Name: glycan node analysis: a bottom-up approach to glycomics
Uploaded: 2016-05-22T15:00:00.000+00:00
Duration: 11 min 36 s
Description: Watch this Scientific Journal Video about Glycan Node Analysis: A Bottom-up Approach to Glycomics at JoVE.com

This work was supported by the College of Liberal Arts and Sciences of Arizona State University in the form of laboratory startup funds to CRB. It was also supported by a grant from Flinn Foundation (Grant No. 1977) and by the National Cancer Institute of the National Institutes of Health under Award Number R33CA191110. JA was supported by the National Institute of General Medical Sciences of the National Institutes of Health Postbaccalalureate Research Education Program (PREP) under award number R25GM071798. The content is solely the responsibility of the authors and does not necessarily represent the official views of the National Institutes of Health.

<ol>
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The authors have nothing to disclose.

일반적으로, 탄당에서 부분적으로 메틸화 된 알디 톨 아세테이트의 성공적인 생산 (PMAAs)는 적은 어려움을 내포하고 N의 -acetylhexosamine (HexNAc) PMAAs의 성공적인 생산보다 더 강력합니다. 이 절차의 모든 단계에서 재생 될 때,이 현상의 뒤에 정확한 메커니즘은 알려져 있지만 탄당에 HexNAcs 대하여 고유 (오히려 수산기 이상)이 N의 아세틸 기의 고유 화학에 관련한다. 이 산 가수 분해에 관?...

