化学阻断抗体芯片复用高通量的复杂样品中特定蛋白糖基化的剖析
Summary
在这项研究中，我们描述了一个复用高通量抗体芯片与外源凝集素的检测方法，可以在特定的蛋白质糖基化分析改进协议。该协议提供了新的可靠的试剂和显着降低，相比以前的程序的时间，成本和实验室设备的要求。
Abstract
在这项研究中，我们描述了一个用于在复用高通量抗体芯片与糖结合蛋白检测，允许特定蛋白质的糖基化分析的有效协议。蛋白质糖基化是最常见的翻译后修饰，对蛋白质的发现，导致多元化的修改的物理，化学，生物学特性和蛋白质。由于糖基化机制是特别容易受到疾病的进展和恶性转化，异常糖基化已被确认为癌症和其他疾病的早期检测标志物。然而，目前的方法来研究蛋白糖基化通常是在最普通的实验室或临床的设置和使用更实用的方法来研究蛋白糖基化需要过于复杂或昂贵。在这项研究中所描述的新的协议，使得与糖结合使用的化学阻断抗体芯片蛋白（GBP）检测，并显着减少的时间，成本和实验室设备，需要研究蛋白糖基化的要求。在此方法中，多个固定的糖蛋白特异性抗体直接打印到芯片的幻灯片和抗体的N-糖链被封锁。受阻，固定糖蛋白特异性抗体能够捕获和隔离从一个复杂的样品，直接应用到芯片幻灯片糖蛋白。聚糖检测，然后可以进行生物素标记的凝集素和其他Gbps的芯片幻灯片的应用，而具有约束力的水平可以决定使用Dylight 549-链霉。通过使用多个生物素标记的凝集素抗体面板和探测，这种方法可以有效的发展，在一个特定的人或动物样品中发现的不同的蛋白质糖基化的文件。
介绍
蛋白糖基化，这是我S的 最普遍的对蛋白质翻译后的修改，修改的物理，化学，生物特性的一种蛋白质，在1-6各种生物过程中起着根本性的作用。由于糖基化机制是特别容易受到疾病的进展和恶性转化已被公认为早期发现癌症和其他疾病的7-12生物标志物，异常糖基化。事实上，目前大多数肿瘤标志物，如三级分数的α-1，甲胎蛋白（法新社）13-15肝癌，胰腺癌16 CA199检测，有17个是所有异常的糖蛋白的糖基。然而，研究蛋白糖基化的方法已经复杂，不适合常规实验室和临床设置。 Chen 等最近发明了一种化学阻断抗体芯片1糖结合蛋白（GBP），高吞吐量的检测方法在一个复杂的样品18届复轮廓本土糖蛋白的糖基化。多个固定的糖蛋白特异性抗体的亲和力在此基础芯片的方法，捕捉和隔离从复杂的混合物，直接对芯片的幻灯片的糖蛋白，对每一个人捕获的蛋白聚糖GBPS测量。因为所有正常的抗体含有N-糖链最Gbps的，可以确认，这种方法的关键步骤是化学阻断抗体聚糖结合英镑。在此过程中，CI S-二醇组抗体聚糖被首先氧化成醛基使用醋酸钠缓冲液避光NAIO 4。醛基肼组的交联剂，4 – （4 N-MaleimidoPhenyl的）丁酸盐酸肼（MPBH），随后的二肽，半胱氨酸，甘氨酸，酰亚胺组，共轭共轭MPBH。从而被转换成笨重没有羟基，这阻碍了外源凝集素和其他Gbps的绑定捕获抗体，聚糖对抗体的顺式二醇组。这堵过程使Gbps和外源凝集素结合，只捕获蛋白聚糖。阻断后，这个化学，血清标本聚糖检测，通过使用不同的生物素标记的凝集素和Gbps的抗体芯片孵育，用Cy3-链霉亲和观察。抗体面板和探测多个外源凝集素的并行使用提供了在一个给定的样本18-20离散多种蛋白质糖基化型材。该方法已成功地应用于多个不同的实验室1，7，13，19-31。然而，影响稳定MPBH和半胱氨酸，甘氨酸，复杂性和扩展的过程，在这个方法的重复性，该方法的有效性和效率。在这个新的协议，我们更换一个更加稳定MPBH和半胱氨酸，甘氨酸试剂谷氨酸肼（GLU-肼），从而显着提高了简化的方法，重现性和缩短整个程序，以便它可以在一个工作日内完成。在这个新的协议，我们描述的详细程序的协议，可以很容易地通过正常的实验室常规蛋白糖基化研究和技术，这是要获得可重复性和可重复的结果。
Protocol
1。打印检测抗体芯片所有的抗体稀释至0.5毫克/毫升磷酸盐缓冲液，pH值7.2（PBS）的。 40μl每个抗体的分装成384井源板。 ，Scienion sciFLEXARRAYER的微阵列加载到384井源板。 20 PATH芯片幻灯片加载到微阵列为目标。 设置打印48个相同的子阵发现在一个9×9的模式（ 图1E，1F）一式三份，其中27个抗体和控制蛋白质微阵列。 启动微阵列打印抗体芯片的?…
Discussion
1。目标蛋白质和捕获抗体的选择
抗体芯片检测之前，一些试剂和材料都需要考虑和准备。聚糖分析或聚糖的生物标志物的筛选，抗体的特定糖候选人的面板设计的抗体芯片，根据文献，或从先前的结果，应确定。购买来自不同厂商，如R＆D系统等的IgG，这些抗体通常是首选，因为我们以前的测试表明，一些IgM和IgE可能完全失去了化学修饰后，其抗原的亲和力捕获抗体。
<p cla…Disclosures
The authors have nothing to disclose.
