Name: chemo-enzymatic synthesis of n-glycans for array development and hiv antibody profiling
Uploaded: 2018-02-05T13:00:00.000+00:00
Duration: 11 min 8 s
Description: Watch this Scientific Journal Video about Chemo-enzymatic Synthesis of N-glycans for Array Development and HIV Antibody Profiling at JoVE.com

저자는 얇은 영화 기술, 계측 기술 연구 센터 (ITRC)와 국가 적용 연구 실험실, Hsinchu 과학 공원, 대만 감사합니다. 이 작품은 국가 과학 위원회에 의해 지원 되었다 (no를 부여 합니다. 대부분 105-0210-01-13-01) 및 학계 Sinica.

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저자는 공개 없다.

에이즈-1 bNAbs PG9, PG16, 및 PGTs 128, 141-145를 포함 한 클래스 중화 HIV-1 분리 순환의 70-80%에서 매우 강력한 보고 했다. 이러한 bNAbs epitopes는 높은 전체 HIV-1 그룹 M의 변종 중 보존, 그러므로 그들은 유도 중화 항 체23,24,25 에이즈 백신에 대 한 효과적인 immunogen 디자인을 안내할 수 있습니다. . 에이즈-1 광범위 하 게 항 체를 중화의 glyca...

N-glycans glycoproteins에 covalently에 연결 되는 아스파라긴 (Asn) 잔류물 합의 Asn-Xxx-Ser/Thr sequon의 단백질 구조, antigenicity, 가용성, 및 lectin 인식 등 여러 생물 학적 과정에 영향을 1 , 2. 화학 합성 N-연결 된 oligosaccharides 그들의 거 대 한 구조 마이크로 고 높은 분기 건축 때문에 중요 한 합성 챌린지를 나타냅니다. 보호의 신중한 선택 그룹 빌딩 블록, anomeric 센터, 및 발기인의 적절 한 사용에서 선택 달성의 반응성을 조정/activator(s) 복잡 한 oligosaccharides의 합성에서 핵심 요소입니다. 복잡 한이 문제를 해결 하려면 사전 N-glycan 합성 작품의 좋은 금액으로 알려졌다 최근3,4. 이러한 강력한 접근에도 불구 하 고 찾는 다양 한 N-glycans의 준비에 대 한 효과적인 방법 주요 도전 (~ 20000) 남아 있다.
에이즈-1 광범위 한 유전적 다양성 및 중화 항 체 응답에서 탈출 하는 능력의 급속 한 돌연변이 율 에이즈-15,6 에 대 한 안전 하 고 예방 백신을 개발 하는 가장 큰 과제 중 하나입니다. , 7. 에이즈를 사용 하 여 호스트 면역 반응을 피하기 위해 하나의 효과적인 전술 이다 봉투 당단백질 gp120 다양 한 N의 닢 glycosylation-호스트 glycosylation 기계8에서 파생 된 glycans 연결 9. 인간 미 발달 신장 (HEK) 293T 세포에서 재조합 단위체 HIV-1 gp120 glycosylation의 정확한 분석에 대 한 최근 보고서 제안 특성 셀 전용 패턴10 구조 microheterogeneity의 발생 , 11 , 12. 따라서 이해의 HIV-1 bNAbs glycan 특이성 필요 잘 특징이 gp120 N-glycan 구조 분석에 대 한 충분 한 수량에 관련.
Glycan microarray 기술 발견 제공 다양 한 탄수화물 묶는 단백질, 바이러스/박테리아 adhesins, 독 소, 항 체, 및 lectines13,14의 특이성의 높은 처리량 기반 탐사 . 기준점과 칩 기반 형태로 체계적인 glycans 배열 문제가 낮은 선호도 단백질 glycan 상호 작용 multivalent 프레 젠 테이 션15,16,,1718을 통해 결정 합니다. 이 칩 기반 glycan 배열 셀 인터페이스를 효과적으로 모방을 편리 하 게 나타납니다. 기술을 풍부 관련 된 기존의 배열 형식 고르지 못한 문제를 극복 하 고, 우리의 그룹 최근 개발 phosphonic 산-엔드 glycans를 사용 하 여 신호 강도 향상 하는 알루미늄 산화물 코팅 유리 (ACG) 슬라이드에 glycan 배열 동질성, 그리고 감도19,20.
Glycan epitopes 광범위 중화 항 체 (bNAbs) 새로 격리 된 에이즈-1의에 대 한 현재 이해를 개선 하기 위해, 우리는 N의 광범위의 준비에 대 한 매우 효율적인 모듈형 전략 개발-glycans21 연결 22 는 ACG에 인쇄할 슬라이드 ( 그림 1참조). 매우 강력한 bNAb PG9 HIV 분리 되었던의 이종 glycan 바인딩 동작의 특이 한 검출 감염 개인23,,2425를 제공 하는 프로 파일링 연구 에이즈-1 bNAbs ACG 배열에의 한 특이성