당지질, 당 단백질, 프로테오글리칸과 글리코 사 미노 글리 칸 일괄 글리 칸으로 알려진 복잡한 이기종 탄수화물의 네 가지 주요 클래스를 구성한다. 마찬가지로 세포막, 글리코 칼 릭스, 세포 외 매트릭스와 유체 유비쿼터스 적분 성분, 글리 칸은 세포 내 이입 세포 내 피킹, 세포 운동성, 신호 전달 분자 인식, 수용체 활성화, 세포 부착, 호스트 병원체 상호 작용 등의 다양한 생화학 적 과정에 참여 삶의 거의 모든 영역에서 세포 간 통신, immunosurveillance 및 면역 반응 개시. 1 현재는, 글리 칸 폴리머를 구축 글리코 실 트랜스퍼 라 아제로 알려진 효소 글리코 시드 구축 (글리 칸을 무너 뜨리는 또한 글리코시다 아제로 알려진) 가수 분해, 개조와 협력하여 행동 궁극적 글리 칸 폴리머 2 최종 생산하고 있습니다. 각 글 라이코 실 트랜스퍼 라제는 다른 복합 당질에서 작동 할 수 있지만하는 글 라이코 실 트랜스퍼 라제일반적 핵성 수용체의 특정 카테고리 (예를 들어, 지질, 폴리펩티드, 핵산, 또는 성장에 특히 활성 뉴클레오티드 당 공여체 (예, GDP 푸 코스)의 단당 잔기를 전송하여 linkage-과 아노 머 특정 글리코 시드 결합을 위조 올리고당). 단백질 (특히 막과 분비 단백질)의 50 % 이상이-병진 후 당화에 의해 수정 된 것으로 추정 된 3 초보 조합 계산이 당화에 의해 당 단백질에게 부여 상당한 변화, 다양성과 특이성에 대한 감사를 제공합니다.; 폴리펩티드 기판 10 글리코 실화 부위를 가지고 있으며, 각 사이트는 단지 3 개의 단당류 환원 말단 1 글리코 시드 결합을 형성 할 경우, 예를 들어, 이론적 최종 당단백 3 10 = 59049 고유 ID를 가정 할 수있다. 당 단백질, 글리코 시드 결합은 일반적으로 측쇄 질소 O를 형성시퀀스에서 Asn-X-빼앗아에서 F 아스파라긴 잔기 /의 Thr N이 O 4 -glycans 수득 세린 및 트레오닌 잔기의 2 측쇄 수산기를 -glycans 수득하는 (X는 프롤린을 제외한 임의의 아미노산 일 수있다). 거의 예외없이, 글리코 엄격한 도너, 억 셉터, 및 결합 특이성을 나타내는 때문에 셀의 glycome의 조성 (즉, 당화 제품의 보수)은 고유 제한된다. 5 중요 풍부한 혈장 당 단백질은 하류 결과로서 비정상적인 당화 고통 이상 글 라이코 실 트랜스퍼 라제의 발현과 활동으로 인해 다수의 병리 적 상태로, 특히 암과 염증성 질환이다. 6-24 주로 후생 유전 학적 요인 탓으로 glycome는 포유류의 게놈의 약 1 %가 형성, 수정을 인코딩하는 동안. 크게 25, 26 프로테옴 및 사체보다 더 다양한 동적, 복잡하고,글리 칸의 조립체는 비 구동 방식 템플릿 - 현저한 콘트라스트 27 당화 진행 폴리펩티드 및 핵산 생합성한다. 