.jove.com/files/ftp_upload/53735/53735fig4large.jpg" target="_blank">Please click here to view a larger version of this figure.</a>
Retention of the deglycosylated proteins by the molecular weight filter enabled in-line proteome wide analysis and glycosylation site identification. Multiple protocols for the combined purification of glycans and proteins were compared to a traditional FASP preparation without deglycosylation (Table 5). Our typical 18 hr filter based PNGase F digestion, followed by FASP trypsin digest (Std protocol) resulted in significant levels of non-specific deamidation, which can interfere with the identification of glycosites. Therefore, a method utilizing a shorter incubation time at elevated temperature (50 &#176;C) and a PNGase F spike-in step (SPI protocol) was explored as an alternative, along with a microwave digestion protocol (MD protocol), to minimize non-specific deamidation. The glycans observed in the new digestion methods were not significantly different in compositions or abundances from the standard 18 hr, 37 &#176;C filter PNGase F digest (Figure 5). Combined with a short trypsin incubation, non-specific deamidation was significantly reduced, with a false positive rate for glycosites &#60;5% (Table 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">Proteins</td>
			<td></td>
			<td colspan="4">Peptides</td>
			<td></td>
			<td colspan="4">Deamidated Peptides</td>
			<td></td>
			<td colspan="4">Glycosites</td>
			<td></td>
			<td colspan="4">Glycoproteins</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="2">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>
Table 5: Comparisons of proteomic data from a standard trypsin digest versus the in-line FANGS-P2GPN tagging method were made. Preparations were performed with (+) and without (-) PNGase F to determine background rates of non-specific deamidation and estimate glycosite false positive rates. Proteins were identified with 1% false discovery rate (FDR); peptides were filtered based on &#34;high&#34; peptide confidence in Proteome Discoverer 1.4 (q &#60;0.01); glycosites were identified based on unique sequences containing deamidation of the conserved glycosylation motif (N-X-S/T); and glycoproteins were defined as identified proteins containing at least one identified glycosite.
<img alt="Figure 5" src="/files/ftp_upload/53735/53735fig5.jpg" /> 
Figure 5: Shortened protocols for glycans were developed to minimize deamination in the samples and compared to the 18hr FANGS PNGase F digest. The average differences in abundances were 10% and 5% for the MD (2.2.3) and SPI (2.2.2) protocols, respectively. Of the 48 glycans identified, 90% displayed less than 1.5-fold variation.&#160; <a href="https://www.jove.com/files/ftp_upload/53735/53735fig5large.jpg" target="_blank">Please click here to view a larger version of this 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.

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