<table><tbody><tr><td>Acetic acid</td><td>Sigma Aldrich</td><td>64197</td><td></td></tr><tr><td>Acetonitrile</td><td>Sigma Aldrich</td><td>75058</td><td></td></tr><tr><td>Acetic anhydride</td><td>Sigma Aldrich</td><td>108247</td><td></td></tr><tr><td>Anhydrous magnesium sulfate</td><td>Sigma Aldrich</td><td>7487889</td><td></td></tr><tr><td>Boron trifluoride ethyl etherate</td><td>Sigma Aldrich</td><td>109637</td><td></td></tr><tr><td>Bovine serum albumin</td><td>Sigma Aldrich</td><td>9048468</td><td></td></tr><tr><td>Bio-Gel P2 polyacrylamide</td><td>Bio-Rad</td><td>1504118</td><td></td></tr><tr><td>Bis(cyclopentadienyl)hafnium(IV) dichloride</td><td>Sigma Aldrich</td><td>12116664</td><td></td></tr><tr><td>&amp;beta;-1, 4 Galactosyl transferases from bovine milk</td><td>Sigma Aldrich</td><td>48279</td><td></td></tr><tr><td>BioDot Cartesion technology with robotic pin SMP3 (Stealth Micro Spotting Pins)</td><td>Arrayit</td><td></td><td></td></tr><tr><td>Cerium ammonium molybdate</td><td>TCI</td><td>C1794</td><td></td></tr><tr><td>Cerium ammonium nitrate</td><td>Sigma Aldrich</td><td>16774213</td><td></td></tr><tr><td>Clean glass slide&amp;nbsp;</td><td>Schott&amp;nbsp;</td><td></td><td></td></tr><tr><td>Cytidine-5&amp;prime;-monophospho-N-acetylneuraminic acid</td><td>Sigma Aldrich</td><td>3063716</td><td></td></tr><tr><td>Deuterated chloroform</td><td>Sigma Aldrich</td><td>865496</td><td></td></tr><tr><td>Donkey Anti-Human IgG (Alexa Fluor647 conjugated</td><td>Jackson Immuno Research, USA</td><td>709605098</td><td></td></tr><tr><td>Dichloromethane</td><td>Sigma Aldrich</td><td>75092</td><td></td></tr><tr><td>Diethylaminosulfur trifluoride</td><td>Sigma Aldrich</td><td>38078090</td><td></td></tr><tr><td>Dimethylformamide</td><td>Sigma Aldrich</td><td>68122</td><td></td></tr><tr><td>Ethyl acetate</td><td>Sigma Aldrich</td><td>141786</td><td></td></tr><tr><td>Ethylene glycol</td><td>Acros Organic</td><td>107211</td><td></td></tr><tr><td>FAST frame slide incubation chambers</td><td>Sigma Aldrich</td><td></td><td></td></tr><tr><td>Guanosine 5&#039;-diphospho-b-L-fucose disodium salt&amp;nbsp;</td><td>Sigma Aldrich</td><td>15839700</td><td></td></tr><tr><td>Lab tracer 2.0 software&amp;nbsp;</td><td></td><td></td><td>Section 4 of the Protocol</td></tr><tr><td>GenePix Pro 4300A reader (microarray image analysis)</td><td>moleculardevices</td><td></td><td>www.moleculardevices.com</td></tr><tr><td>GraphPad Prism Software (Image processing )</td><td>GraphPad Software, Inc</td><td></td><td>http://www.graphpad.com/guides/prism/6/user-guide/</td></tr><tr><td>Lithium hydroxide</td><td>Sigma Aldrich</td><td>1310652</td><td></td></tr><tr><td>Manganese chloride</td><td>Sigma Aldrich</td><td>7773015</td><td></td></tr><tr><td>Methanol</td><td>Sigma Aldrich</td><td>67561</td><td></td></tr><tr><td>N-butanol</td><td>Sigma Aldrich</td><td>71363</td><td></td></tr><tr><td>Oxalic acid</td><td>Acros Organic</td><td>144627</td><td></td></tr><tr><td>Palladium hydroxide</td><td>Sigma Aldrich</td><td>12135227</td><td></td></tr><tr><td>Phosphate Buffered Saline</td><td>Thermo Fisher Scientific&amp;nbsp;</td><td>10010023</td><td></td></tr><tr><td>Pyridine</td><td>Sigma Aldrich</td><td>110861</td><td></td></tr><tr><td>P-Toluene sulfonic acid monohydrate</td><td>Sigma Aldrich</td><td>773476</td><td></td></tr><tr><td>Silver triflate</td><td>Sigma Aldrich</td><td>2923286</td><td></td></tr><tr><td>Sodium bicarbonate</td><td>Sigma Aldrich</td><td>144558</td><td></td></tr><tr><td>Sodium chloride</td><td>Sigma Aldrich</td><td>7647145</td><td></td></tr><tr><td>Sodium hydrogen carbonate</td><td>Sigma Aldrich</td><td>144558</td><td></td></tr><tr><td>Sodium methoxide&amp;nbsp;</td><td>Sigma Aldrich</td><td>124414</td><td></td></tr><tr><td>Sodium sulfate</td><td>Sigma Aldrich</td><td>7757826</td><td></td></tr><tr><td>Toluene&amp;nbsp;</td><td>Sigma Aldrich</td><td>108883</td><td></td></tr><tr><td>Tris buffer&amp;nbsp;</td><td>Amresco</td><td>N/A</td><td>Ultra-pure grade</td></tr><tr><td>Tween-20</td><td>Amresco</td><td>9005645</td><td></td></tr><tr><td>Uridine diphosphate galactose (UDP-galactose)</td><td>Sigma Aldrich</td><td>137868521</td><td></td></tr></tbody></table>

