Summary

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

Published: May 04, 2012
doi:

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 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。

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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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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).

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