Name: analysis of n-glycans from raphanus sativus cultivars using pngase h+
Uploaded: 2018-06-25T15:00:00.000+00:00
Duration: 8 min 26 s
Description: Watch this Scientific Journal Video about Analysis of N-glycans from Raphanus sativus Cultivars Using PNGase H+ at JoVE.com

이 작품은 중국의 자연 과학 재단에 의해 부분적으로 지원 (부여 번호 31471703, A0201300537 및 31671854 J.V. 하 L.L., 번호 31470435 G.Y.에 부여), 그리고 100 외국 재능 계획 (보조금 번호 JSB2014012 J.V.).

1. 샘플 컬렉션
<ol><li>신선한 무 (Raphanus sativus L.)의 다른 cultivars을 구입.</li>
</ol>2입니다. 무에서 단백질의 격리
<ol><li>10 분 동안 부엌 믹서와 신선한 무의 약 100 g을 균질.</li>
	<li>50 mL 원심 분리기 튜브 및 불용 성 물질을 제거 20 분 4 ° C에서 14000 × g 에서 원심 슬러리를 전송 합니다.</li>
	<li>새로운 50 mL 원심 분리기 튜브에는 상쾌한을 신중 하 게 전송 하 고 2m trichloroacetic 산 (TCA) 솔루션의 동일한 볼륨을 추가 합니다. 참고: TCA를 추가 수용 성 (glyco) 단백질 침전 됩니다.</li>
	<li>30 분 동안 4 ° C에서 14000 × g 에서 centrifuge 고 수송과 (glyco) 단백질에서는 상쾌한을 제거 합니다.</li>
	<li>녹는 oligosaccharides 및 다 당 류를 제거 하 5 분 동안 4 ° C에서 14000 × g 에서 이온된 수와 원심 분리기의 20 mL와 펠 릿을 세척.</li>
	<li>2.5 단계를 반복 합니다. 4 번입니다.</li>
	<li>다시 1 ml의 이온을 제거 된 물과 신선한 1.5 mL 원심 분리기 튜브에 펠 릿을 일시 중단 합니다.</li>
</ol>3. N-Glycans의 준비
<ol><li>2.7 단계에서 단백질 해결책의 믹스 50 µ L. (신선한 무의 5 g와 같음) 10 mM 아세트산에 재조합 PNGase H+ 의 0.2 뮤와 함께.</li>
	<li>12 h 37 ° C에서 반응 혼합물을 품 어. 부 화 후 5 분 여분의 단백질 및 효소 제거 14000 × g 에서 원심.</li>
	<li>신선한 1.5 mL 원심 분리기 튜브는 상쾌한 전송.</li>
</ol>4. NGlycans의 정화
<ol><li>Nglycans 풍부와 Nglycan에 대 한 fluorogenic 2 aminobenzamide (2-AB) 레이블 에이전트의 선택도 증가 하는 반응 혼합물에서 소금 및 기타 불순물을 제거 고체 상 추출 (SPE) 열을 준비 derivatization입니다.<ol>
<li>씻어 열 이온된 물 3 mL를 추가 합니다.</li>
		<li>SPE-열 활성 trifluoroacetic 산 (TFA, 0.1 %v / v)을 포함 하는 80% 이기 솔루션의 3 mL를 추가 합니다.</li>
		<li>SPE-열 equilibrate 하 이온된 물 3 mL를 추가 합니다.</li>
	</ol>
</li>
	<li>3.3 열에 단계에서 샘플을 전송 하 고 흐름을 통해 삭제.</li>
	<li>이온된 물 흐름을 통해 삭제를 열을 씻어 1.5 mL를 추가 합니다.</li>
	<li>0.1%를 포함 20% 이기 용액의 1.5 mL를 사용 하 여 열에서 출시 된 Nglycans elute TFA (v/v) 2 mL 원심 분리기 관으로.</li>
	<li>상 온에서 원심 증발에 의해 용 매를 제거 합니다. 이 샘플은 완전히 건조 될 때까지 증발.</li>
</ol>5. 형광 Derivatization NGlycans의
<ol><li>2 AB 솔루션, 디 메 틸 ⁚ / 아세트산 산 성 용액 (7:3, v/v)에 35 m m 2-AB와 0.1 M 나트륨 cyanoborohydride의 구성의 1 mL를 준비 합니다.</li>