chemo-enzymatic synthesis of n-glycans for array development and hiv antibody profiling

우리 N의 광범위의 빠른 준비를 위한 매우 효율적인 방법 제시-oligosaccharides (초과 20000 구조 추정) 인간 glycoproteins에 일반적으로 발견 되는 연결. 원하는 구조 다양성을 달성 하기 위해 전략 뒤에 3-O 및 6-O 에 그들의 stepwise α 선택적 glycosylations oligosaccharyl 불 소 단위의 세 종류의 화학 효소 합성 시작의 위치는 중요 한 β-mannoside 결합 하는 데 일반적인 코어 trisaccharide의만 노 오 스 잔류물. 우리는 더 HIV 항 체와 이종 ligand 상호 작용의 분석에 대 한 공유 혼합된 배열을 만드는 알루미늄 산화물 코팅 유리 (ACG) 슬라이드의 표면에 N-glycans를 첨부. 특히, 남자 (5)는 새로 고립 된 HIV-1 광범위 중화 항 체 (bNAb), PG9, 밀접 하 게 간격 둔된 남자5GlcNAc2 의 혼합물에의 바인딩 동작 및 2, 6-디-sialylated bi antennary 복잡 한 입력 N-glycan (SCT ) ACG 배열, 에이즈 백신 개발을 위한 효과적인 immunogen 디자인 가이드에 새로운 길을 엽니다. 또한, 우리의 ACG 배열 hetero ligand 바인딩 동작에 대 한 다른 HIV 항 체를 연구 하는 강력한 도구를 구현 합니다.

