Kemisk blokeret antistof Microarray for Multiplexed Højkapacitetsforskning Profilering af specifikke protein Glycosylering i komplekse prøver

Summary

I denne undersøgelse beskriver vi en forbedret protokol til et multiplekset high-throughput-antistof mikroarray med lectin påvisningsmetode, der kan anvendes i glycosylering profilering af specifikke proteiner. Denne protokol indeholder nye pålidelige reagenser og reducerer den tid, omkostninger og lab krav til udstyr i forhold til den tidligere 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. Udskrive en Antistof Microarray for assayet Fortyndes alle antistoffer til 0,5 mg / ml i phosphatbufret saltvand, pH 7,2 (PBS). Portion 40 ul af hvert antistof i 384-brønds kilde plade. Læg 384-brønds source plade på Scienion sciFLEXARRAYER microarrayer. Læg 20 PATH microarray glider på microarrayer som mål. Indstille microarrayer at udskrive 48 identiske subarrays, hvor 27-antistoffer og kontrol proteiner plettede in triplo i en 9×9 mønster (fig. 1E,…

Discussion

1. Target protein og capture Antibody Selection

Forud for antistoffet mikroarray assayet nogle reagenser og materialer for at blive betragtet og fremstilles. At designe et antistof microarray for glycan profilering eller glycan biomarkør screening, et panel af antistoffer specifikke for glycoprotein ansøgere skal fastlægges i henhold til litteraturen eller fra tidligere resultater. Disse antistoffer var normalt købt fra forskellige leverandører, såsom R & D Systems mv IgG foretrækkes…

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.