당화 효소 및 영양소 및 전구체 가용성과 같은 환경 적 요인의 상대적인 양 및 활성 사이의 상호 작용은 궁극적으로 포도당의 특성상, 속도 및 정도를 결정한다. 5,28 배아 (예를 들어, 판정 및 분화) 세포 활성화 및 진행을 세포주기에 영향을 미치는 유전자 발현 (즉, 전사 및 번역) 및 그 활성 세포의 글리 칸 프로파일의 바로 상류 인 결정 가능한 글리코의 동일성과 양을 변경. (의 일부) 증식하기 때문에, 접착제 및 암 세포의 침략적 속성은 일반 배아 세포, 글리 칸 생합성 경로 (예를 들면, 전구체 축적, 규제 완화 표현, aberran의 특정 변화들과 유사. t 수정, 구조 절단, 또는 소설 형성) 글리코 실화는 분명히 당화에 몇 변경이 발암과 전이를 활성화 할 수 있습니다, 매우 복잡하지만 종양 형성, 진행, 마이그레이션 및 침략 (29)의 다양한 단계를 나타내는 등의 보편적 인 암 바이오 마커를 제공; 분명히, 특정 &quot;비정상적인&quot;글리코 실화 제품이 실제로 면역 인식을 회피하고 험한 혈관 및 전이성 환경에서 마이그레이션의 요구를 생존 할 수 있도록함으로써 암 세포를 혜택을 누릴 수 있습니다. 28,30,31 당연히 실험 변경의 패턴을 방해하거나 방지하는 것이 밝혀졌다 유전자 발현 및 비정상적인 글리 칸 형성은 종양을 중지 할 수 있습니다. (29) 그럼에도 불구하고 biofluid 샘플 (예를 들어, 소변, 타액 및 혈장 또는 혈청)에서 검출 된 비정상적인 글리 칸은 직접 암의 지표 (또는 다른 질병), 오히려 하류하지 않을 수 있습니다 미묘하지만 중요한의 결과면역 시스템 또는 예측 기관의 악성 상태의 정량화 파급 효과의 변화. (32) 그들은 glycome 대한 범용 정보 많은 분자 상호 작용을 기반 글라 이코믹스 기술 (예를 들면, 렉틴 / 항체 어레이 및 대사 / 공유 라벨)를 제공하지만 전체 글리 칸 구조의 검출에 의존 개별 글리 칸에 대한 상세한 구조 정보를 제공하지 않는다. 현저하게 대조적으로, 질량 분석기 (MS)를 식별하고 각각의 글리 칸 구조를 정량화 폴리펩티드 코어의 결합 부위와 같은 구조적 정보를 노출 할 수있다. 규제 완화 식 또는 하나의 글 라이코 실 트랜스퍼 라제의 활동은 여러 당화 경로에 해로운 분자 사건의 폭포를 시작할 수 있습니다. 각 전이 효소가 하나 이상의 glycoconjugate 기판 상에 상이한 성장 글리 칸 중합체에서 작동 할 수 있기 때문에, 규제 완화 생합성 캐스케이드는 dispropor을 수득레이 터는 단 하나의 글리 칸 제품의 양하지만 인트라 또는 세포 외 체액의 비정상적 글리 칸의 여러 이기종 클래스 증가했다. (33) 그러나, 고유의 비정상적인 글리 칸은 때때로 암 또는 때문에 다른 글리 칸 - 정서적 병리에 대한 바이오 마커로 실용적 고려, 대형 수영장에 비해 의 글리 칸을 잘 조절이 비정상적 글리 칸은 수도가 자주도 질량 분석과 같은 매우 민감한 기술에 의해 탐지 남아 매우 작은 부분을 나타냅니다. 예를 들어, 인트라 - 및 세포 외 체액 (크기 여덟 주문 걸쳐) 광범위한 단백질 농도 스펙트럼은 더욱 풍부한 종에 의해 마스크된다 희소 당 단백질의 검출을 방지 할 수있다. (32) 또한, 당 전이 효소의 활성을 측정하는 것이 실용적 상당한 유지 많은 글리코 실 트랜스퍼 라 아제 임상 biofluids에 결석 또는 생체 전 비활성 상태이기 때문에 이론적 도전. consisten의 어려움에도 불구하고TLY 감지하고 독특한 전체 글리 칸의 울트라 분 수량을 정량화, 질량 분석의 실무자는 임상 마커로 그대로 글리 칸을 사용으로 엄청난 진보를 만들었습니다. 