다양 한 N-glycans의 합성에 대 한 모듈형 화학 효소 전략은 그림 1에 표시 됩니다. 전략은 다양성 α-특정 mannosylation는 3-O 및 6-O 에 다음 세 가지 중요 한 모듈의 화학 효소 합성 하 여 처음에 만들 수 있습니다는 사실에 근거한 일반적인만 노 오 스 잔류물의 위치 trisaccharide Nglycans의 핵심. Bi-트라이-, tetra antennary 복합 형식 N<...

Watch this Scientific Journal Video about Chemo-enzymatic Synthesis of N-glycans for Array Development and HIV Antibody Profiling at JoVE.com

Chemo-enzymatic Synthesis of N-glycans for Array Development and HIV Antibody Profiling

Glycan microarray를 개발 하 고 광범위 하 게 항 체를 중화 하는 HIV의 프로 파일링에 대 한 사용 설명 되는 산화 알루미늄 코팅 유리에 첨부 파일에 대 한 N-glycans의 합성에 모듈 접근 (ACG 슬라이드) 슬라이드.

N-glycans 배열 개발 및 HIV 항 체의 항 암 치료 효소 합성 프로 파일링

We present a highly efficient way for the rapid preparation of a wide range of N-linked oligosaccharides (estimated to exceed 20,000 structures) that are ...

chemo-enzymatic-synthesis-n-glycans-for-array-development-hiv

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JoVE Journal

Chemistry

Chemo-enzymatic Synthesis of N-glycans for Array Development and HIV Antibody Profiling

8.7K 조회수.  Academia Sinica. Glycan microarray를 개발 하 고 광범위 하 게 항 체를 중화 하는 HIV의 프로 파일링에 대 한 사용 설명 되는 산화 알루미늄 코팅 유리에 첨부 파일에 대 한 N-glycans의 합성에 모듈 접근 (ACG 슬라이드) 슬라이드.

비디오: Chemo-enzymatic Synthesis of N-glycans for Array Development and HIV Antibody Profiling

Profiling Anti-Neu5Gc IgG in Human Sera with a Sialoglycan Microarray Assay

Cells are covered with a cloak of carbohydrate chains (glycans) that is commonly altered in cancer and that includes variations in sialic acid (Sia) expression. These are acidic sugars that have a 9-carbon backbone and that cap vertebrate glycans on cell surfaces. Two of the major Sia forms in mammals are N-acetylneuraminic acid (Neu5Ac) and its hydroxylated form, N-glycolylneuraminic acid (Neu5Gc). Humans cannot produce endogenous Neu5Gc due to the inactivation of the gene encoding cytidine 5&#39;monophosphate-Neu5Ac (CMP-Neu5Ac) hydroxylase (CMAH). Foreign Neu5Gc is acquired by human cells through the dietary consumption of red meat and dairy and subsequently appears on diverse glycans on the cell surface, accumulating mostly on carcinomas. Consequently, humans have circulating anti-Neu5Gc antibodies that play diverse roles in cancer and other chronic inflammation-mediated diseases and that are becoming potential diagnostic and therapeutic targets. Here, we describe a high-throughput sialoglycan microarray assay to assess such anti-Neu5Gc antibodies in the human sera. Neu5Gc-containing glycans and their matched pairs of controls (Neu5Ac-containing glycans), each with a core primary amine, are covalently linked to epoxy-coated glass slides. We exemplify the printing of 56 slides in a 16-well format using a specific nano-printer capable of generating up to 896 arrays per print. Each slide can be used to screen 16 different human sera samples for the evaluation of anti-Neu5Gc antibody specificity, intensity, and diversity. The protocol describes the complexity of this robust tool and provides a basic guideline for those aiming to investigate the response to Neu5Gc dietary carbohydrate antigen in diverse clinical samples in an array format.

A sialoglycan microarray assay can be used to evaluate anti-Neu5Gc antibodies in human sera, making it a potential high-throughput diagnostic assay for cancer and other chronic inflammation-mediated human diseases.