Acknowledgements
这项工作是由肝炎病毒研究的研究所支持。
Materials
| ID | Name of the reagent | Abbreviation | Company | Catalog # |
| L1 | Biotinylated Concanavalin A | ConA | Vector Laboratories | BK-1000 |
| L2 | Biotinylated Sambucus Nigra Lectin | SNA | Vector Laboratories | B-1305 |
| L3 | Biotinylated Lens Culinaris Agglutinin | LCA | Vector Laboratories | BK-2000 |
| L4 | Biotinylated Ricinus Communis Agglutinin I | RCA | Vector Laboratories | BK-1000 |
| L5 | Biotinylated Aleuria Aurantia Lectin | AAL | Vector Laboratories | B-1395 |
| L6 | Biotinylated Erythrina Cristagalli Lectin | ECL | Vector Laboratories | BK-3000 |
| L7 | Biotinylated Griffonia (Bandeiraea) Simplicifolia Lectin II | GSL II | Vector Laboratories | BK-3000 |
| L8 | Biotinylated Wheat Germ Agglutinin | WGA | Vector Laboratories | BK-1000 |
| L9 | Biotinylated Phaseolus vulgaris Erythroagglutinin | PHA-E | Vector Laboratories | BK-2000 |
| L10 | Biotinylated Phaseolus vulgaris Leucoagglutinin | PHA-L | Vector Laboratories | BK-2000 |
| L11 | Biotinylated Peanut Agglutinin | PNA | Vector Laboratories | BK-1000 |
| L12 | Biotinylated Pisum Sativum Agglutinin | PSA | Vector Laboratories | BK-2000 |
| L13 | Biotinylated Dolichos Biflorus Agglutinin | DBA | Vector Laboratories | BK-1000 |
| L14 | Biotinylated Datura Stramonium Lectin | DSL | Vector Laboratories | BK-3000 |
| L15 | Biotinylated Sophora Japonica Agglutinin | SJA | Vector Laboratories | BK-2000 |
| L16 | Biotinylated Soybean Agglutinin | SBA | Vector Laboratories | BK-1000 |
| L17 | Biotinylated Solanum Tuberosum (Potato) Lectin | STL | Vector Laboratories | BK-3000 |
| L18 | Biotinylated Griffonia (Bandeiraea) Simplicifolia Lectin I | GSL I | Vector Laboratories | BK-2000 |
| L19 | Biotinylated Vicia Villosa Lectin | VVL | Vector Laboratories | BK-2000 |
| L20 | Biotinylated Lycopersicon Esculentum (Tomato) Lectin | LEL | Vector Laboratories | BK-3000 |
| L21 | Biotinylated Ulex Europaeus Agglutinin I | UEA I | Vector Laboratories | BK-1000 |
| L22 | Biotinylated Jacalin | JACALIN | Vector Laboratories | BK-3000 |
| A1 | Goat F(ab’)2 Fragment anti-human IgM, Fc5μ antibody | IgM | Jackson Immuno Research | 109-006-129 |
| A2 | Donkey F(ab’)2 Frag anti-human IgG (H+L) antibody | AB1 | Jackson Immuno Research | 709-006-149 |
| A3 | Mouse anti-human IgG F(ab’)2 monoclonal antibody | AB3 | Jackson Immuno Research | 209-005-097 |
| A4 | Goat anti-human alpha 2 macroglobulin polyclonal antibody | A2M | GeneTex | GTX62924 |
| A5 | Rabbit anti-human alpha-1-antitrypsin polyclonal antibody | A1AT | Lee Biosiences | CA1T-80A |
| A6 | Mouse anti-human alpha-1-antitrypsin monoclonal antibody | A1AT | Sigma Aldrich | SAB4200198 |
| A7 | Rabbit anti-human alpha-1-antitrypsin polyclonal antibody | ACT | NeoMarkers | RB-367-A1 |
| A8 | Rabbit anti-human alpha-1-antichymotrypsin polyclonal antibody | ACT | Fisher Scientific | RB9213R7 |
| A9 | Mouse anti-human transferrin monoclonal antibody | Transferrin | GeneTex | GTX101035 |
| A10 | Rabbit anti-human transferrin polyclonal antibody | Transferrin | GeneTex | GTX77130 |
| A11 | Goat anti-human apolipoprotein J polyclonal antibody | ApoJ | Abcam | ab7610 |
| A12 | Mouse anti-human GP73 monoclonal antibody | GP73 | Abbott | 14H4-23 |
| A13 | Mouse anti-human GP73 monoclonal antibody | GP73 | SANTA CRUZ BIOTECHNOLOGY INC | sc-101275 |
| A14 | Rabbit anti-human alpha-1 fetoprotein polyclonal antibody | AFP | GenWay | GWB-41C966 |
| A15 | Mouse anti-human alpha-1 fetoprotein monoclonal antibody | AFP | Fitzgerald | 10-A05A |