	<li>6 다른 무에서 파생 된 말린 샘플 (4.5 단계.) 2-AB 솔루션의 5 µ L를 추가 합니다. 와 동 각 샘플까지 완전히 해산.</li>
	<li>2 h 65 ° c.에 대 한 혼합물을 품 어</li>
	<li>5 분 동안 실내 온도 아래로 샘플 식 고 이온된 수의 5 µ L 및 이기의 40 µ L를 추가 합니다. 3 분 14000 x g에서 튜브 원심</li>
	<li>300 µ L 높은 복구 HPLC 유리병에는 상쾌한에서 48 µ L를 전송. 최대 한 달 동안-20 ° C에서 derivatized N-glycan 샘플을 저장 합니다.</li>
</ol>6. HILIC UPLC Nglycans의 프로 파일링
<ol><li>각각 330 nm/420 nm의 여기/방출 파장에서 형광 온라인 detectorset에 연결 된 표준 UPLC 시스템을 사용 하 여 샘플을 분석 합니다.</li>
	<li>소수 성 상호 작용 액체 크로마토그래피 (HILIC)를 사용 하 여-UPLC glycan 열 분석을 위한 60 ° C의 열 온도에.</li>
	<li>950 mL의 액체 크로마토그래피-질량 분석 (LCMS)와 재고 솔루션 (1m 암모늄 편대 솔루션, pH 4.5) 50 mL를 희석 하 여 준비 하는 용 매 A-등급 물. 준비 재고 솔루션을 다음과 같습니다.<ol>
<li>LCMS 급 H2o.의 700 mL에 포 름 산의 43 개 mL를 추가</li>
		<li>4.5 pH 수성 암모니아 솔루션 (25 %w / w)의 dropwise 추가 조정.</li>
		<li>측정 실린더 용 매를 전송 하 고 LCMS 급 H2o. 저장소 4-6 ° C에서 최대 3 개월까지 최대 1000 mL 채우기.</li>
	</ol>
</li>
	<li>솔벤트 B. LCMS 급 이기 사용</li>
	<li>N-glycans 다음 그라데이션 차입 구분:<ol>
<li>44.5 분을 0에서 세트로 용 A. 0.5 mL/min 유량 용 매 B의 95%를 추가 하 여 시작 합니다.</li>
		<li>0 분-6 분에서 점차적으로 용 매 B 선형 그라데이션 to78%의 비율을 감소.</li>
		<li>44.5 분 6 분에서 점차적으로 55.9%를 선형 그라데이션으로 용 매 B의 비율을 감소.</li>
		<li>46.5 분 44.5 분에서 급속 하 게 용 매 B 0 %55.9 %에서 선형 그라데이션으로 비율을 감소 하 고 2 분 동안 0%에서 누른 열 세척을 시작. 55 분 44.5에서 0.25 mL/분 유량을 줄일 수 있습니다.</li>
		<li>50.5 분 48.5 분에서 용 매 B 95% 증가 하 여 추가 열을 세척 하 고 4.5 분 95%에서 개최.</li>
		<li>다시-57 분 50.5에서 95% 용 매 B 0.5 mL/min의 시작 조건에 열을 equilibrate.</li>
	</ol>
</li>
	<li>UPLC 시스템으로 샘플의 45 µ L를 주사.</li>
	<li>N-glycans UPLC 여 15 그리고 40 분 사이 보존 시간 elute</li>
	<li>2 mL 원심 분리기 튜브를 사용 하 여 각 관찰된 UPLC 피크 분수를 수집 하 고 샘플 원심 증발에 의해 건조.</li>
	<li>2 AB dextran 표준 (2−20 포도 당 단위) 식물-N-glycan 파일링, 보정 하 고 표준화 된 포도 당 단위로 그들의 차입 시간을 할당 하 라는의 약 1 pmol 주사.</li>
</ol>7. MALDI TOF MS 분석
<ol><li>LCMS의 단계 5에서 6.8 µ L에서 샘플을 분해-등급 물. 샘플 솔루션 및 6-아 자-2-thiothymine (ATT) 솔루션 (0.3 w/v %70 %v / v 수성 이기에), 그리고 피 펫 MALDI TOF 샘플 캐리어에 혼합물의 1 µ L의 믹스 1 µ L.</li>
	<li>긍정적인 이온 모드 시작 버튼을 누르면 MALDI TOF 질량 분석기 악기를 사용 하 여 샘플을 분석 합니다.</li>
	<li>동반 MALDI TOF MS 분석 소프트웨어를 사용 하 여 질량 스펙트럼을 분석 합니다.</li>
	<li>오픈 소스 glycan 해석 소프트웨어19를 사용 하 여 스펙트럼을 해석 합니다. 2-AB, [M + 나]+에 대 한 검색 매개 변수를 설정 하 고 2.0 다를 정확도 매개 변수를 설정 합니다.</li>
</ol>

