이 작품은 인간 프론티어 과학 프로그램기구 (HFSP 수상 2007 년 RGP0058/2007-C)에 의해 지원되었다. 우리는 프로젝트를 시작을위한 유익한 토론을위한 교수 리차드 로버츠 감사합니다.

<ol>
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M., Marappan, S., Yip, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Capture+compound+mass+spectrometry:+a+technology+for+the+investigation+of+small+molecule+protein+interactions.">Capture compound mass spectrometry: a technology for the investigation of small molecule protein interactions.</a> Assay Drug Dev. Technol. 5, 381-390 (2007).</li><li>Dalhoff, C., Hueben, M., Lenz, T., Poot, P., Nordhoff, E., Koster, H., Weinhold, E. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Synthesis+of+S-Adenosyl-L-homocysteine+Capture+Compounds+for+Selective+Photoinduced+Isolation+of+Methyltransferases.">Synthesis of S-Adenosyl-L-homocysteine Capture Compounds for Selective Photoinduced Isolation of Methyltransferases.</a> ChemBioChem. 11, 256-265 (2010).</li><li>Lu, S. C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=S-Adenosylmethionine.">S-Adenosylmethionine.</a> Int. J. Biochem. Cell. Biol. 32, 391-3952 (2000).</li><li>Cantoni, G. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Biological+methylation:+selected+aspects.">Biological methylation: selected aspects.</a> Annu. Rev. Biochem. 44, 435-451 (1975).</li><li>Jeltsch, A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Beyond+Watson+and+Crick:+DNA+methylation+and+molecular+enzymology+of+DNA+methyltransferases.">Beyond Watson and Crick: DNA methylation and molecular enzymology of DNA methyltransferases.</a> ChemBioChem. 3, 274-293 (2002).</li><li>Chow, C. S., Lamichhane, T. N., Mahto, S. K. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Expanding+the+nucleotide+repertoire+of+the+ribosome+with+post-transcriptional+modifications.">Expanding the nucleotide repertoire of the ribosome with post-transcriptional modifications.</a> ACS Chem. Biol. 2, 610-619 (2007).</li><li>Grillo, M. A., Colombatto, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=S-Adenosylmethionine+and+protein+methylation.">S-Adenosylmethionine and protein methylation.</a> Amino Acids. 28, 357-362 (2005).</li><li>Fujioka, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Mammalian+small+molecule+methyltransferases:+their+structural+and+functional+features.">Mammalian small molecule methyltransferases: their structural and functional features.</a> Int. J. Biochem. 24, 1917-1924 (1992).</li><li>Borchardt, R. T., Salvatore, F., Borek, E., Zappia, V., Williams-Ashman, H. G., Schlenk, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> The Biochemistry of S-Adenosylmethionine. , 151-171 (1977).</li><li>Hoffman, J. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Chromatographic+analysis+of+the+chiral+and+covalent+instability+of+S-adenosyl-l-methionine.">Chromatographic analysis of the chiral and covalent instability of S-adenosyl-l-methionine.</a> Biochemistry. 25, 4444-4449 (1986).</li><li>Keller, A., Nesvizhskii, A. I., Kolker, E., Aebersold, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Empirical+statistical+model+to+estimate+the+accuracy+of+peptide+identifications+made+by+MS/MS+and+database+search.">Empirical statistical model to estimate the accuracy of peptide identifications made by MS/MS and database search.</a> Anal. Chem. 74, 5383-5392 (2002).</li><li>Nesvizhskii, A. I., Keller, A., Kolker, E., Aebersold, R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+statistical+model+for+identifying+proteins+by+tandem+mass+spectrometry.">A statistical model for identifying proteins by tandem mass spectrometry.</a> Anal. Chem. 75, 4646-4658 (2003).</li></ol>

ThomasLenz, OliviaGrabner, MirkoGlinski, MathiasDreger 및 HubertKosterare이 문서에서 사용되는 시약의 일부를 생산 caprotec bioanalytics GmbH의, Inc의 고용.