최근 GC-MS를 이용 함께 각 당쇄에 많은 고유성 부여 모든 구성 분 지점과 결합 특이 단당류 ( &quot;당쇄 노드&quot;)의 검출을 용이하게 그대로 글리 칸의 분석에 상보적인 방법을 개발 경우는 직접 과실 글 라이코 실 트랜스퍼 라제 (들)의 상대 활성을 정량화 분자 대리를 제공합니다. 첫 번째는 1958 년에 글리 칸 분석에 직접 응용 프로그램을보고 있기 때문에, 가스 크로마토 그래피 (GC)는 34 당 메틸화 모노와 이당류를 분석 자신의 anomericity 절대 구성을 결정하고, 이후의 질량 분석 분석을 분리하는 강력한 기술을 입증했다. (35) 1984 년과 2007 년, Ciucanu와 동료 사이도입 된 2005 년과 2008 년 사이에 물과 클로로포름. (35, 36)를 이용하여 메틸화 글리 칸의 액체 / 액체 추출하여 수산화 나트륨 및 아이오도 메탄을 사용 고상 글리 칸과 메틸화 기술, 정제, 강 및 동료는 시간을 절약 스핀 통합 메틸화 단계에 칼 럼 접근. 2008 년 37, 38는 게츠의 연구 그룹은 비교하고 잠재적으로 침습적 및 비 구별하는 매트릭스 보조 레이저 탈착 이온화 (MALDI) 질량 분석기를 사용하여 글리 칸 프로파일 링 방법과 메틸화 정량적 인 고체상을 고안 -invasive 유방암 세포 39는, 2009 년 게츠 팀 합성 효소, 화학적 분리 기술은 높은 알칼리성 고상 메틸화 방식으로 O -glycans 그대로 당 단백질로부터을 절단하는 40을 게츠 절차 동시 메틸화 화학적 분리를 용이 않는다. O의 -glycans, 그것은 단지 미리 절연 글리코에 도포단백질. 우리는 2013 년이 기술을 수정 트리 플루오로 아세트산을 통합하여 전체 미분 획 biofluids 및 균질 조직 샘플을 위해 적응 (TFA)을 가수 분해, 감소, 및 아세틸 화 단계. 또한 당지질 및 N에서 글리 칸을 해제 (33)이 추가 단계는 당 단백질에서 글리 칸을 -linked 및 변환 특유의 메틸화 - 및 - 아세틸 패턴 고유 GC-MS에 의해 분석을 용이하게 원래 그대로 글리 칸 폴리머 (41) (그림 2)로 구성 글리 칸 노드의 특성을 부분적으로 메틸화 된 알디 톨 아세테이트 (PMAAs, 그림 1),로. 33 궁극적으로,이 절차는 &quot;코어 푸코 실화」, 「6 시알 릴&quot;, &quot;한 GlcNAc를 이등분&quot;및 &quot;베타 1-6 분기&quot;고유 당쇄 기능 직접 상대 정량에 기초하여 복소 biofluid 모든 글리 칸의 합성 세로 생성 - 각 하나의 GC-MS의 chromatograp에서 파생 된HIC 피크. 이 문서는 또한 메틸화의 최적화, 아세틸, 격리 및 상대 정량 모드의 개선과 함께 청소 단계를 제공합니다.