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저자는 공개 없다.

우리가 여기 제시 하는 프로토콜 무의 다양 한 재배의 N-glycan 프로 파일의 비교를 허용 한다. 기존 프로토콜에 비해이 방법의 중요 한 장점은 Nglycans의 효소 출시와 2 AB와 derivatization 반응 사이의 버퍼 변경할 필요가 없습니다입니다. 이 절차의 가장 중요 한 단계는 N-glycans SPE 열을 사용 하 여 염 분을 제거 하는 실패의 정화 또는 반응 혼합물에 다른 불순물 형광 derivatization 효율?...

식물에서 N-glycans 그린 관심 증가 최근 몇 년 동안, 이전 연구로 알레르기 성 응답1,2 유도할 수 있는 면역 cross-reactions의 N-glycans을 강조 표시 . 그것은 입증 되었습니다 이전 촉매 활동3,4, 내열성 및 접는5,6 또는 subcellular 지 방화 N-glycans 공장 glycoproteins에 영향을 미칠 수 있는 고 분 비7. Glycan 구조 그들의 각각 기능 연관, 위하여 N-glycans 먼저 해제 되어야 합니다 glycoproteins에서 화학적 또는 효소. N-및 O-glycans 방출에 대 한 고전적인 화학 방법은 β-제거, 알칼리 샘플 치료 borohydride alditol8를 가진 감소에 의해 동반입니다. 그러나,이 절차는 fluorophore로 하는 걸로 하 고 glycan 구조의 줄이는 끝에서 단 당 류 단위의 상당한 감퇴의 원인. 수산화 암모늄/탄산 치료에 따라 화학 deglycosylation는 또한 일반적으로 사용 되는 대체 방법9. 이러한 화학 릴리스 방법의 그대로 단백질 같은 대량 범위에 레이블이 없는 glycan 풀 펩 티 드 파편의 간섭 없이 대량 spectrometric 분석을 수 있는 성능이 저하 됩니다. 그러나, 이러한 방법의 한 가지 단점이 일반적인 탄수화물 구조 발견 식물10에서 α1, glycans-3-fucosylated N의 증가 저하 속도입니다. 또는, 펩 티 드를 사용 하 여 효소 분리 방법:N-glycanases (PNGases, EC 3.5.1.52) 또한 광범위 하 게 적용 됩니다. 재조합 PNGase F ( Flavobacterium meningosepticum)에서 가장 일반적인 선택 하 고 N-glycans, 코어 α1, fucose 311,12베어링 구조를 제외 하 고 모든 종류의 릴리스 합니다. 따라서, PNGase A (아몬드 씨에서 분리)으로 식물 N-glycans13의 분석을 위해 사용 됩니다. 그러나,이 효소 deglycosylates만 proteolytically-파생 된 glycopeptides, deglycosylate 기본 glycoproteins14수입니다. 따라서, 다단계 샘플 검사 결과 추가 분석, glycans, 특히 낮은 풍부15의 그의 광범위 한 손실이 발생 하기 전에 필요 합니다. N-glycan 릴리스 및 형광 간단 하 고 강력한 방식으로 라벨에 대 한 최적화 된 워크플로우를 제시 하는 방법의 전반적인 목표가입니다. 기본 근거 PNGase H+는 Terriglobus roseus 에서 최근에 발견 되었다 recombinantly 대장균에 표현 될 수 있다, N-glycans 산 성에서 단백질 비 계에서 직접은 수은 조건16. PNGase H+ 를 사용 하 여 대체 방법의 주요 장점은입니다 형광 라벨 반응 반응 버퍼17,18를 변경 하지 않고 동일한 반응 관에서 수행할 수 있습니다. 간단한 준비 조건 및 낮은 풍부 oligosaccharides의 높은 복구 확인이 방법은 유용한 도구 Nglycans의 분석에. 이 프로토콜은 다양 한 식물 종에서 N-glycans의 분석에 적합 합니다.