설명 프로토콜을 다음과 같은 경우 다음과 같은주의 사항 및 의견은 유용할 수 있습니다 :이 이후 엄격한 세척 조건을 허용하는 등 CCS의) 가장 큰 장점은 CC와 MTase 사이에 공유 결합의 형성에 자리잡고 있습니다. 공유 결합 crosslink는 자외선 (310 nm의 최대.)에 의해 실행 photoreaction에 의해 이루어진다. 일반 오버헤드 빛이 자외선의 작은 파편만이 포함되어 있습니다 단, caproBox의 제어와 냉각 조사까지 오버헤드에 더 이상 노출 또는 일 빛으로부터 SAH - CC를 보호합니다. B) MTases가 격리해야하는에서 생물 학적 샘플이 변성하는 경향이 단백질을 포함할 수 있습니다, 따라서 그것은 샘플이 시원한 유지하고 항상 얼굴이 볼만 피하기 위해 필수입니다. C) caproBox는 0-4로 샘플을 냉각 ° C, UV 빛을 발하는 램프가, 그러나 또한 열을 방출. 따라서 그것은 간략하게 조사하기 전에 튜브를 원심 필요하므로 대문자 또는 유리 벽면을 준수하는 단백질 침전의 씨앗을 형성 수 없습니다. 자석 구슬을 다시 중단이 (세척 솔루션의 예) 손으로 불가 능할 경우 D), 곧 초음파 욕조에 샘플을 배치하여 초음파를 적용합니다. E) 갓 0.2 % TFA/60 %의 ACN 솔루션을 준비합니다. 우리는 달리 캡처된 단백질이 구슬에서 죽습되지 않을 수 있습니다는 사실을 발견했습니다. F) 캡처 단백질의 최종 분석 LC-MS/MS에 의해 수행됩니다. 질량 분석법은 매우 민감한 방법입니다. 이것은 마지막 단계 (단계 더 나아가 2.11)에서 독점적으로 LC - MS 등급 시약을 사용해야합니다. 외부 단백질 소스, 먼지 또는 실험자에서 발생하는 등 각질에 의해 실험의 오염을 피하십시오. 특히 최종 소화 단계 동안, 그것은 장갑과 실험 복과 가능한 고정시켜 또는 이상 깨끗한 벤치에서 최종 단계를 수행하기 위해, 깨끗한 작업 공간에 대한 관심을 지불하는 것이 좋습니다. LC MS 학년 물에 tryptic 소화에 사용되는 50 MM의 탄산수 소 암모늄 버퍼, -20 ° C에서 0.22 μm의 필터, 나누어지는, 저장을 통해 필터를 준비하고, contaminations가 발생하지 않도록 한 번만 각 나누어지는을 사용합니다. 트립신 용액 (시퀀싱 학년, 로체, 동결 건조된 트립신 1 MM HCL을 추가하여 0.5 μg / μl 솔루션을 준비)를 4 ° C 몇 주 동안 저장할 수 있습니다. G) 신뢰할 수있는 질량 스펙트럼을 얻으려면 그것은 ESI-MS/MS 분석에서 안정 스프레이를하는 것이 필수적입니다. H) 현재 연구에 사용되는 이외의 LC-MS/MS 시스템의 경우, 측정 매개 변수와 펩티드 식별 알고리즘은 개별적으로 조정해야합니다. 다음 수정은 설명 프로토콜과 관련하여 가능합니다 :)이 또는 60% ACN/0.2 %의 TFA (단계 2.11)에 의해 비즈에서 단백질을 발표하기 위해 단백질 직접 tryptically 구슬 중지 (이내 소화 수있는 동일한 볼륨으로 단계 5.1)이나, SDS - PAGE에 대해, 단백질은 95 단계로 2.9 이후 수집한 비즈를 보류하고 가열에 의해 발표 수 있습니다 ° C SDS 샘플 버퍼에 10 분 (모두 전체 정지 또는 유일한 뜨는 로드할 수에 대한 겔 주머니에). 우리는 비즈 천천히에도 여러 수성 세척 단계 후 온 - 비드 tryptic 소화하는 동안 수성 tryptic 다이제스트 솔루션에 폴리머 소량의 릴리스는 사실을 발견했습니다. 폴리머 오염 MS 펩티드 식별을 방해하고 80 % ACN을 (적어도 세 번)를 사용하여 비즈 씻어 수 있습니다. 80 % ACN 세척 단계 후, 비즈 전에 온 - 비드 tryptic 소화 물에 한번 세탁해야합니다. B) streptavidin - horseraddish 퍼옥시데이즈 및 ECL 기판을 사용하여 서양 blots 또한 단백질에 비오틴이 포함된 CC의 성공 crosslinking을 시각화하는 데 사용할 수 있습니다. 감도가 젤의 은색 얼룩보다 약 10 배 이상이기 때문에 따라서, 단계 3.2 이후 얻은 젤이, blotted 또는 수 있습니다 중, 10 μl 샘플 단계 2.7 이후에도 서양 얼룩으로 분석 수 있습니다. 후자의 경우에는 endogeneously biotinylated 단백질도 인위적으로 SAH - CC로 biotinylated 단백질 이외에 감지됩니다 잊었습니다. C) 우리는, 특별한 시스템 (lysate, 대상 단백질, 선택성 그리고 CC의 반응 기능을 주소)에 따라 사실을 발견 여부 crosslinking 반응은 무료 CC 및 솔루션 4 단백질 사이에 일어난 일입니다 &quot;오프 구슬&quot;구성, 또는 현재 설명 &quot;에 - 구슬 &#39;구성 (그림 1B)은 더 나은 수행합니다. 일반적으로 메서드는 또한와 호환되어야하는 국가 수준의 예술 안정 동위 원소 단백질이나 펩타이드의 라벨 기술, 또는 2D 겔 전기 영동에 의해 캡처 샘플 평가. &quot;오프 구슬&quot;구성에서 전체 셀 (게시되지 않은 결과) 내에 단백질을 포착하는 것도 가능합니다. 또한, 약물이나 단백질의 cofactor 구속력이 사이트는 MS 펩타이드 배열에 의해 단백질 시퀀스 내에서 CC의 가까이별로 crosslinking 위치를 결정하여 설명하실 수 있습니다. 단백질을 작은 분자의 바인딩 모드는 다른 사용자를 사용하여 탐험 수 있습니다택 기능과 다양한 링커 길이에 hemical 첨부 파일 위치. 으로 현재 연구에 나타난 단백질 또한 단백질 바인딩 파트너는 선택도 기능 (PrmA의 기판으로 RplK) 또는 알려지지 않은 작은 분자의 단백질 상호 작용 (GlnA에 SAH) 확인할 수 있습니다하여 해결. 요약, CCS, 사진 crosslinking의 반응의 추가 기능, 고감도와 복잡한 단백질 혼합물에서 낮은 풍부한 단백질이나 기능성 단백질 가족의 격리 및 식별 가능하며 작은 분자를 연구를위한 추가 도구를 사용하여 과학자를 제공 - 단백질 상호 작용 .