Sialoglycan Microarray Assay를 사용하여 Human Sera에서 Anti-Neu5Gc IgG 프로파일 링

Cells are covered with a cloak of carbohydrate chains (glycans) that is commonly altered in cancer and that includes variations in sialic acid (Sia) ...

연구

행동분석학

A Miniaturized Glycan Microarray Assay for Assessing Avidity and Specificity of Influenza A Virus Hemagglutinins

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the natural reservoir for IAV, but IAV can cross the species barrier to poultry, swine, horses and humans. Avian viruses recognize sialic acid attached to a penultimate galactose by a &#945;2-3 linkage (avian-type receptors) whereas human viruses preferentially recognize sialic acid with a &#945;2-6 linkage (human-type receptors). To monitor if avian viruses are adapting to human type receptors, several methods can be used. Glycan microarrays with diverse libraries of synthetic sialosides are increasingly used to evaluate receptor specificity. However, this technique is not used for measuring avidities. Measurement of avidity is typically achieved by evaluating the binding of serially diluted hemagglutinin or virus to glycans adsorbed to conventional polypropylene 96-well plates. In this assay, glycans with &#945;2-3 or &#945;2-6 sialic acids are coupled to biotin and adsorbed to streptavidin plates, or are coupled to polyacrylamide (PAA) which directly adsorb to the plastic. We have significantly miniaturized this assay by directly printing PAA-linked sialosides and their non PAA-linked counterparts on micro-well glass slides. This set-up, with 48 arrays on a single slide, enables simultaneous assays of 6 glycan binding proteins at 8 dilutions, interrogating 6 different glycans, including two non-sialylated controls. This is equivalent to 18x 96-well plates in the traditional plate assay. The glycan array format decreases consumption of compounds and biologicals and thus greatly enhances efficiency.

Using a printed glycan microarray strategy, a conventional 96-well plate assay was miniaturized for analysis of influenza A virus hemagglutinin avidity and specificity for sialic acid containing receptors.

인플루엔자 바이러스 Hemagglutinins의 결합력 및 특이도를 평가하기위한 소형화 글리 칸 마이크로 어레이 분석

Influenza A virus (IAV) hemagglutinins recognize sialic acids on the cell surface as functional receptors to gain entry into cells. Wild waterfowl are the ...

면역학 및 감염병학

<meta charset="utf8"></head><body>Glycomics-Guided Glycoproteomics를 사용한 대장암 조직의 프로파일링

Glycomics-Guided Glycoproteomics Facilitates Comprehensive Profiling of the Glycoproteome in Complex Tumor Microenvironments

Glycosylation is a common and structurally diverse protein modification that impacts a wide range of tumorigenic processes. Mass spectrometry-driven glycomics and glycoproteomics have emerged as powerful approaches to analyze liberated glycans and intact glycopeptides, respectively, offering a deeper understanding of the heterogeneous glycoproteome in the tumor microenvironment (TME). This protocol details the glycomics-guided glycoproteomics method, an integrated omics technology, which firstly employs porous graphitized carbon-LC-MS/MS-based glycomics to elucidate the glycan structures and their quantitative distribution in the glycome of tumor tissues, cell populations, or bodily fluids being investigated. This allows for a comparative glycomics analysis to identify altered glycosylation across patient groups, disease stages, or conditions, and, importantly, serves to enhance the downstream glycoproteomics analysis of the same sample(s) by creating a library of known glycan structures, thus reducing the data search time and the glycoprotein misidentification rate. Focusing on N-glycoproteome profiling, the sample preparation steps for the glycomics-guided glycoproteomics method are detailed in this protocol, and key aspects of the data collection and analysis are discussed. The glycomics-guided glycoproteomics method provides quantitative information on the glycoproteins present in the TME and their glycosylation sites, site occupancy, and site-specific glycan structures. Representative results are presented from formalin-fixed paraffin-embedded tumor tissues from colorectal cancer patients, demonstrating that the method is sensitive and compatible with tissue sections commonly found in biobanks. Glycomics-guided glycoproteomics, therefore, offers a comprehensive view into the heterogeneity and dynamics of the glycoproteome in complex TMEs, generating robust biochemical data required to better understand the glycobiology of cancers.