<table><tbody><tr><td>&lt;strong&gt;Chemicals:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Trichloroacetic acid&amp;nbsp;</td><td>SCR, Shanghai</td><td>80132618</td><td></td></tr><tr><td>Acetic acid glacial</td><td>Huada, Guangzhou</td><td>64-19-7</td><td></td></tr><tr><td>Acetonitrile</td><td>General-reagent</td><td>G80988C</td><td></td></tr><tr><td>Trifluoroacetic acid</td><td>Energy chemical</td><td>W810031</td><td></td></tr><tr><td>2-aminobenzamide</td><td>Heowns, Tianjin</td><td>A41900</td><td></td></tr><tr><td>Sodium cyanoborohydride</td><td>J&amp;amp;K Scientific Ltd</td><td>314162</td><td></td></tr><tr><td>Dimethyl sulfoxide</td><td>Huada, Guangzhou</td><td>67-68-5</td><td></td></tr><tr><td>2AB-labeled dextran ladder, 200 pmol</td><td>Agilent Technologies</td><td>AT-5190-6998</td><td></td></tr><tr><td>6-Aza-2-thiothymine&amp;nbsp;</td><td>Sigma</td><td>275514</td><td></td></tr><tr><td>&lt;strong&gt;Tools/Materials:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Kitchen blender</td><td>Bear, Guangzhou</td><td>LLJ-A10T1</td><td></td></tr><tr><td>Centrifuge</td><td>Techcomp</td><td>CT15RT</td><td></td></tr><tr><td>Centrifugal Evaporator</td><td>Hualida, Taicang</td><td>LNG-T120</td><td></td></tr><tr><td>SPE column</td><td>Supelco</td><td>Supelclean ENVI Carb SPE column</td><td></td></tr><tr><td>MALDI-TOF mass spectrometer</td><td>Bruker</td><td>Autoflex</td><td></td></tr><tr><td>&lt;strong&gt;HPLC Analysis:&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>High-recovery HPLC vial</td><td>Agilent Technologies</td><td>&amp;nbsp;# 5188-2788</td><td></td></tr><tr><td>HPLC System</td><td>Shimadzu</td><td>Nexera</td><td></td></tr><tr><td>Fluorescence Detector for HPLC</td><td>Shimadzu</td><td>RF-20Axs&amp;nbsp;</td><td></td></tr><tr><td>Column oven</td><td>Hengxin</td><td>CO-2000</td><td></td></tr><tr><td>HPLC Column</td><td>Waters</td><td>Acquity UPLC BEH glycan column</td><td>2.1 &amp;times; 150 mm, 1.7 &amp;mu;m particle size</td></tr><tr><td>LCMS-grade Water</td><td>Merck Millipore</td><td>#WX00011</td><td></td></tr><tr><td>LCMS-grade Acetonitrile</td><td>Merck Millipore</td><td># 100029</td><td></td></tr><tr><td>Formic acid</td><td>Aladdin</td><td>F112034</td><td></td></tr><tr><td>Ammonia solution</td><td>Aladdin</td><td>A112080</td><td></td></tr></tbody></table>