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<html xmlns="http://www.w3.org/1999/xhtml" xml:lang="en" lang="en">	
		
		<div>
		<table border="1">
		<thead>
		<tr>
		<th>Material Name</th>
		<th>Type</th>
		<th>Company</th>
		<th>Catalogue Number</th>
		<th>Comment</th>
		</tr>
		</thead>
		<tbody>
		
<tr>
<td>SAH caproKit&trade;</td>
<td>&nbsp;</td>
<td>caprotec bioanalytics GmbH</td>
<td>1-1010-050 (50 reactions) 1-1010-010 (10 reactions)</td>
<td>Includes the SAH-CC, SAH competitor, streptavidin coated magnetic beads, capture buffer, and wash buffer</td>
</tr>
<tr>
<td>caproBox&trade;</td>
<td>&nbsp;</td>
<td>caprotec bioanalytics GmbH</td>
<td>1-5010-003 (110 V) 1-5010-004 (230 V)</td>
<td>For reproducible photo-activation while cooling the samples</td>
</tr>
<tr>
<td>caproMag&trade;</td>
<td>&nbsp;</td>
<td>caprotec bioanalytics GmbH</td>
<td>1-5100-001</td>
<td>For easy handling of magnetic particles without pipetting</td>
</tr>		</tbody>
		</table>
		</div>

profiling of methyltransferases and other s-adenosyl-l-homocysteine-binding proteins by capture compound mass spectrometry (ccms)

