Name: chemically-blocked antibody microarray for multiplexed high-throughput profiling of specific protein glycosylation in complex samples
Uploaded: 2012-05-04T13:00:00
Description: Watch this Scientific Journal Video about Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples at JoVE.com

This work was supported by the Institute for Hepatitis and Virus Research.

1. Print an Antibody Microarray for the Assay
<ol>
	<li>Dilute all antibodies to 0.5 mg/ml in phosphate buffer saline, ph 7.2 (PBS).</li>
	<li>Aliquot 40 &mu;l of each antibody into the 384-well source plate.</li>
	<li>Load the 384-well source plate onto the Scienion sciFLEXARRAYER microarrayer.</li>
	<li>Load 20 PATH microarray slides onto the microarrayer as target.</li>
	<li>Set the microarrayer to print 48 identical subarrays, in which 27 antibodies and control proteins are spotted in triplicate in a 9x9 pattern (<...

<ol>
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K., Wingren, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=High-throughput+proteomics+using+antibody+microarrays:+an+update.">High-throughput proteomics using antibody microarrays: an update.</a> Expert Review of Molecular Diagnostics. 7, 673-686 (2007).</li><li>Sanchez-Carbayo, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Antibody+array-based+technologies+for+cancer+protein+profiling+and+functional+proteomic+analyses+using+serum+and+tissue+specimens.">Antibody array-based technologies for cancer protein profiling and functional proteomic analyses using serum and tissue specimens.</a> Tumor Biology. 31, 103-112 (2010).</li><li>Porter, 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+motif-based+analysis+of+glycan+array+data+to+determine+the+specificities+of+glycan-binding+proteins.">A motif-based analysis of glycan array data to determine the specificities of glycan-binding proteins.</a> Glycobiology. 20, 369-380 (2010).</li><li>Maupin, K. 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1. Target protein and capture antibody selection
Prior to the antibody microarray assay, some reagents and materials are needed to be considered and prepared. To design an antibody microarray for glycan profiling or glycan biomarker screening, a panel of antibodies specific to glycoprotein candidates should be determined according to the literature or from previous results. These antibodies were usually purchased from different vendors such as R&amp;D Systems etc. IgGs are preferred capture antib...

