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.

プロテオミクスの分野では、フィルタ支援試料調製（FASP）が広く、出発材料の量を最小化する試料調製のアーティファクトを減少させる、およびサンプルスループット1を最大化する能力のために採用されています。しかしながら、このような方法は未だ出現し、グライコミクスの分野のための牽引力を獲得しています。高スループットの開発は、定量的なワークフローがあるため生体防御および癌または疾患2,3によってその変調におけるグリコシル化の重要な役割で必要とされています。哺乳類では、N個の -glycansは、コア構造を飾る糖単位（ヘキソース（16進数）、ヘキソサミン（のHexNAc）、シアル酸（NeuAcで）、およびフコース（Fucは））、（16進数3のHexNAc 2）4共有結合した繰り返しで構成されていますアスパラギンへ。異性体がカウントされるときglycospaceがかなり大きいですが（&gt; 10 12）、それは、組成に基づいて非常に小さく、分子量は、典型的には1,000-8,000ダ5 &lt;範囲/ SUP&gt;。クラスの組成均一性およびグリカンの親水性は、精製、分離、および質量分析（MS）ワークフロー6に特有の課題を提起します。 伝統的には、Nの -glycansはF（PNGアーゼF）は、ペプチドのNによるタンパク質またはペプチドから-glycosidase消化され、次いで、ヒドラジドビーズ8によって、レクチンアフィニティークロマトグラフィー7によって濃縮捕捉、または固相抽出（SPE）9,10を介して精製しました。これらの方法は全て非常に有効であるが、これらは、脱塩のための余分な工程を導入し、同時に処理されたサンプルの数を制限します。過去10年間、グライコミクスのためのハイスループットプラットフォームの数が提案されています。 Kim らは、真空操作、SPE 96ウエルプレート11を用いて、半自動化された方法を発表しました。また、アフィニティーフィルター法は、（N -glyco-FASP）をFi接続の初期誘導体化を必要とマン・グループ、によって開発されましたレクチン12の合成とLTER。最後に、トーマス・オーツのグループは、半定量法、glycospace 13の狭い組成サイズを悪用フィルター支援N -Glycan分離（牙）を、提案しました。分子量カットオフフィルターに依存する、小さな汚染物質が第一無駄に洗浄し、次いで、N個の -glycansを消化し ​​、溶出しました。脱グリコシル化タンパク質は、このプロトコルでフィルター上に残存すると、インラインFASPに供することができます。 エレクトロスプレーイオン化（ESI）MSによる同定およびグリカンの定量化は、異性体および低豊富な種の検出のための誘導体化の（部分的な）解決のためにオフライン分離を必要とします。個性で標識グリカンヒドラジドタグ戦略正規逆相液体クロマトグラフィー（RPLC）14,15との互換性を与えます。 4フェネチル - ベンゾヒドラジド（P2PGN）疎水性タグはENH、グリカンの親水性を仲介します、平均して、4倍の16でイオン化をancing。容易な条件で化学量論的に1：例えば過メチル化17またはアミン反応性タグ付け化学18のような他の技術が、同様の利点を提供するが、ヒドラジド反応において、グリカンは1を反応させます。相対的定量化は、天然の（NAT）または13 C 6安定同位体ラベル（SIL）で誘導体化された試料のタンデム分析によって達成されます。 以下の方法は、正確な相対的定量化のためのP2GPN疎水性タグにプラズマアプリケーションやカップル、それをするための牙を展開します。更に、分析の完全性を損​​なうことなく、サンプルの単一のアリコートにショットガンプロテオミクス、脱アミド化プロファイル、および定量的グライコミクスを実行するように設計されました。