<meta charset="utf8"></head><body>Glycomics-guided glycoproteomics는 복잡한 종양 미세환경에서 당단백질체의 포괄적인 프로파일링을 용이하게 합니다.

This protocol details the glycomics-guided glycoproteomics method, an integrated omics technology that offers comprehensive insights into the heterogeneous glycoproteome in complex tumor microenvironments required to better understand the glycobiology of cancers.

Glycomics-guided glycoproteomics는 복잡한 종양 미세환경에서 당단백질체의 포괄적인 프로파일링을 용이하게 합니다. 

Glycosylation is a common and structurally diverse protein modification that impacts a wide range of tumorigenic processes. Mass spectrometry-driven ...

암 연구학

Purification and Analytics of a Monoclonal Antibody from Chinese Hamster Ovary Cells Using an Automated Microbioreactor System

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using Chinese hamster ovary (CHO) cells, culture and process conditions must be optimized to maximize antibody titers and achieve target quality profiles. Typically, this optimization uses automated microscale bioreactors (15 mL) to screen multiple process conditions in parallel. Optimization criteria include culture performance and the critical quality attributes (CQAs) of the monoclonal antibody (mAb) product, which may impact its efficacy and safety. Culture performance metrics include cell growth and nutrient consumption, while the CQAs include the mAb's N-glycosylation and aggregation profiles, charge variants, and molecular weight. This detailed protocol describes how to purify and subsequently analyze HCCF samples produced by an automated microbioreactor system to gain valuable performance metrics and outputs. First, an automated protein A fast protein liquid chromatography (FPLC) method is used to purify the mAb from harvested cell culture samples. Once concentrated, the glycan profiles are analyzed by mass spectrometry using a specific platform (refer to the Table of Materials). Antibody molecular weights and aggregation profiles are determined using size exclusion chromatography-multiple angle light scattering (SEC-MALS), while charge variants are analyzed using microchip capillary zone electrophoresis (mCZE). In addition to the culture performance metrics captured during the bioreactor process (i.e., culture viability, cell counts, and common metabolites including glutamine, glucose, lactate, and ammonia), spent media is analyzed to identify limiting nutrients to improve the feeding strategies and overall process design. Therefore, a detailed protocol for the absolute quantification of amino acids by liquid chromatography-mass spectrometry (LC-MS) of spent media is also described. The methods used in this protocol take advantage of high-throughput platforms that are compatible for large numbers of small-volume samples.

A detailed protocol for the purification and subsequent analysis of a monoclonal antibody from harvested cell culture fluid (HCCF) of automated microbioreactors has been described. Use of analytics to determine critical quality attributes (CQAs) and maximizing limited sample volume to extract vital information is also presented.

자동화된 미생물체 시스템을 사용하여 중국 햄스터 난소 세포의 단일 클론 항체의 정화 및 분석

Monoclonal antibodies (mAbs) are one of the most popular and well-characterized biological products manufactured today. Most commonly produced using ...

생체공학

Printed Glycan Array: A Sensitive Technique for the Analysis of the Repertoire of Circulating Anti-carbohydrate Antibodies in Small Animals

The repertoire of circulating anti-carbohydrate antibodies of a given individual is often associated with its immunological status. Not only the individual immune condition determines the success in combating internal and external potential threat signals, but also the existence of a particular pattern of circulating anti-glycan antibodies (and their serological level variation) could be a significant marker of the onset and progression of certain pathological conditions. Here, we describe a Printed Glycan Array (PGA)-based methodology that offers the opportunity to measure hundreds of glycan targets with very high sensitivity; using a minimal amount of sample, which is a common restriction present when small animals (rats, mice, hamster, etc.) are used as models to address aspects of human diseases. As a representative example of this approach, we show the results obtained from the analysis of the repertoire of natural anti-glycan antibodies in BALB/c mice. We demonstrate that each BALB/c mouse involved in the study, despite being genetically identical and maintained under the same conditions, develops a particular pattern of natural anti-carbohydrate antibodies. This work claims to expand the use of PGA technology to investigate repertoire (specificities) and the levels of circulating anti-carbohydrates antibodies, both in health and during any pathological condition.