<table border="1">
	<tbody>
		<tr>
			<td>ID</td>
			<td>Name of the reagent</td>
			<td>Abbreviation</td>
			<td>Company</td>
			<td>Catalog #</td>
		</tr>
		<tr>
			<td>L1</td>
			<td>Biotinylated Concanavalin A</td>
			<td>ConA</td>
			<td>Vector Laboratories</td>
			<td>BK-1000</td>
		</tr>
		<tr>
			<td>L2</td>
			<td>Biotinylated Sambucus Nigra Lectin</td>
			<td>SNA</td>
			<td>Vector Laboratories</td>
			<td>B-1305</td>
		</tr>
		<tr>
			<td>L3</td>
			<td>Biotinylated Lens Culinaris Agglutinin</td>
			<td>LCA</td>
			<td>Vector Laboratories</td>
			<td>BK-2000</td>
		</tr>
		<tr>
			<td>L4</td>
			<td>Biotinylated Ricinus Communis Agglutinin I</td>
			<td>RCA</td>
			<td>Vector Laboratories</td>
			<td>BK-1000</td>
		</tr>
		<tr>
			<td>L5</td>
			<td>Biotinylated Aleuria Aurantia Lectin</td>
			<td>AAL</td>
			<td>Vector Laboratories</td>
			<td>B-1395</td>
		</tr>
		<tr>
			<td>L6</td>
			<td>Biotinylated Erythrina Cristagalli Lectin</td>
			<td>ECL</td>
			<td>Vector Laboratories</td>
			<td>BK-3000</td>
		</tr>
		<tr>
			<td>L7</td>
			<td>Biotinylated Griffonia (Bandeiraea) Simplicifolia Lectin II</td>
			<td>GSL II</td>
			<td>Vector Laboratories</td>
			<td>BK-3000</td>
		</tr>
		<tr>
			<td>L8</td>
			<td>Biotinylated Wheat Germ Agglutinin</td>
			<td>WGA</td>
			<td>Vector Laboratories</td>
			<td>BK-1000</td>
		</tr>
		<tr>
			<td>L9</td>
			<td>Biotinylated Phaseolus vulgaris Erythroagglutinin</td>
			<td>PHA-E</td>
			<td>Vector Laboratories</td>
			<td>BK-2000</td>
		</tr>
		<tr>
			<td>L10</td>
			<td>Biotinylated Phaseolus vulgaris Leucoagglutinin</td>
			<td>PHA-L</td>
			<td>Vector Laboratories</td>
			<td>BK-2000</td>
		</tr>
		<tr>
			<td>L11</td>
			<td>Biotinylated Peanut Agglutinin</td>
			<td>PNA</td>
			<td>Vector Laboratories</td>
			<td>BK-1000</td>
		</tr>
		<tr>
			<td>L12</td>
			<td>Biotinylated Pisum Sativum Agglutinin</td>
			<td>PSA</td>
			<td>Vector Laboratories</td>
			<td>BK-2000</td>
		</tr>
		<tr>
			<td>L13</td>
			<td>Biotinylated Dolichos Biflorus Agglutinin</td>
			<td>DBA</td>
			<td>Vector Laboratories</td>
			<td>BK-1000</td>
		</tr>
		<tr>
			<td>L14</td>
			<td>Biotinylated Datura Stramonium Lectin</td>
			<td>DSL</td>
			<td>Vector Laboratories</td>
			<td>BK-3000</td>
		</tr>
		<tr>
			<td>L15</td>
			<td>Biotinylated Sophora Japonica Agglutinin</td>
			<td>SJA</td>
			<td>Vector Laboratories</td>
			<td>BK-2000</td>
		</tr>
		<tr>
			<td>L16</td>
			<td>Biotinylated Soybean Agglutinin</td>
			<td>SBA</td>
			<td>Vector Laboratories</td>
			<td>BK-1000</td>
		</tr>
		<tr>
			<td>L17</td>
			<td>Biotinylated Solanum Tuberosum (Potato) Lectin</td>
			<td>STL</td>
			<td>Vector Laboratories</td>
			<td>BK-3000</td>
		</tr>
		<tr>
			<td>L18</td>
			<td>Biotinylated Griffonia (Bandeiraea) Simplicifolia Lectin I</td>
			<td>GSL I</td>
			<td>Vector Laboratories</td>
			<td>BK-2000</td>
		</tr>
		<tr>
			<td>L19</td>
			<td>Biotinylated Vicia Villosa Lectin</td>
			<td>VVL</td>
			<td>Vector Laboratories</td>
			<td>BK-2000</td>
		</tr>
		<tr>
			<td>L20</td>
			<td>Biotinylated Lycopersicon Esculentum (Tomato) Lectin</td>
			<td>LEL</td>
			<td>Vector Laboratories</td>
			<td>BK-3000</td>
		</tr>
		<tr>
			<td>L21</td>
			<td>Biotinylated Ulex Europaeus Agglutinin I</td>
			<td>UEA I</td>
			<td>Vector Laboratories</td>
			<td>BK-1000</td>
		</tr>
		<tr>
			<td>L22</td>
			<td>Biotinylated Jacalin</td>
			<td>JACALIN</td>
			<td>Vector Laboratories</td>
			<td>BK-3000</td>
		</tr>
		<tr>
			<td>A1</td>