analysis of n-glycans from raphanus sativus cultivars using pngase h+

최근 몇 년 동안, 식물의 탄수화물 moieties 받은 관심, 그들은 cross-reactive, 알레르기 자극 면역 반응의. 또한, 탄수화물 구조 또한 식물 물질 대사에 중요 한 역할을 담당할. 여기, 우리가 준비 하 고 사용 하는 N-glycanase 특정 식물에서 파생 된 탄수화물 구조의 릴리스에 대 한 무 (Raphanus sativus)의 다른 재배 품 정에서 N-glycans를 분석 하는 간단 하 고 빠른 방법을 제시. 이 위해 무 homogenates의 원유 trichloroacetic 산 침전 PNGase H+로 치료 되었고 형광 태그 2-aminobenzamide를 사용 하 여 표시. 레이블이 지정 된 N-glycan 샘플 이후 울트라 성능 액체 크로마토그래피 (UPLC) 분리 및 세부 구조에 대 한 비행 (TOF MALDI) 질량 분석의 매트릭스 보조 레이저 탈 착 이온화 시간으로 분석 되었다 평가 및 무 파생 N-glycan 구조 상대 abundancies 척도를. 이 프로토콜 사용할 수 있습니다 또한 다양 한 다른 식물 종에서 N-glycans의 분석 그리고 함수 추가 조사 및 인간의 건강에 N-glycans의 효과 대 한 유용할 수 있습니다.

그림 1 (glyco-) 무, N-glycans의 준비, UPLC 분석 및 이러한 구성 요소의 MALDI TOF MS 분석에서 단백질의 격리를 포함 하 여 설명된 프로토콜의 구조 개요를 보여 줍니다. 그림 2 분석된 무 재배의 derivatized Nglycans의 대표 UPLC chromatograms를 보여 줍니다. 그림 3 2AB derivatized N-glycan 구조 MALDI TOF 질량 ?...

Watch this Scientific Journal Video about Analysis of N-glycans from Raphanus sativus Cultivars Using PNGase H+ at JoVE.com

Analysis of N-glycans from Raphanus sativus Cultivars Using PNGase H+

N-glycans Raphanus sativus Cultivars 사용 PNGase H+ 에서 분석

우리는 준비 및 N-glycans 무 (Raphanus sativus)의 다른 재배에서의 분석을 위한 간단 하 고 빠른 방법을 설명합니다.

In recent years, the carbohydrate moieties of plants have received considerable attention, as they are a potential source of cross-reactive, ...

analysis-of-n-glycans-from-raphanus-sativus-cultivars-using-pngase-h

Research

JoVE Journal

Biochemistry

Analysis of N-glycans from Raphanus sativus Cultivars Using PNGase H+

6.5K 조회수.  Nanjing Agricultural University. 우리는 준비 및 N-glycans 무 (Raphanus sativus)의 다른 재배에서의 분석을 위한 간단 하 고 빠른 방법을 설명합니다.

비디오: Analysis of N-glycans from Raphanus sativus Cultivars Using PNGase H+

Structural Characterization of Mannan Cell Wall Polysaccharides in Plants Using PACE

Plant cell wall polysaccharides are notoriously difficult to analyze, and most methods require expensive equipment, skilled operators, and large amounts of purified material. Here, we describe a simple method for gaining detailed polysaccharide structural information, including resolution of structural isomers. For polysaccharide analysis by gel electrophoresis (PACE), plant cell wall material is hydrolyzed with glycosyl hydrolases specific to the polysaccharide of interest (e.g., mannanases for mannan). Large format polyacrylamide gels are then used to separate the released oligosaccharides, which have been fluorescently labeled. Gels can be visualized with a modified gel imaging system (see Table of Materials). The resulting oligosaccharide fingerprint can either be compared qualitatively or, with replication, quantitatively. Linkage and branching information can be established using additional glycosyl hydrolases (e.g., mannosidases and galactosidases). Whilst this protocol describes a method for analyzing glucomannan structure, it can be applied to any polysaccharide for which characterized glycosyl hydrolases exist. Alternatively, it can be used to characterize novel glycosyl hydrolases using defined polysaccharide substrates.

A protocol for the structural analysis of polysaccharides by gel electrophoresis (PACE), using mannan as an example, is described.

속도 사용 하 여 식물에 Mannan 세포 벽 다 당 류 구조 특성

Plant cell wall polysaccharides are notoriously difficult to analyze, and most methods require expensive equipment, skilled operators, and large amounts ...