<table><tbody><tr><td>Sodium hydroxide beads&amp;nbsp;</td><td>367176</td><td>Sigma-Aldrich&amp;nbsp;</td><td>20-40 mesh, reagent grade 97%</td></tr><tr><td>0.9 ml Spin column</td><td>69705</td><td>Pierce division of ThermoFisher Scientific&amp;nbsp;</td><td>Includes&amp;nbsp; plugs and polyethylene frits&amp;nbsp;</td></tr><tr><td>GC-MS autosampler vial (silanized)*</td><td>C4000-9</td><td>ThermoFisher Scientific</td><td>Target DP High Recovery Vial, 1.5&amp;nbsp;ml, 12&amp;nbsp;mm x 32&amp;nbsp;mm, includes Teflon-lined pierceable caps</td></tr><tr><td>1.5 ml polypropylene test tubes</td><td>05-402-25</td><td>ThermoFisher Scientific&amp;nbsp;</td><td>Snap-cap lid</td></tr><tr><td>2 ml polypropylene test tubes</td><td>05-408-138</td><td>ThermoFisher Scientific&amp;nbsp;</td><td>Snap-cap lid</td></tr><tr><td>Dimethyl Sulfoxide (DMSO)</td><td>D8418</td><td>Sigma-Aldrich&amp;nbsp;</td><td>BioReagent for molecular biology, reagent grade &gt;99.0%</td></tr><tr><td>Iodomethane&amp;nbsp;</td><td>I8507</td><td>Sigma-Aldrich&amp;nbsp;</td><td>Contains copper as stabilizer, ReagentPlus 99%</td></tr><tr><td>Acetonitrile</td><td>A955-4</td><td>ThermoFisher Scientific&amp;nbsp;</td><td>Optima LC/MS</td></tr><tr><td>Microcentrifuge</td><td>75002436: Sorvall Legend Micro 17 Centrifuge</td><td>ThermoFisher Scientific&amp;nbsp;</td><td>24 x 1.5/2.0 rotor with ClickSeal biocontainment lid. Rotor catalog number: 75003424</td></tr><tr><td>13 x 100 glass test tube (silanized)*</td><td>53283-800</td><td>VWR</td><td>13 mm x 100 mm borosilicate glass test tubes with screw-cap finish</td></tr><tr><td>Caps for glass test tubes</td><td>14-930-15D</td><td>ThermoFisher Scientific&amp;nbsp;</td><td>Kimble&amp;trade; Black Phenolic Screw Caps; 13&amp;nbsp;mm-415 GPI thread; PTFE-faced rubber liner.</td></tr><tr><td>Sodium chloride&amp;nbsp;</td><td>S7653</td><td>Sigma-Aldrich&amp;nbsp;</td><td>&gt;99.5% pure</td></tr><tr><td>Chloroform</td><td>4440-08</td><td>Macron Fine Chemicals&amp;nbsp;</td><td></td></tr><tr><td>Trifluoroacetic acid&amp;nbsp;</td><td>299537</td><td>Sigma-Aldrich&amp;nbsp;</td><td>99% purified by redistillation for protein sequencing&amp;nbsp;</td></tr><tr><td>Sodium borohydride</td><td>71321</td><td>Fluka Analytical&amp;nbsp;</td><td>99%</td></tr><tr><td>Ammonium hydroxide solution&amp;nbsp;</td><td>320145</td><td>Sigma-Aldrich&amp;nbsp;</td><td>NH&lt;sub&gt;3&lt;/sub&gt; content: 28.0-30.0%</td></tr><tr><td>Methanol</td><td>AH230-4</td><td>Honeywell Burdick &amp;amp; Jackson</td><td>HPLC grade</td></tr><tr><td>Acetic acid&amp;nbsp;</td><td>320099</td><td>Sigma-Aldrich&amp;nbsp;</td><td>99.70%</td></tr><tr><td>Plastic vacuum desiccator&amp;nbsp;</td><td>Any model of adequate size</td><td>FoodSaver</td><td></td></tr><tr><td>Acetic anhydride&amp;nbsp;</td><td>539996</td><td>Sigma-Aldrich&amp;nbsp;</td><td>99.50%</td></tr><tr><td>Dichloromethane&amp;nbsp;</td><td>D143SK-4</td><td>ThermoFisher Scientific&amp;nbsp;</td><td>Stabilized HPLC grade</td></tr><tr><td>Acetone&amp;nbsp;</td><td>9006-03</td><td>J.T.Baker</td><td>Baker Analyzed&amp;nbsp;</td></tr><tr><td>Heated evaporation manifold (main unit)</td><td>pi18823</td><td>ThermoFisher Scientific&amp;nbsp;</td><td>Thermo Scientific* Reacti-Therm* Heating and Stirring Module; Triple-block Model with Heating and Stirring Function</td></tr><tr><td>Heated evaporation manifold (overhead evaporator)</td><td>pi18826</td><td>ThermoFisher Scientific&amp;nbsp;</td><td>ThermoScientific* Reacti-Vap Evaporator, 27-port; For use with triple-block Reacti-Therm heating module</td></tr><tr><td>Aluminum sample-holder blocks for evaporation manifold</td><td>pi18816</td><td>ThermoFisher Scientific&amp;nbsp;</td><td>Block, Aluminum, Reacti-Block S-1, Holds 13&amp;nbsp;mm dia test tubes, 13 holes (14&amp;nbsp;mm dia. x 45&amp;nbsp;mm deep)</td></tr><tr><td>Gas chromatograph</td><td>Model A7890</td><td>Agilent&amp;nbsp;</td><td>Equipped with CTC PAL autosampler</td></tr><tr><td>Mass spectrometer&amp;nbsp;</td><td>GCT Premier (Time-of-Flight)</td><td>Waters&amp;nbsp;</td><td></td></tr><tr><td>Split-mode liner (deactivated / silanized)</td><td>5183-4647</td><td>Agilent</td><td>Containing a small plug of silanized glass wool</td></tr><tr><td>DB-5ms GC column</td><td>122-5532</td><td>Agilent</td><td>30 m x 0.25 mm ID x 0.25 micron film thickness</td></tr><tr><td>Chlorotrimethylsilane</td><td>95541</td><td>Sigma-Aldrich&amp;nbsp;</td><td></td></tr><tr><td>Glass vacuum desiccator (for glassware silanization)</td><td>EW-06536-30</td><td>Cole-Parmer</td><td>12&quot; wide; 230 mm plate size</td></tr><tr><td colspan="4">*Glassware silanization is carried out in-house, overnight using chlorotrimethylsilane vapor in a large glass vacuum desiccator.</td></tr></tbody></table>