			<td>Goat F(ab')2 Fragment anti-human IgM, Fc5&mu; antibody</td>
			<td>IgM</td>
			<td>Jackson Immuno Research</td>
			<td>109-006-129</td>
		</tr>
		<tr>
			<td>A2</td>
			<td>Donkey F(ab')2 Frag anti-human IgG (H+L) antibody</td>
			<td>AB1</td>
			<td>Jackson Immuno Research</td>
			<td>709-006-149</td>
		</tr>
		<tr>
			<td>A3</td>
			<td>Mouse anti-human IgG F(ab')2 monoclonal antibody</td>
			<td>AB3</td>
			<td>Jackson Immuno Research</td>
			<td>209-005-097</td>
		</tr>
		<tr>
			<td>A4</td>
			<td>Goat anti-human alpha 2 macroglobulin polyclonal antibody</td>
			<td>A2M</td>
			<td>GeneTex</td>
			<td>GTX62924</td>
		</tr>
		<tr>
			<td>A5</td>
			<td>Rabbit anti-human alpha-1-antitrypsin polyclonal antibody</td>
			<td>A1AT</td>
			<td>Lee Biosiences</td>
			<td>CA1T-80A</td>
		</tr>
		<tr>
			<td>A6</td>
			<td>Mouse anti-human alpha-1-antitrypsin monoclonal antibody</td>
			<td>A1AT</td>
			<td>Sigma Aldrich</td>
			<td>SAB4200198</td>
		</tr>
		<tr>
			<td>A7</td>
			<td>Rabbit anti-human alpha-1-antitrypsin polyclonal antibody</td>
			<td>ACT</td>
			<td>NeoMarkers</td>
			<td>RB-367-A1</td>
		</tr>
		<tr>
			<td>A8</td>
			<td>Rabbit anti-human alpha-1-antichymotrypsin polyclonal antibody</td>
			<td>ACT</td>
			<td>Fisher Scientific</td>
			<td>RB9213R7</td>
		</tr>
		<tr>
			<td>A9</td>
			<td>Mouse anti-human transferrin monoclonal antibody</td>
			<td>Transferrin</td>
			<td>GeneTex</td>
			<td>GTX101035</td>
		</tr>
		<tr>
			<td>A10</td>
			<td>Rabbit anti-human transferrin polyclonal antibody</td>
			<td>Transferrin</td>
			<td>GeneTex</td>
			<td>GTX77130</td>
		</tr>
		<tr>
			<td>A11</td>
			<td>Goat anti-human apolipoprotein J polyclonal antibody</td>
			<td>ApoJ</td>
			<td>Abcam</td>
			<td>ab7610</td>
		</tr>
		<tr>
			<td>A12</td>
			<td>Mouse anti-human GP73 monoclonal antibody</td>
			<td>GP73</td>
			<td>Abbott</td>
			<td>14H4-23</td>
		</tr>
		<tr>
			<td>A13</td>
			<td>Mouse anti-human GP73 monoclonal antibody</td>
			<td>GP73</td>
			<td>SANTA CRUZ BIOTECHNOLOGY INC</td>
			<td>sc-101275</td>
		</tr>
		<tr>
			<td>A14</td>
			<td>Rabbit anti-human alpha-1 fetoprotein polyclonal antibody</td>
			<td>AFP</td>
			<td>GenWay</td>
			<td>GWB-41C966</td>
		</tr>
		<tr>
			<td>A15</td>
			<td>Mouse anti-human alpha-1 fetoprotein monoclonal antibody</td>
			<td>AFP</td>
			<td>Fitzgerald</td>
			<td>10-A05A</td>
		</tr>
		<tr>
			<td>A16</td>
			<td>Mouse anti-human hemopexin monoclonal antibody</td>
			<td>Hemopexin</td>
			<td>Assaypro</td>
			<td>60190-05011</td>
		</tr>
		<tr>
			<td>A17</td>
			<td>Mouse anti-human glypican-3(1G12) monoclonal antibody</td>
			<td>GPL3</td>
			<td>Santa Cruz Bio</td>
			<td>sc-65443</td>
		</tr>
		<tr>
			<td>A18</td>
			<td>Mouse anti-human Kininogen (LMW) monoclonal antibody</td>
			<td>Kininogen</td>
			<td>Assaypro</td>
			<td>20333-05011</td>
		</tr>
		<tr>
			<td>A19</td>
			<td>Rabbit anti-human MMP-21 monoclonal antibody</td>
			<td>MMP21</td>
			<td>Epitomic</td>
			<td>1955-1</td>
		</tr>
		<tr>
			<td>A20</td>
			<td>Mouse anti-human CEACAM-1 monoclonal antibody</td>
			<td>CEACAM</td>
			<td>R&amp;D Systems</td>
			<td>MAB1180</td>
		</tr>
		<tr>
			<td>A21</td>
			<td>Rat anti-human DPPIV/CD26 monoclonal antibody</td>
			<td>DPPIV</td>
			<td>R&amp;D Systems</td>
			<td>MAB22441</td>
		</tr>
		<tr>
			<td>A22</td>
			<td>Mouse anti-human PIVKA II monoclonal antibody</td>
			<td>PIVICA</td>
			<td>Crystal chem</td>
			<td>8040</td>
		</tr>
		<tr>
			<td>A23</td>
			<td>Mouse anti-carcinoembryonic antigen</td>