| A16 | Mouse anti-human hemopexin monoclonal antibody | Hemopexin | Assaypro | 60190-05011 |
| A17 | Mouse anti-human glypican-3(1G12) monoclonal antibody | GPL3 | Santa Cruz Bio | sc-65443 |
| A18 | Mouse anti-human Kininogen (LMW) monoclonal antibody | Kininogen | Assaypro | 20333-05011 |
| A19 | Rabbit anti-human MMP-21 monoclonal antibody | MMP21 | Epitomic | 1955-1 |
| A20 | Mouse anti-human CEACAM-1 monoclonal antibody | CEACAM | R&D Systems | MAB1180 |
| A21 | Rat anti-human DPPIV/CD26 monoclonal antibody | DPPIV | R&D Systems | MAB22441 |
| A22 | Mouse anti-human PIVKA II monoclonal antibody | PIVICA | Crystal chem | 8040 |
| A23 | Mouse anti-carcinoembryonic antigen | CEA | US biological | C1300 |
| A24 | Mouse anti-CA125 Cancer Antigen | CA125 | US biological | C0050-01D |
| A25 | Mouse anti -CA19-9 Cancer antigen | CA19-9 | US biological | C0075-18 |
| A26 | Mouse anti-Lewis x monoclonal antibody | Lewis X | Calbiochem | 434631 |
| bio | Biotinylated BSA (positive control) | Bio | Home-made | N/A |
Table 1. List of lectins and antibodies used in this protocol.
| Name of the reagent s/equipments | Company | Catalogue number |
| Non contact microarrayer | BioDot Inc | sciFLEXARRAYER |
| 384 microplate | Fisher | 14-230-243 |
| FoodSaver | FoodSaver | V3835 |
| Ultrathin nitrocellulose coate microarray slides | Gentel | PATH |
| Slide Imprinter (optional) | The Gel Company | WSP60-1 |
| Shaker | Fisher | 15-453-211 |
| Centrifuge | Eppendorf | 5804 000.013 |
| Slide washing basin/Slide Staining Dish with Removable Rack | Fisher | 08-812 |
| Slide incubation chamber/microscope slide box | Fisher | 03-448-5 |
| Brij 35, 30 w/v% solution in water | Acros Organics | AC32958-0025 |
| Tween-20 | Fisher | P337-100 |
| Sodium Periodate (NaIO4) | Sigma | 311448 |
| L-Glutamic acid γ-hydrazide | Sigma | G-7257 |
| Sodium Acetate Anhydrous (CH3COONa) | Sigma | S2889 |
| Bovine Serum Albumin (BSA) | Lampire Biological Labs | 7500804 |
| Phosphate Buffer Saline (PBS) (10X) | Denville Scientific | CP4390-48 |
| Dylight 549 conjugated NeutrAvidin | Thermo | 22837 |
| Protease Inhibitor Cocktail Tablets | Roche | 4693159001 |
| ChromPure Human IgG, Fc fragment | Jackson Immunoresearch | 009-000-008 |
| ChromPure Human IgG, whole molecule | Jackson Immunoresearch | 009-000-003 |
| ChromPure Mouse IgG, whole molecule | Jackson Immunoresearch | 015-000-003 |
| ChromPure Mouse IgG, Fc fragment | Jackson Immunoresearch | 015-000-008 |
| ChromPure Rabbit IgG, whole molecule | Jackson Immunoresearch | 011-000-003 |
| ChromPure Donkey IgG, whole molecule | Jackson Immunoresearch | 017-000-003 |
| Microarray Scanner | Tecan | LS Reloaded |
Table 2. List of equipments and reagents used in this protocol.
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Tags
Chemically-blocked Antibody MicroarrayMultiplexed High-throughput ProfilingSpecific Protein GlycosylationComplex SamplesGlycan Binding Protein DetectionPost-translational ModificationPhysical Properties Of ProteinsChemical Properties Of ProteinsBiological Properties Of ProteinsDisease ProgressionMalignant TransformationEarly Detection BiomarkersCancer BiomarkersPractical MethodLaboratory SettingsClinical SettingsChemically Blocked Antibody MicroarrayGlycan-binding Protein DetectionTime ReductionCost ReductionLab Equipment Requirements
Cite This Article
Lu, C., Wonsidler, J. L., Li, J., Du, Y., Block, T., Haab, B., Chen, S. Chemically-blocked Antibody Microarray for Multiplexed High-throughput Profiling of Specific Protein Glycosylation in Complex Samples. J. Vis. Exp. (63), e3791, doi:10.3791/3791 (2012).