glycan node analysis: a bottom-up approach to glycomics

Synthesized in a non-template-driven process by enzymes called glycosyltransferases, glycans are key players in various significant intra- and extracellular events. Many pathological conditions, notably cancer, affect gene expression, which can in turn deregulate the relative abundance and activity levels of glycoside hydrolase and glycosyltransferase enzymes. Unique aberrant whole glycans resulting from deregulated glycosyltransferase(s) are often present in trace quantities within complex biofluids, making their detection difficult and sometimes stochastic. However, with proper sample preparation, one of the oldest forms of mass spectrometry (gas chromatography-mass spectrometry, GC-MS) can routinely detect the collection of branch-point and linkage-specific monosaccharides ("glycan nodes") present in complex biofluids. Complementary to traditional top-down glycomics techniques, the approach discussed herein involves the collection and condensation of each constituent glycan node in a sample into a single independent analytical signal, which provides detailed structural and quantitative information about changes to the glycome as a whole and reveals potentially deregulated glycosyltransferases. Improvements to the permethylation and subsequent liquid/liquid extraction stages provided herein enhance reproducibility and overall yield by facilitating minimal exposure of permethylated glycans to alkaline aqueous conditions. Modifications to the acetylation stage further increase the extent of reaction and overall yield. Despite their reproducibility, the overall yields of N-acetylhexosamine (HexNAc) partially permethylated alditol acetates (PMAAs) are shown to be inherently lower than their expected theoretical value relative to hexose PMAAs. Calculating the ratio of the area under the extracted ion chromatogram (XIC) for each individual hexose PMAA (or HexNAc PMAA) to the sum of such XIC areas for all hexoses (or HexNAcs) provides a new normalization method that facilitates relative quantification of individual glycan nodes in a sample. Although presently constrained in terms of its absolute limits of detection, this method expedites the analysis of clinical biofluids and shows considerable promise as a complementary approach to traditional top-down glycomics.

상대 두 가지 중요한 메틸화 단계가 제대로 실행 된있는 경우에 성공적으로 메틸화, 가수 분해, 감소, 인간 혈장 샘플의 아세틸 화를 보여주는 총 이온 전류 크로마토 그램 (TIC)는 그림 3에 표시됩니다. 탄당에 HexNAcs의 상대의 절대 수율 : N의 -acetylhexosamin...

Watch this Scientific Journal Video about Glycan Node Analysis: A Bottom-up Approach to Glycomics at JoVE.com

Glycan Node Analysis: A Bottom-up Approach to Glycomics

This article presents an enhanced form of a novel bottom-up glycomics technique designed to analyze the pooled compositional profile of glycans in unfractionated biofluids through the chemical breakdown of glycans into their constituent linkage-specific monosaccharides for detection by GC-MS. Potential applications include early detection of cancer and other glycan-affective disorders.

글리 칸 노드 분석 : 글리코 믹스에 대한 상향식 접근

Synthesized in a non-template-driven process by enzymes called glycosyltransferases, glycans are key players in various significant intra- and ...

glycan-node-analysis-a-bottom-up-approach-to-glycomics

Research

JoVE Journal

Chemistry

10.3K 조회수.  Arizona State University. This article presents an enhanced form of a novel bottom-up glycomics technique designed to analyze the pooled compositional profile of glycans in unfractionated biofluids through the chemical breakdown of glycans into their constituent linkage-specific monosaccharides for detection by GC-MS. Potential applications include early detection of cancer and other glycan-affective disorders.

비디오: Glycan Node Analysis: A Bottom-up Approach to Glycomics

Improved In-gel Reductive &#946;-Elimination for Comprehensive O-linked and Sulfo-glycomics by Mass Spectrometry

Separation of proteins by SDS-PAGE followed by in-gel proteolytic digestion of resolved protein bands has produced high-resolution proteomic analysis of biological samples. Similar approaches, that would allow in-depth analysis of the glycans carried by glycoproteins resolved by SDS-PAGE, require special considerations in order to maximize recovery and sensitivity when using mass spectrometry (MS) as the detection method. A major hurdle to be overcome in achieving high-quality data is the removal of gel-derived contaminants that interfere with MS analysis. The sample workflow presented here is robust, efficient, and eliminates the need for in-line HPLC clean-up prior to MS. Gel pieces containing target proteins are washed in acetonitrile, water, and ethyl acetate to remove contaminants, including polymeric acrylamide fragments. O-linked glycans are released from target proteins by in-gel reductive &#946;-elimination and recovered through robust, simple clean-up procedures. An advantage of this workflow is that it improves sensitivity for detecting and characterizing sulfated glycans. These procedures produce an efficient separation of sulfated permethylated glycans from non-sulfated (sialylated and neutral) permethylated glycans by a rapid phase-partition prior to MS analysis, and thereby enhance glycomic and sulfoglycomic analyses of glycoproteins resolved by SDS-PAGE.

