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

Summary

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.

Abstract

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 processes^1-6. 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 α-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.

Protocol

1. Print an Antibody Microarray for the Assay

1. Dilute all antibodies to 0.5 mg/ml in phosphate buffer saline, ph 7.2 (PBS).
2. Aliquot 40 μl of each antibody into the 384-well source plate.
3. Load the 384-well source plate onto the Scienion sciFLEXARRAYER microarrayer.
4. Load 20 PATH microarray slides onto the microarrayer as target.
5. Set the microarrayer to print 48 identical subarrays, in which 27 antibodies and control proteins are spotted in triplicate in a 9x9 pattern (Figure 1E, 1F).
6. Start the microarrayer to print the antibody microarray slides.
7. Collect the antibody microarray slide....

Discussion

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&D Systems etc. IgGs are preferred capture antib.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Biotinylated Concanavalin A	Vector Laboratories	BK-1000	ConA
Biotinylated Sambucus Nigra Lectin	Vector Laboratories	B-1305	SNA
Biotinylated Lens Culinaris Agglutinin	Vector Laboratories	BK-2000	LCA
Biotinylated Ricinus Communis Agglutinin I	Vector Laboratories	BK-1000	RCA
Biotinylated Aleuria Aurantia Lectin	Vector Laboratories	B-1395	AAL
Biotinylated Erythrina Cristagalli Lectin	Vector Laboratories	BK-3000	ECL
Biotinylated Griffonia (Bandeiraea) Simplicifolia Lectin II	Vector Laboratories	BK-3000	GSL II
Biotinylated Wheat Germ Agglutinin	Vector Laboratories	BK-1000	WGA
Biotinylated Phaseolus vulgaris Erythroagglutinin	Vector Laboratories	BK-2000	PHA-E
Biotinylated Phaseolus vulgaris Leucoagglutinin	Vector Laboratories	BK-2000	PHA-L
Biotinylated Peanut Agglutinin	Vector Laboratories	BK-1000	PNA
Biotinylated Pisum Sativum Agglutinin	Vector Laboratories	BK-2000	PSA
Biotinylated Dolichos Biflorus Agglutinin	Vector Laboratories	BK-1000	DBA
Biotinylated Datura Stramonium Lectin	Vector Laboratories	BK-3000	DSL
Biotinylated Sophora Japonica Agglutinin	Vector Laboratories	BK-2000	SJA
Biotinylated Soybean Agglutinin	Vector Laboratories	BK-1000	SBA
Biotinylated Solanum Tuberosum (Potato) Lectin	Vector Laboratories	BK-3000	STL
Biotinylated Griffonia (Bandeiraea) Simplicifolia Lectin I	Vector Laboratories	BK-2000	GSL I
Biotinylated Vicia Villosa Lectin	Vector Laboratories	BK-2000	VVL
Biotinylated Lycopersicon Esculentum (Tomato) Lectin	Vector Laboratories	BK-3000	LEL
Biotinylated Ulex Europaeus Agglutinin I	Vector Laboratories	BK-1000	UEA I
Biotinylated Jacalin	Vector Laboratories	BK-3000	JACALIN
Goat F(ab')2 Fragment anti-human IgM, Fc5μ antibody	Jackson Immuno Research	109-006-129	IgM
Donkey F(ab')2 Frag anti-human IgG (H+L) antibody	Jackson Immuno Research	709-006-149	AB1
Mouse anti-human IgG F(ab')2 monoclonal antibody	Jackson Immuno Research	209-005-097	AB3
Goat anti-human alpha 2 macroglobulin polyclonal antibody	GeneTex	GTX62924	A2M
Rabbit anti-human alpha-1-antitrypsin polyclonal antibody	Lee Biosiences	CA1T-80A	A1AT
Mouse anti-human alpha-1-antitrypsin monoclonal antibody	Sigma-Aldrich	SAB4200198	A1AT
Rabbit anti-human alpha-1-antitrypsin polyclonal antibody	NeoMarkers	RB-367-A1	ACT
Rabbit anti-human alpha-1-antichymotrypsin polyclonal antibody	Fisher Scientific	RB9213R7	ACT
Mouse anti-human transferrin monoclonal antibody	GeneTex	GTX101035	Transferrin
Rabbit anti-human transferrin polyclonal antibody	GeneTex	GTX77130	Transferrin
Goat anti-human apolipoprotein J polyclonal antibody	Abcam	ab7610	ApoJ
Mouse anti-human GP73 monoclonal antibody	Abbott Laboratories	14H4-23	GP73
Mouse anti-human GP73 monoclonal antibody	SANTA CRUZ BIOTECHNOLOGY INC	sc-101275	GP73
Rabbit anti-human alpha-1 fetoprotein polyclonal antibody	GenWay	GWB-41C966	AFP
Mouse anti-human alpha-1 fetoprotein monoclonal antibody	Fitzgerald	10-A05A	AFP
Mouse anti-human hemopexin monoclonal antibody	Assaypro	60190-05011	Hemopexin
Mouse anti-human glypican-3(1G12) monoclonal antibody	Santa Cruz Bio	sc-65443	GPL3
Mouse anti-human Kininogen (LMW) monoclonal antibody	Assaypro	20333-05011	Kininogen
Rabbit anti-human MMP-21 monoclonal antibody	Epitomic	1955-1	MMP21
Mouse anti-human CEACAM-1 monoclonal antibody	R&D Systems	MAB1180	CEACAM
Rat anti-human DPPIV/CD26 monoclonal antibody	R&D Systems	MAB22441	DPPIV
Mouse anti-human PIVKA II monoclonal antibody	Crystal chem	8040	PIVICA
Mouse anti-carcin–mbryonic antigen	US biological	C1300	CEA
Mouse anti-CA125 Cancer Antigen	US biological	C0050-01D	CA125
Mouse anti -CA19-9 Cancer antigen	US biological	C0075-18	CA19-9
Mouse anti-Lewis x monoclonal antibody	Calbiochem	434631	Lewis X
Biotinylated BSA (positive control)	Home-made	N/A	Bio
Table 1. List of lectins and antibodies used in this protocol.
Non contact microarrayer	BioDot Inc	sciFLEXARRAYER
384 microplate	Fisher Scientific	14-230-243
FoodSaver	FoodSaver	V3835
Ultrathin nitrocellulose coate microarray slides	Gentel	PATH
Slide Imprinter (optional)	The Gel Company	WSP60-1
Shaker	Fisher Scientific	15-453-211
Centrifuge	Eppendorf	5804 000.013
Slide washing basin/Slide Staining Dish with Removable Rack	Fisher Scientific	08-812
Slide incubation chamber/microscope slide box	Fisher Scientific	03-448-5
Brij 35, 30 w/v% solution in water	Acros Organics	AC32958-0025
Tween-20	Fisher Scientific	P337-100
Sodium Periodate (NaIO4)	Sigma-Aldrich	311448
L-Glutamic acid γ-hydrazide	Sigma-Aldrich	G-7257
Sodium Acetate Anhydrous (CH3COONa)	Sigma-Aldrich	S2889
Bovine Serum Albumin (BSA)	Lampire Biological Labs	7500804
Phosphate Buffer Saline (PBS) (10X)	Denville Scientific	CP4390-48
Dylight 549 conjugated NeutrAvidin	Thermo Fisher Scientific, Inc.	22837
Protease Inhibitor Cocktail Tablets	Roche Group	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 Group Ltd.	LS Reloaded
Table 2. List of equipments and reagents used in this protocol.