<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="図1" src="/files/ftp_upload/53735/53735fig1.jpg" /> 図1：組み合わせたプロテオミクスとグライコミクス解析のための牙-P2GPN疎水性タグ付け結合された方法（方法A）が与えられたのスキーム手順グリコシル化部位の同定と、グライコミクスのみの処理とタンデムグライコミクスおよびプロテオミクス解析の間で異なる、強調表示されます。 <a href="https://www.jove.com/files/ftp_upload/53735/53735fig1large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> インラインプロテオミクスとグライコミクス、フィルターベースP2GPN疎水性タグ付け方法（方法A）は、プールされた鶏の血漿サンプル（ 図1）を使用して識別、定量化、および分子量のバイアスのために検証されました。もっぱらグライコミクス実験のために、Nの存在量で相互比較は法によって抽出された-glycansSPE（ゴールドスタンダード、方法B）作製した（ 図2）を carbographします。 2つの方法のグリカンの存在量に有意差はなかったです。同様の方法で抽出されたグリカンのNAT比内ばらつきもSILを定量することによって評価しました。両方の方法のログ2 -distributionsはゼロを中心に、ガウスた、と（ 図3A-B）に有意差はなかっ。分子量は、フィルタは、Nは分子量範囲及び親水性（ 図4）に基づいて-glycans区別しなかったことを示唆し、完全に重なった2つのプロトコルの間の範囲です。 <img alt="図2" src="/files/ftp_upload/53735/53735fig2.jpg" /> 図2：NAT およびSIL Nの等モル混合物を、方法AまたはB法により抽出された-glycansは鶏血漿2.5μlのから調製した（N = 4）の試料は肛門ました。各NAT / SILチャネルで、セクション6に各グリカンのための存在量を示唆した推奨パラメータに従ってUPLC-MSによってyzed、分子を補正し、抽出イオンクロマトグラム（MMA = 3 ppm）で下の面積を積分することによって計算されました重量が重なり、TGNFによって調整します。これらの比率は、その標準誤差で示しています。方法Bの存在量：方法A Nの -glycansはた（p&gt; 0.05）で有意差は認められなかった<a href="https://www.jove.com/files/ftp_upload/53735/53735fig2large.jpg" target="_blank">。この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図3" src="/files/ftp_upload/53735/53735fig3.jpg" /> 図3（A）方法A（N = 133）または（B）方法Bにおける内変動性（N = 123）の戦略を比較しました。同じ抽出方式からNの -glycansのNATとSIL等モル混合物で、、3つの技術の反復にわたって分析しました。ログ2 -distributionsは、ゼロを中心と2つのワークフローの間で有意差は認められなかったた。 <a href="https://www.jove.com/files/ftp_upload/53735/53735fig3large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</a> <img alt="図4" src="/files/ftp_upload/53735/53735fig4.jpg" /> 図4：各戦略からのグリカンの分子量範囲を比較しました。 2つのワークフローは、同じ分子量の範囲に及ぶグリカンを生成した。N -glycans他対1のプロトコルで検出されたばかりの検出（1×10 5豊富）の限界を超えて下落したと系統的なバイアスを反映するものではありません。 <a href="https://www.jove.com/files/ftp_upload/53735/53735fig4large.jpg" target="_blank">拡大表示はこちらをクリックしてくださいこの図のバージョン。</a> 表5）と比較しました。 FASPトリプシン消化物（STDプロトコル）に続いて私たちの典型的な18時間のフィルタベースのPNGase F消化は、glycositesの識別を妨害することができるの非特異的脱アミド化の有意なレベルをもたらしました。従って、高温（50℃）で、より短いインキュベーション時間を利用する方法およびPNGアーゼFのスパイクステップ（SPIプロトコル）は、非最小化するために、マイクロ波消化プロトコル（MDプロトコル）と共に、代替として調査しました。特定の脱アミド。新しい消化法で観察されたグリカンは、標準の18時間からの組成物または存在量における有意差は認められなかった37°CフィルタのPNGase Fダイジェスト（ <strong&gt;図5）。短いトリプシンインキュベーションと組み合わせることで、非特異的脱アミド化が著しくglycosites &lt;5％（ 表5）のための偽陽性率で、減少しました。 <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">タンパク質</td><td></td><td colspan="4">ペプチド</td><td></td><td colspan="4">脱アミド化ペプチド</td><td></td><td colspan="4"> Glycosites </td><td></td><td colspan="4">糖タンパク質</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"> 18時間</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> 表5： 標準トリプシンからのプロテオームデータの比較のインライン牙-P2GPNタギング法に対するダイジェストが行われた準備が（+）とし、せずに行った-非特 ​​異的脱アミド化および推定の背景率を決定するために、PNGアーゼF（）。偽陽性率をglycosite。タンパク質は、1％の偽発見率（FDR）で同定されました。ペプチドは、プロテオームのDiscoverer 1.4（Q &lt;0.01）で「高」ペプチドの信頼に基づいて、濾過しました。 glycositesはidentifiました保存されたグリコシル化モチーフ（NXS / T）の脱アミド化を含むユニークな配列に基づいてED。および糖タンパク質は、少なくとも1つの識別glycositeを含む同定されたタンパク質として定義されていました。 <img alt="図5" src="/files/ftp_upload/53735/53735fig5.jpg" /> 図5：グリカンのための短縮プロトコルは、試料中の脱アミノ化を最小限に抑えるために開発され、18時間牙のPNGase F消化物と比較しました。存在量の平均の差は10％とそれぞれMD（2.2.3）およびSPI（2.2.2）プロトコル、5％でした。同定された48グ ​​リカンのうち、90％未満1.5倍の変化を示した。 <a href="https://www.jove.com/files/ftp_upload/53735/53735fig5large.jpg" target="_blank">この図の拡大版をご覧になるにはこちらをクリックしてください。</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.

定量的グライコミクスおよびプロテオミクス複合精製戦略

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 ビュー.  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.

ビデオ: 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.

超高速液体クロマトグラフィー - 質量分析（UPLC-MS）による血清中の大規模非標的メタボロームプロファイリング

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

化学

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.

安定同位体標識するための還元ジメチル化を用いた定量的プロテオミクス

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.

クロマトグラフィーのための最適な固定相の定量的な選択を伴う合成とIodoaziridinesの精製

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.

質量分析法による総合的なO-結合およびスルホグライコミクスのための改良されたゲル内還元β脱離

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  - クリア天然ゲル溶出液フラクションエントラップメント電気泳動

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.

糖鎖ノード分析：グライコミクスへのボトムアップアプローチ

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.

ペプチドおよびタンパク質定量を使用して自動免疫-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 ...

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.

原金ナノ粒子合成による急速なナノプローブ信号強調

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.

キャピラリー ゾーン電気泳動タンデム質量分析法による大規模なトップダウン プロテオミクス

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トレーサー製造のための固相11C-メチル化、精製および製剤

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