이러한 친화도 크로마 토그래피 1 또는 활동 기반 단백질 프로파일이 같은 기능을 작은 분자 - 단백질 상호 작용에 기초하여 프로테옴의 복잡도를 줄이기 위해 접근의 다양한 방법이 있습니다. Trifunctional 캡처 화합물 (CCS, 그림 1A) 3은 일반적인 접근 방식을 바탕으로하는 최초의 평형 중심의 작은 분자 프로브 (여기 선택성 기능, S - adenosyl - L - 호모 시스테인, SAH, 그림 1A) 사이의 상호 작용 및 대상 단백질은 irreversibly CC의 독립적인 사진 activable 반응 기능 (여기 phenylazide)와 대상 단백질의 표면 사이에 사진 crosslinking에 고정됩니다. 고체 상 (여기서 streptavidin 자성 구슬)의 도움으로 복잡한 생물학 혼합물에서 단백질 conjugates - 정렬 기능 (여기 비오틴)는 CC를 분리하는 역할을합니다. 실험의 두 가지 구성이 가능합니다 : &quot;오프 구슬&quot;4 또는 현재 설명 &quot;에 - 구슬 &#39;구성 (그림 1B). 택 기능은 관심의 거의 모든 작은 분자 (기판, 억제제, 약물 분자) 수 있습니다. (SAM, 그림 1A) S - Adenosyl - L - 메티오닌은 아마도 ATP, 자연 5, 6에 가장 널리 사용 cofactor의 두번째입니다. 이것은 화학 반응은 DNA 7, RNA 8, 단백질 9, 또는 작은 분자 10 methylate SAM - 종속 methyltransferases (MTases)에 의해 촉매되는 모든 살아있는 유기체의 주요 메틸 그룹 기증자로 사용됩니다. 다양한 생리 시나리오에서 methylation 반응의 중요한 역할 (유전자 조절, epigenetics, 신진 대사)을 감안할 때, MTases의 프로파일 링은 kinases의 프로 파일링 기능으로 proteomics의 유사한 중요성 될 것으로 기대하실 수 있습니다. 자신의 프로파일에 대한 분석 도구는 그러나, 사용할 수 없습니다. 우리는 최근이 기술 격차 (그림 1A)을 채우기 위해 선택성 그룹으로 SAH과 CC를 소개했다. SAH, 메틸이 완료된 후 SAM의 제품은 알려진 일반 MTase 제품 억제제 11. 이런 이유 때문에 자연 cofactor SAM은 그 화학적 불안정성 12 때문에 SAH가 MTases을 타겟으로 CC를위한 이상적인 선택도 기능입니다 cofactor의 다른 부분을 전송하거나 극단적인 반응뿐만 아니라를 시작 더 효소에 의해 사용됩니다. 여기, 우리는 기본 모델로 하였다 대장균 (E. 대장균), 가장 특징 prokaryotes 중 하나의 변형 DH5α에서 프로 파일링 MTases 및 기타 SAH 결합 단백질에 의해 SAH - CC 및 CCMS의 유틸리티를보고 수많은, 생화학 생물 학적 및 biotechnological 연구 유기체. 사진 활성 crosslinking는 실험의 수율과 감도를 강화하고, 특이성이 쉽게 무료로 SAH을 초과 사용하여 경쟁 실험에 대해 테스트할 수 있습니다.

Watch this Scientific Journal Video about Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry CCMS at JoVE.com

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry CCMS

캡처 화합물 사진 crosslinking 및 정화하여 다음 기능을 가역 작은 분자 - 단백질 상호 작용에 의한 프로테옴의 복잡도를 줄이기 위해 trifunctional 작은 분자 수 있습니다. 여기있는 캡처 컴파 운드를 사용하여<em&gt; S</em&gt; - adenosyl -<sub&gt; L</sub에서 methyltransferases를 분리 선택도의 함수로&gt; - 호모 시스테인 바인딩<em&gt; 대장균</em&gt; 전체 세포는 lysate와 MS에 의해 그들을 식별합니다.

Methyltransferases의 프로 파일링 및 기타 S - adenosyl - L</sub캡처 컴파 운드 질량 분광법 (CCMS)에서> - 호모 시스테인 결합 단백질

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity ...