In order to comprehensively explore the diversity of O-linked glycans, a new procedure for in-gel reductive &#946;-elimination, combined with permethylation and a rapid phase-partition method, is applied to the analysis of O-linked glycans directly released from glycoproteins resolved by SDS-PAGE and amenable to subsequent glycomic analysis by mass spectrometry.

질량 분석에 의해 - 겔 개선 환원 적 β-제거 포괄적 인 O가 연결된과에 대한 설포 글라 이코믹스

Separation of proteins by SDS-PAGE followed by in-gel proteolytic digestion of resolved protein bands has produced high-resolution proteomic analysis of ...

연구

화학

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 ...

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

In recent years, the carbohydrate moieties of plants have received considerable attention, as they are a potential source of cross-reactive, allergy-provoking immune responses. In addition, carbohydrate structures also play a critical role in plant metabolism. Here, we present a simple and rapid method for preparing and analyzing N-glycans from different cultivars of radish (Raphanus sativus) using an N-glycanase specific for the release of plant-derived carbohydrate structures. To achieve this, crude trichloroacetic acid precipitates of radish homogenates were treated with PNGase H+, and labeled using 2-aminobenzamide as a fluorescent tag. The labeled N-glycan samples were subsequently analyzed by ultra performance liquid chromatography (UPLC) separation and matrix-assisted laser desorption ionization-time of flight (MALDI-TOF) mass spectrometry for a detailed structural evaluation and to quantify relative abundancies of the radish-derived N-glycan structures. This protocol can also be used for the analysis of N-glycans from various other plant species, and may be useful for further investigation of the function and effects of N-glycans on human health.

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

We describe a simple and rapid method for the preparation and analysis of N-glycans from different cultivars of radish (Raphanus sativus).

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

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

생화학

Mass Spectrometric Analysis of Glycosphingolipid Antigens

Glycosphingolipids (GSL's) belong to the glycoconjugate class of biomacromolecules, which bear structural information for significant biological processes such as embryonic development, signal transduction, and immune receptor recognition1-2. They contain complex sugar moieties in the form of isomers, and lipid moieties with variations including fatty acyl chain length, unsaturation, and hydroxylation. Both carbohydrate and ceramide portions may be basis of biological significance. For example, tri-hexosylceramides include globotriaosylceramide (Gal&alpha;4Gal&beta;4Glc&beta;1Cer) and isoglobotriaosylceramide (Gal&alpha;3Gal&beta;4Glc&beta;1Cer), which have identical molecular masses but distinct sugar linkages of carbohydrate moiety, responsible for completely different biological functions3-4. In another example, it has been demonstrated that modification of the ceramide part of alpha-galactosylceramide, a potent agonist ligand for invariant NKT cells, changes their cytokine secretion profiles and function in animal models of cancer and auto-immune diseases5. The difficulty in performing a structural analysis of isomers in immune organs and cells serve as a barrier for determining many biological functions6.
Here, we present a visualized version of a method for relatively simple, rapid, and sensitive analysis of glycosphingolipid profiles in immune cells7-9. This method is based on extraction and chemical modification (permethylation, see below Figure 5A, all OH groups of hexose were replaced by MeO after permethylation reaction) of glycosphingolipids10-15, followed by subsequent analysis using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS) and ion trap mass spectrometry. This method requires 50 million immune cells for a complete analysis. The experiments can be completed within a week. The relative abundance of the various glycosphingolipids can be delineated by comparison to synthetic standards. This method has a sensitivity of measuring 1% iGb3 among Gb3 isomers, when 2 fmol of total iGb3/Gb3 mixture is present9.
Ion trap mass spectrometry can be used to analyze isomers. For example, to analyze the presence of globotriaosylceramide and isoglobtriaosylceramide in the same sample, one can use the fragmentation of glycosphingolipid molecules to structurally discriminate between the two (see below Figure 5). Furthermore, chemical modification of the sugar moieties (through a permethylation reaction) improves the ionization and fragmentation efficiencies for higher sensitivity and specificity, and increases the stability of sialic acid residues. The extraction and chemical modification of glycosphingolipids can be performed in a classic certified chemical hood, and the mass spectrometry can be performed by core facilities with ion trap MS instruments.