This work shows the potential of printed glycan array (PGA) technology for the analysis of circulating anti-carbohydrate antibodies in small animals.

인쇄 Glycan 배열: 작은 동물에 반대로 탄수화물 항 체 순환의 레 퍼 토리의 분석에 대 한 민감한 기술

The repertoire of circulating anti-carbohydrate antibodies of a given individual is often associated with its immunological status. Not only the ...

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.

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.

단지 샘플의 특정 단백질 글리코 실화의 다중 높은 처리량 프로파일을위한 화학을 차단 항체 Microarray

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

생물학

Tetrameric Fluorescent Antigen Arrays for Single-Step Identification of Antigen-Specific B Cells

Fluorescent antigen production is a critical step in the identification of antigen-specific B cells. Here, we detailed the preparation, purification, and the use of four-arm, fluorescent PEG-antigen conjugates to selectively identify antigen-specific B cells through avid engagement with cognate B cell receptors. Using modular click chemistry and commercially available fluorophore kit chemistries, we demonstrated the versatility of preparing customized fluorescent PEG-conjugates by creating distinct arrays for proteolipid protein (PLP139-151) and insulin, which are important autoantigens in murine models of multiple sclerosis and type 1 diabetes, respectively. Assays were developed for each fluorescent conjugate in its respective disease model using flow cytometry. Antigen arrays were compared to monovalent autoantigen to quantify the benefit of multimerization onto PEG backbones. Finally, we illustrated the utility of this platform by isolating and assessing anti-insulin B cell responses after antigen stimulation ex vivo. Labeling insulin-specific B cells enabled the amplified detection of changes to co-stimulation (CD86) that were otherwise dampened in aggregate B cell analysis. Together, this report enables the production and use of fluorescent antigen arrays as a robust tool for probing B cell populations.

<meta charset="utf8"></head><body>항원 특이적 B 세포의 단일 단계 식별을 위한 Tetrameric Fluorescent Antigen Array

This protocol details the customizable production and use of fluorescent probes for labeling antigen-specific B cells.

항원 특이적 B 세포의 단일 단계 식별을 위한 Tetrameric Fluorescent Antigen Array

Fluorescent antigen production is a critical step in the identification of antigen-specific B cells. Here, we detailed the preparation, purification, and ...

N-glycans 배열 개발 및 HIV 항 체의 항 암 치료 효소 합성 프로 파일링

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Sialoglycan Microarray Assay를 사용하여 Human Sera에서 Anti-Neu5Gc IgG 프로파일 링

인플루엔자 바이러스 Hemagglutinins의 결합력 및 특이도를 평가하기위한 소형화 글리 칸 마이크로 어레이 분석

Glycomics-guided glycoproteomics는 복잡한 종양 미세환경에서 당단백질체의 포괄적인 프로파일링을 용이하게 합니다.

Glycomics-guided glycoproteomics는 복잡한 종양 미세환경에서 당단백질체의 포괄적인 프로파일링을 용이하게 합니다.

Glycomics-guided glycoproteomics는 복잡한 종양 미세환경에서 당단백질체의 포괄적인 프로파일링을 용이하게 합니다.

자동화된 미생물체 시스템을 사용하여 중국 햄스터 난소 세포의 단일 클론 항체의 정화 및 분석

인쇄 Glycan 배열: 작은 동물에 반대로 탄수화물 항 체 순환의 레 퍼 토리의 분석에 대 한 민감한 기술

단지 샘플의 특정 단백질 글리코 실화의 다중 높은 처리량 프로파일을위한 화학을 차단 항체 Microarray

항원 특이적 B 세포의 단일 단계 식별을 위한 Tetrameric Fluorescent Antigen Array

항원 특이적 B 세포의 단일 단계 식별을 위한 Tetrameric Fluorescent Antigen Array