연구

생화학

A Quantitative Glycomics and Proteomics Combined Purification Strategy

There is a growing desire in the biological and clinical sciences to integrate and correlate multiple classes of biomolecules to unravel biology, define pathways, improve treatment, understand disease, and aid biomarker discovery. N-linked glycosylation is one of the most important and robust post-translational modifications on proteins and regulates critical cell functions such as signaling, adhesion, and enzymatic function. Analytical techniques to purify and analyze N-glycans have remained relatively static over the last decade. While accurate and effective, they commonly require significant expertise and resources. Though some high-throughput purification schemes have been developed, they have yet to find widespread adoption and often rely on the enrichment of glycopeptides. One promising method, developed by Thomas-Oates et al., filter aided N-glycan separation (FANGS), was qualitatively demonstrated on tissues. Herein, we adapted FANGS to plasma and coupled it to the individuality normalization when labeling with glycan hydrazide tags strategy in order to achieve accurate relative quantification by liquid chromatography mass spectrometry and enhanced electrospray ionization. Furthermore, we designed new functionality to the protocol by achieving tandem, shotgun proteomics and glycosylation site analysis on hen plasma. We showed that N-glycans purified on filter and derivatized by hydrophobic hydrazide tags were comparable in terms of abundance and class to those by solid phase extraction (SPE); the latter is considered a gold standard in the field. Importantly, the variability in the two protocols was not statistically different. Proteomic data that was collected in-line with glycomic data had the same depth compared to a standard trypsin digest. Peptide deamidation is minimized in the protocol, limiting non-specific deamidation detected at glycosylation motifs. This allowed for direct glycosylation site analysis, though the protocol can accommodate 18O site labeling as well. Overall, we demonstrated a new in-line high-throughput, unbiased, filter based protocol for quantitative glycomics and proteomics analysis.

A high-throughput protocol was developed for combined proteomics and glycomics purification and LC-MS/MS quantification in plasma. Deamidation analysis of N-linked glycosylation motifs was specific to deglycosylated sites. Accurate quantitation of N-glycans was achieved by coupling filter aided N-glycan separation to the individuality normalization when labeling with glycan hydrazide tags strategy.

양적 글리코 믹스와 단백질 체학 복합 정제 전략

There is a growing desire in the biological and clinical sciences to integrate and correlate multiple classes of biomolecules to unravel biology, define ...

화학

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

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 commonly found on human glycoproteins. To achieve the desired structural diversity, the strategy began with the chemo-enzymatic synthesis of three kinds of oligosaccharyl fluoride modules, followed by their stepwise &#945;-selective glycosylations at the 3-O and 6-O positions of the mannose residue of the common core trisaccharide having a crucial &#946;-mannoside linkage. We further attached the N-glycans to the surface of an aluminum oxide-coated glass (ACG) slide to create a covalent mixed array for the analysis of hetero-ligand interaction with an HIV antibody. In particular, the binding behavior of a newly isolated HIV-1 broadly neutralizing antibody (bNAb), PG9, to the mixture of closely spaced Man5GlcNAc2 (Man5) and 2,6-di-sialylated bi-antennary complex type N-glycan (SCT) on an ACG array, opens a new avenue to guide the effective immunogen design for HIV vaccine development. In addition, our ACG array embodies a powerful tool to study other HIV antibodies for hetero-ligand binding behavior.

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

A modular approach to the synthesis of N-glycans for attachment to an aluminum oxide-coated glass slide (ACG slide) as a glycan microarray has been developed and its use for the profiling of an HIV broadly neutralizing antibody has been demonstrated.

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 ...