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Biology

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (CCMS)

15.5K 조회수.  caprotec bioanalytics GmbH. 캡처 화합물 사진 crosslinking 및 정화하여 다음 기능을 가역 작은 분자 - 단백질 상호 작용에 의한 프로테옴의 복잡도를 줄이기 위해 trifunctional 작은 분자 수 있습니다. 여기있는 캡처 컴파 운드를 사용하여 S - adenosyl - L - 호모 시스테인 바인딩 대장균 전체 세포는 lysate와 MS에 의해 ?...

비디오: Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry CCMS

MALDI Sample Preparation: the Ultra Thin Layer Method

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) 1,2. The ultra-thin layer method involves the production of a substrate layer of matrix crystals (alpha-cyano-4-hydroxycinnamic acid) on the sample plate, which serves as a seeding ground for subsequent crystallization of a matrix/analyte mixture. Advantages of the ultra-thin layer method over other sample deposition approaches (e.g. dried droplet) are that it provides (i) greater tolerance to impurities such as salts and detergents, (ii) better resolution, and (iii) higher spatial uniformity. This method is especially useful for the accurate mass determination of proteins. The protocol was initially developed and optimized for the analysis of membrane proteins and used to successfully analyze ion channels, metabolite transporters, and receptors, containing between 2 and 12 transmembrane domains 2. Since the original publication, it has also shown to be equally useful for the analysis of soluble proteins. Indeed, we have used it for a large number of proteins having a wide range of properties, including those with molecular masses as high as 380 kDa 3. It is currently our method of choice for the molecular mass analysis of all proteins. The described procedure consistently produces high-quality spectra, and it is sensitive, robust, and easy to implement.

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS).

든 샘플 준비 : 울트라 씬 레이어 방법

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption ...

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Antibody Profiling by Luciferase Immunoprecipitation Systems (LIPS)

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease mechanisms and may elucidate new markers to substratify disease with different clinical features and better understand pathogenesis. We have developed a highly quantitative method called Luciferase Immunoprecipitation Systems (LIPS) for profiling patient sera antibody responses to autoantigens and pathogen antigens associated with infection. Unlike ELISAs, the highly sensitive LIPS is easily implemented to survey humoral serological response profiles to different antigens in a universal format and produces dynamic antibody titer ranges up to 6 log10 for some antigens. In these studies, quantitative profiling by LIPS of patient humoral responses against panels of antigens or even the entire proteome of some pathogens (i.e. HIV), is typically more informative than testing a single antigen by ELISA. In addition, LIPS also eliminates time and effort needed to produce highly purified antigens as well as the labor-intensive assay optimization steps needed for standard ELISAs. Here we provide a detailed protocol describing the technical aspects of performing LIPS assays for readily profiling antibody responses to single or multiple antigens.

Antibody Profiling by Luciferase Immunoprecipitation Systems LIPS

The technical aspects of performing LIPS (Luciferase Immunoprecipitation Systems) are described. The overall approach involves expressing chimeric genes encoding antigens fused to Renilla luciferase (Ruc) in mammalian cells. Crude Ruc-antigen extracts are then prepared and, without purification, employed in immunoprecipitation assays to quantify antibodies.

루시페라제 Immunoprecipitation 시스템 (입술)에 의해 항체 프로파일

Technologies for comprehensively understanding and quantifying antibody profiles to autoantigens and infectious agents may yield new insights into disease ...

Analyzing Large Protein Complexes by Structural Mass Spectrometry

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an array of dynamic, multi-protein complexes1. To gain a mechanistic understanding of the various cellular processes, it is crucial to determine the structure of such protein complexes, and reveal how their structural organization dictates their function. Many aspects of multi-protein complexes are, however, difficult to characterize, due to their heterogeneous nature, asymmetric structure, and dynamics. Therefore, new approaches are required for the study of the tertiary levels of protein organization.
One of the emerging structural biology tools for analyzing macromolecular complexes is mass spectrometry (MS)2-5. This method yields information on the complex protein composition, subunit stoichiometry, and structural topology. The power of MS derives from its high sensitivity and, as a consequence, low sample requirement, which enables examination of protein complexes expressed at endogenous levels. Another advantage is the speed of analysis, which allows monitoring of reactions in real time. Moreover, the technique can simultaneously measure the characteristics of separate populations co-existing in a mixture. 
Here, we describe a detailed protocol for the application of structural MS to the analysis of large protein assemblies. The procedure begins with the preparation of gold-coated capillaries for nanoflow electrospray ionization (nESI). It then continues with sample preparation, emphasizing the buffer conditions which should be compatible with nESI on the one hand, and enable to maintain complexes intact on the other. We then explain, step-by-step, how to optimize the experimental conditions for high mass measurements and acquire MS and tandem MS spectra. Finally, we chart the data processing and analyses that follow. Rather than attempting to characterize every aspect of protein assemblies, this protocol introduces basic MS procedures, enabling the performance of MS and MS/MS experiments on non-covalent complexes. Overall, our goal is to provide researchers unacquainted with the field of structural MS, with knowledge of the principal experimental tools.