			<td>CEA</td>
			<td>US biological</td>
			<td>C1300</td>
		</tr>
		<tr>
			<td>A24</td>
			<td>Mouse anti-CA125 Cancer Antigen</td>
			<td>CA125</td>
			<td>US biological</td>
			<td>C0050-01D</td>
		</tr>
		<tr>
			<td>A25</td>
			<td>Mouse anti -CA19-9 Cancer antigen</td>
			<td>CA19-9</td>
			<td>US biological</td>
			<td>C0075-18</td>
		</tr>
		<tr>
			<td>A26</td>
			<td>Mouse anti-Lewis x monoclonal antibody</td>
			<td>Lewis X</td>
			<td>Calbiochem</td>
			<td>434631</td>
		</tr>
		<tr>
			<td>bio</td>
			<td>Biotinylated BSA (positive control)</td>
			<td>Bio</td>
			<td>Home-made</td>
			<td>N/A</td>
		</tr>
	</tbody>
</table>
Table 1. List of lectins and antibodies used in this protocol.
<table border="1">
	<tbody>
		<tr>
			<td>Name of the reagent s/equipments</td>
			<td>Company</td>
			<td>Catalogue number</td>
		</tr>
		<tr>
			<td>Non contact microarrayer</td>
			<td>BioDot Inc</td>
			<td>sciFLEXARRAYER</td>
		</tr>
		<tr>
			<td>384 microplate</td>
			<td>Fisher</td>
			<td>14-230-243</td>
		</tr>
		<tr>
			<td>FoodSaver</td>
			<td>FoodSaver</td>
			<td>V3835</td>
		</tr>
		<tr>
			<td>Ultrathin nitrocellulose coate microarray slides</td>
			<td>Gentel</td>
			<td>PATH</td>
		</tr>
		<tr>
			<td>Slide Imprinter (optional)</td>
			<td>The Gel Company</td>
			<td>WSP60-1</td>
		</tr>
		<tr>
			<td>Shaker</td>
			<td>Fisher</td>
			<td>15-453-211</td>
		</tr>
		<tr>
			<td>Centrifuge</td>
			<td>Eppendorf</td>
			<td>5804 000.013</td>
		</tr>
		<tr>
			<td>Slide washing basin/Slide Staining Dish with Removable Rack</td>
			<td>Fisher</td>
			<td>08-812</td>
		</tr>
		<tr>
			<td>Slide incubation chamber/microscope slide box</td>
			<td>Fisher</td>
			<td>03-448-5</td>
		</tr>
		<tr>
			<td>Brij 35, 30 w/v% solution in water</td>
			<td>Acros Organics</td>
			<td>AC32958-0025</td>
		</tr>
		<tr>
			<td>Tween-20</td>
			<td>Fisher</td>
			<td>P337-100</td>
		</tr>
		<tr>
			<td>Sodium Periodate (NaIO4)</td>
			<td>Sigma</td>
			<td>311448</td>
		</tr>
		<tr>
			<td>L-Glutamic acid &gamma;-hydrazide</td>
			<td>Sigma</td>
			<td>G-7257</td>
		</tr>
		<tr>
			<td>Sodium Acetate Anhydrous (CH3COONa)</td>
			<td>Sigma</td>
			<td>S2889</td>
		</tr>
		<tr>
			<td>Bovine Serum Albumin (BSA)</td>
			<td>Lampire Biological Labs</td>
			<td>7500804</td>
		</tr>
		<tr>
			<td>Phosphate Buffer Saline (PBS) (10X)</td>
			<td>Denville Scientific</td>
			<td>CP4390-48</td>
		</tr>
		<tr>
			<td>Dylight 549 conjugated NeutrAvidin</td>
			<td>Thermo</td>
			<td>22837</td>
		</tr>
		<tr>
			<td>Protease Inhibitor Cocktail Tablets</td>
			<td>Roche</td>
			<td>4693159001</td>
		</tr>
		<tr>
			<td>ChromPure Human IgG, Fc fragment</td>
			<td>Jackson Immunoresearch</td>
			<td>009-000-008</td>
		</tr>
		<tr>
			<td>ChromPure Human IgG, whole molecule</td>
			<td>Jackson Immunoresearch</td>
			<td>009-000-003</td>
		</tr>
		<tr>
			<td>ChromPure Mouse IgG, whole molecule</td>
			<td>Jackson Immunoresearch</td>
			<td>015-000-003</td>
		</tr>
		<tr>
			<td>ChromPure Mouse IgG, Fc fragment</td>
			<td>Jackson Immunoresearch</td>
			<td>015-000-008</td>
		</tr>
		<tr>
			<td>ChromPure Rabbit IgG, whole molecule</td>
			<td>Jackson Immunoresearch</td>
			<td>011-000-003</td>
		</tr>
		<tr>
			<td>ChromPure Donkey IgG, whole molecule</td>
			<td>Jackson Immunoresearch</td>
			<td>017-000-003</td>
		</tr>
		<tr>
			<td>Microarray Scanner</td>
			<td>Tecan</td>
			<td>LS Reloaded</td>
		</tr>
	</tbody>
</table>
Table 2. List of equipments and reagents used in this protocol.