A specific and sensitive method to gain insight into the expression profile of glycosphingolipid antigens in immune organs and cells is described. The method takes advantage of the ion trap mass spectrometry allowing step-wise fragmentation of glycosphingolipid molecules for structural analysis in comparison to chemically synthesized standards.

Glycosphingolipid 항원의 대량 Spectrometric 분석

Glycosphingolipids (GSL's) belong to the glycoconjugate class of biomacromolecules, which bear structural information for significant biological processes ...

면역학 및 감염병학

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 ...

The Application of Open Searching-based Approaches for the Identification of Acinetobacter baumannii O-linked Glycopeptides

Protein glycosylation is increasingly recognized as a common modification within bacterial organisms, contributing to prokaryotic physiology and optimal infectivity of pathogenic species. Due to this, there is increasing interest in characterizing bacterial glycosylation and a need for high-throughput analytical tools to identify these events. Although bottom-up proteomics readily enables the generation of rich glycopeptide data, the breadth and diversity of glycans observed in prokaryotic species make the identification of bacterial glycosylation events extremely challenging.
Traditionally, the manual determination of glycan compositions within bacterial proteomic datasets made this a largely bespoke analysis restricted to field-specific experts. Recently, open searching-based approaches have emerged as a powerful alternative for the identification of unknown modifications. By analyzing the frequency of unique modifications observed on peptide sequences, open searching techniques allow the identification of common glycans attached to peptides within complex samples. This article presents a streamlined workflow for the interpretation and analysis of glycoproteomic data, demonstrating how open searching techniques can be used to identify bacterial glycopeptides without prior knowledge of the glycan compositions.
Using this approach, glycopeptides within samples can rapidly be identified to understand glycosylation differences. Using Acinetobacter baumannii as a model, these approaches enable the comparison of glycan compositions between strains and the identification of novel glycoproteins. Taken together, this work demonstrates the versatility of open database-searching techniques for the identification of bacterial glycosylation, making the characterization of these highly diverse glycoproteomes easier than ever before.

The Application of Open Searching-based Approaches for the Identification of Acinetobacter baumannii O-linked Glycopeptides

Open searching enables the identification of glycopeptides decorated with previously unknown glycan compositions. Within this article, a streamlined approach for undertaking open searching and subsequent glycan-focused glycopeptide searches are presented for bacterial samples using Acinetobacter baumannii as a model.

Acinetobacter baumannii O 결합 글리코 펩타이드의 확인을위한 개방형 검색 기반 접근법의 적용

Protein glycosylation is increasingly recognized as a common modification within bacterial organisms, contributing to prokaryotic physiology and optimal ...

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 ...

글리 칸 노드 분석 : 글리코 믹스에 대한 상향식 접근

요약

더 많은 비디오 탐색

이 비디오의 챕터

질량 분석에 의해 - 겔 개선 환원 적 β-제거 포괄적 인 O가 연결된과에 대한 설포 글라 이코믹스

양적 글리코 믹스와 단백질 체학 복합 정제 전략

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

Glycosphingolipid 항원의 대량 Spectrometric 분석

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

Acinetobacter baumannii O 결합 글리코 펩타이드의 확인을위한 개방형 검색 기반 접근법의 적용

Nonspecific Proteolysis-Based Glycomics Strategy for Bacterial Glycoproteins

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.