N-glycan Profiling of Glycoproteins by Hydrophilic Interaction Liquid Chromatography with Fluorescence and Mass Spectrometric Detection

Glycosylation is a vital modification found in proteins. N-glycan profiling of glycoproteins is required to detect novel biomarker candidates and determine glycan alterations in diseases. Most commercially available biopharmaceutical proteins are glycoproteins. The efficacy of these drugs is affected by glycosylation patterns. Therefore, an in-depth characterization method for the N-glycans is necessary. Here, we present a comprehensive approach for qualitative and quantitative analysis of N-glycans using hydrophilic interaction liquid chromatography equipped with fluorescence detection and tandem mass spectrometry (HILIC-FLD-MS/MS). N-glycans were released from glycoproteins with a facile method and labeled by a procainamide fluorophore tag in the strategy. Subsequently, the procainamide labeled N-glycans were analyzed by a HILIC-FLD-MS/MS technique. In this approach, N-glycan structures were confirmed by the tandem mass spectrometric analysis, whereas fluorescence detection was used for the quantitative analysis. An application for data analysis of the detected N-glycan peaks is described in the study. This protocol can be applied to any glycoprotein extracted from various species.

N-glycan Profiling of Glycoproteins by Hydrophilic Interaction Liquid Chromatography with Fluorescence and Mass Spectrometric Detection

N-glycan profiling of glycoproteins is essential for discovering novel biomarkers and understanding glycan functions in cellular events. Additionally, N-glycan analysis of protein biopharmaceuticals is very important for human use. In this current article, a high-throughput strategy for identifying and quantifying N-glycan structures was presented using the HILIC-FLD-MS/MS technique.

Glycosylation is a vital modification found in proteins. N-glycan profiling of glycoproteins is required to detect novel biomarker candidates and ...

Pulse-chase Analysis of N-linked Sugar Chains from Glycoproteins in Mammalian Cells

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although this modification was implicated in several biological processes, additional aspects of its function are emerging, with recent evidence of its role in the production of signals for glycoprotein quality control and trafficking. Thus, phenomena related to N-linked glycans and their processing are being intensively investigated. Methods that have been recently developed for proteomic analysis have greatly improved the characterization of glycoprotein N-linked glycans. Nevertheless, they do not provide insight into the dynamics of the sugar chain processing involved. For this, labeling and pulse-chase analysis protocols are used that are usually complex and give very low yields. We describe here a simple method for the isolation and analysis of metabolically labeled N-linked oligosaccharides. The protocol is based on labeling of cells with [2-3H] mannose, denaturing lysis and enzymatic release of the oligosaccharides from either a specifically immunoprecipitated protein of interest or from the general glycoprotein pool by sequential treatments with endo H and N-glycosidase F, followed by molecular filtration (Amicon). In this method the isolated oligosaccharides serve as an input for HPLC analysis, which allows discrimination between various glycan structures according to the number of monosaccharide units comprising them, with a resolution of a single monosaccharide. Using this method we were able to study high mannose N-linked oligosaccharide profiles of total cell glycoproteins after pulse-chase in normal conditions and under proteasome inhibition. These profiles were compared to those obtained from an immunoprecipitated ER-associated degradation (ERAD) substrate. Our results suggest that most NIH 3T3 cellular glycoproteins are relatively stable and that most of their oligosaccharides are trimmed to Man9-8GlcNAc2. In contrast, unstable ERAD substrates are trimmed to Man6-5GlcNAc2 and glycoproteins bearing these species accumulate upon inhibition of proteasomal degradation.

We describe a method for analysis of the alteration of N-linked glycans through the early life of glycoproteins after their biosynthesis in mammalian cells. This is achieved by pulse-chase analysis of metabolically labeled glycans, enzymatic release from glycoproteins and examination by HPLC.

포유 동물 세포에 Glycoproteins의 N - 연결 설탕 체인 펄스 체이스 분석

Attachment of the Glc3Man9GlcNAc2 precursor oligosaccharide to nascent polypeptides in the ER is a common modification for secretory proteins. Although ...

생물학

Nonspecific Proteolysis-Based Glycomics Strategy for Bacterial Glycoproteins

Protein glycosylation is one of the most common and complex post-translational modifications. Many techniques have been developed to characterize the specific roles of glycans, the relationship between their structures and their impact on the functions of proteins. A common method for glycan analysis is to employ exoglycosidase cleavage to release N-linked glycans from glycoproteins or glycopeptides using Peptide-N-Glycosidase F (PNGase F). However, the glycan-protein linkages in bacteria are different and there is no enzyme available to release glycans from bacterial glycoproteins. In addition, free glycans have also been described in mammalian cells, bacteria, yeast, plants, and fish. In this article, we present a method that can characterize the N-linked glycosylation system in Campylobacter jejuni by detecting asparagine (Asn)-linked and free glycans that are not attached to their target proteins. In this method, total proteins from C. jejuni were digested by Pronase E with a higher enzyme to protein ratio (2:1&#8722;3:1) and a longer incubation time (48&#8722;72 h). The resulted Asn-glycans and free glycans were then purified using porous graphitic carbon cartridges, permethylated, and analyzed by mass spectrometry.