chemically-blocked antibody microarray for multiplexed high-throughput profiling of specific protein glycosylation in complex samples

In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that allows for the glycosylation profiling of specific proteins. Glycosylation of proteins is the most prevalent post-translational modification found on proteins, and leads diversified modifications of the physical, chemical, and biological properties of proteins. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases. However, current methods to study protein glycosylation typically are too complicated or expensive for use in most normal laboratory or clinical settings and a more practical method to study protein glycosylation is needed. The new protocol described in this study makes use of a chemically blocked antibody microarray with glycan-binding protein (GBP) detection and significantly reduces the time, cost, and lab equipment requirements needed to study protein glycosylation. In this method, multiple immobilized glycoprotein-specific antibodies are printed directly onto the microarray slides and the N-glycans on the antibodies are blocked. The blocked, immobilized glycoprotein-specific antibodies are able to capture and isolate glycoproteins from a complex sample that is applied directly onto the microarray slides. Glycan detection then can be performed by the application of biotinylated lectins and other GBPs to the microarray slide, while binding levels can be determined using Dylight 549-Streptavidin. Through the use of an antibody panel and probing with multiple biotinylated lectins, this method allows for an effective glycosylation profile of the different proteins found in a given human or animal sample to be developed.
Introduction
Glycosylation of protein, which is the most ubiquitous post-translational modification on proteins, modifies the physical, chemical, and biological properties of a protein, and plays a fundamental role in various biological processes1-6. Because the glycosylation machinery is particularly susceptible to disease progression and malignant transformation, aberrant glycosylation has been recognized as early detection biomarkers for cancer and other diseases 7-12. In fact, most current cancer biomarkers, such as the L3 fraction of &alpha;-1 fetoprotein (AFP) for hepatocellular carcinoma 13-15, and CA199 for pancreatic cancer 16, 17 are all aberrant glycan moieties on glycoproteins. However, methods to study protein glycosylation have been complicated, and not suitable for routine laboratory and clinical settings. Chen et al. has recently invented a chemically blocked antibody microarray with a glycan-binding protein (GBP) detection method for high-throughput and multiplexed profile glycosylation of native glycoproteins in a complex sample 18. In this affinity based microarray method, multiple immobilized glycoprotein-specific antibodies capture and isolate glycoproteins from the complex mixture directly on the microarray slide, and the glycans on each individual captured protein are measured by GBPs. Because all normal antibodies contain N-glycans which could be recognized by most GBPs, the critical step of this method is to chemically block the glycans on the antibodies from binding to GBP. In the procedure, the cis-diol groups of the glycans on the antibodies were first oxidized to aldehyde groups by using NaIO4 in sodium acetate buffer avoiding light. The aldehyde groups were then conjugated to the hydrazide group of a cross-linker, 4-(4-N-MaleimidoPhenyl)butyric acid Hydrazide HCl (MPBH), followed by the conjugation of a dipeptide, Cys-Gly, to the maleimide group of the MPBH. Thus, the cis-diol groups on glycans of antibodies were converted into bulky none hydroxyl groups, which hindered the lectins and other GBPs bindings to the capture antibodies. This blocking procedure makes the GBPs and lectins bind only to the glycans of captured proteins. After this chemically blocking, serum samples were incubated with the antibody microarray, followed by the glycans detection by using different biotinylated lectins and GBPs, and visualized with Cy3-streptavidin. The parallel use of an antibody panel and multiple lectin probing provides discrete glycosylation profiles of multiple proteins in a given sample 18-20. This method has been used successfully in multiple different labs 1, 7, 13, 19-31. However, stability of MPBH and Cys-Gly, complicated and extended procedure in this method affect the reproducibility, effectiveness and efficiency of the method. In this new protocol, we replaced both MPBH and Cys-Gly with one much more stable reagent glutamic acid hydrazide (Glu-hydrazide), which significantly improved the reproducibility of the method, simplified and shorten the whole procedure so that the it can be completed within one working day. In this new protocol, we describe the detailed procedure of the protocol which can be readily adopted by normal labs for routine protein glycosylation study and techniques which are necessary to obtain reproducible and repeatable results.

Watch this Scientific Journal Video about Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples at JoVE.com

Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples

In this study, we describe an improved protocol for a multiplexed high-throughput antibody microarray with lectin detection method that can be used in glycosylation profiling of specific proteins. This protocol features new reliable reagents and significantly reduces the time, cost, and lab equipment requirements as compared to the previous procedure.

 In this study, we describe an effective protocol for use in a multiplexed high-throughput antibody microarray with glycan binding protein detection that ...

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