This study presents a method for glycomics analysis of glycoproteins by a combination of pronase E digestion, permethylation, and mass spectrometry analysis. This method is capable of analyzing all types of N-linked glycans, including bacterial N-glycans.

Protein glycosylation is one of the most common and complex post-translational modifications. Many techniques have been developed to characterize the ...

<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

Identification and Characterization of Protein Glycosylation using Specific Endo- and Exoglycosidases

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes such as cell adhesion, molecular trafficking, clearance, and signal transduction1-4. In eukaryotes, the most common glycosylation modifications in the secretory pathway are additions at consensus asparagine residues (N-linked); or at serine or threonine residues (O-linked) (Figure 1). Initiation of N-glycan synthesis is highly conserved in eukaryotes, while the end products can vary greatly among different species, tissues, or proteins. Some glycans remain unmodified ("high mannose N-glycans") or are further processed in the Golgi ("complex N-glycans"). Greater diversity is found for O-glycans, which start with a common N-Acetylgalactosamine (GalNAc) residue in animal cells but differ in lower organisms1.
The detailed analysis of the glycosylation of proteins is a field unto itself and requires extensive resources and expertise to execute properly. However a variety of available enzymes that remove sugars (glycosidases) makes possible to have a general idea of the glycosylation status of a protein in a standard laboratory setting. Here we illustrate the use of glycosidases for the analysis of a model glycoprotein: recombinant human chorionic gonadotropin beta (hCG&beta;), which carries two N-glycans and four O-glycans 5. The technique requires only simple instrumentation and typical consumables, and it can be readily adapted to the analysis of multiple glycoprotein samples.
Several enzymes can be used in parallel to study a glycoprotein. PNGase F is able to remove almost all types of N-linked glycans6,7. For O-glycans, there is no available enzyme that can cleave an intact oligosaccharide from the protein backbone. Instead, O-glycans are trimmed by exoglycosidases to a short core, which is then easily removed by O-Glycosidase. The Protein Deglycosylation Mix contains PNGase F, O-Glycosidase, Neuraminidase (sialidase), &beta;1-4 Galactosidase, and &beta;-N-Acetylglucosaminidase. It is used to simultaneously remove N-glycans and some O-glycans8 . Finally, the Deglycosylation Mix was supplemented with a mixture of other exoglycosidases (&alpha;-N-Acetylgalactosaminidase, &alpha;1-2 Fucosidase, &alpha;1-3,6 Galactosidase, and &beta;1-3 Galactosidase ), which help remove otherwise resistant monosaccharides that could be present in certain O-glycans.
SDS-PAGE/Coomasie blue is used to visualize differences in protein migration before and after glycosidase treatment. In addition, a sugar-specific staining method, ProQ Emerald-300, shows diminished signal as glycans are successively removed. This protocol is designed for the analysis of small amounts of glycoprotein (0.5 to 2 &mu;g), although enzymatic deglycosylation can be scaled up to accommodate larger quantities of protein as needed.

Using specific glycosidases to remove sugars from glycoproteins followed by SDS-PAGE is a valuable method to detect glycan modifications on protein samples and is a good choice for initial glycobiology studies. Changes following deglycosylation can be detected as shifts in gel mobility or by staining with glycan sensitive reagents.

특정 엔도와 exoglycosidases를 사용하여 단백질 글리코 실화의 식별 및 특성

Glycosylation, the addition of covalently linked sugars, is a major post-translational modification of proteins that can significantly affect processes ...

N-glycans Raphanus sativus Cultivars 사용 PNGase H⁺ 에서 분석

요약

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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.

특정 엔도와 exoglycosidases를 사용하여 단백질 글리코 실화의 식별 및 특성