Mass spectrometry has proven to be a valuable tool for analyzing large protein complexes. This method enables insights into the composition, stoichiometry and overall architecture of multi-subunit assemblies. Here, we describe, step-by-step, how to perform a structural mass spectrometry analysis, and characterize macromolecular structures.

구조 질량 분광법에 의해 큰 단백질 단지 분석

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an ...

OLIgo Mass Profiling (OLIMP) of Extracellular Polysaccharides

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is that they contain complex sugar moieties in form of glycoproteins, proteoglycans, and/or polysaccharides. Examples include the extracellular matrix of humans and animal cells consisting mainly of fibrillar proteins and proteoglycans or the polysaccharide based cell walls of plants and fungi, and the proteoglycan/glycolipid based cell walls of bacteria. All these glycostructures play vital roles in cell-to-cell and cell-to-environment communication and signalling.
An extraordinary complex example of an extracellular matrix is present in the walls of higher plant cells. Their wall is made almost entirely of sugars, up to 75% dry weight, and consists of the most abundant biopolymers present on this planet. Therefore, research is conducted how to utilize these materials best as a carbon-neutral renewable resource to replace petrochemicals derived from fossil fuel. The main challenge for fuel conversion remains the recalcitrance of walls to enzymatic or chemical degradation due to the unique glycostructures present in this unique biocomposite.
Here, we present a method for the rapid and sensitive analysis of plant cell wall glycostructures. This method OLIgo Mass Profiling (OLIMP) is based the enzymatic release of oligosaccharides from wall materials facilitating specific glycosylhydrolases and subsequent analysis of the solubilized oligosaccharide mixtures using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS)1 (Figure 1). OLIMP requires walls of only 5000 cells for a complete analysis, can be performed on the tissue itself2, and is amenable to high-throughput analyses3. While the absolute amount of the solubilized oligosaccharides cannot be determined by OLIMP the relative abundance of the various oligosaccharide ions can be delineated from the mass spectra giving insights about the substitution-pattern of the native polysaccharide present in the wall.
OLIMP can be used to analyze a wide variety of wall polymers, limited only by the availability of specific enzymes4. For example, for the analysis of polymers present in the plant cell wall enzymes are available to analyse the hemicelluloses xyloglucan using a xyloglucanase5, 11, 12, 13, xylan using an endo-&beta;-(1-4)-xylanase 6,7, or for pectic polysaccharides using a combination of a polygalacturonase and a methylesterase 8. Furthermore, using the same principles of OLIMP glycosylhydrolase and even glycosyltransferase activities can be monitored and determined 9.

OLIgo Mass Profiling OLIMP of Extracellular Polysaccharides

A rapid way is described to gain insights into the structure of polysaccharides in an extracellular matrix. The method takes advantage of the specificity of glycosylhydrolases and the sensitivity of mass spectrometry allowing minute amounts of materials to be analyzed. This technique is adaptable to be used directly on tissue itself.

세포외 다당류의 OLIgo 질량 프로파일 (OLIMP)

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is ...

Atmospheric-pressure Molecular Imaging of Biological Tissues and Biofilms by LAESI Mass Spectrometry

Ambient ionization methods in mass spectrometry allow analytical investigations to be performed directly on a tissue or biofilm under native-like experimental conditions. Laser ablation electrospray ionization (LAESI) is one such development and is particularly well-suited for the investigation of water-containing specimens. LAESI utilizes a mid-infrared laser beam (2.94 &mu;m wavelength) to excite the water molecules of the sample. When the ablation fluence threshold is exceeded, the sample material is expelled in the form of particulate matter and these projectiles travel to tens of millimeters above the sample surface. In LAESI, this ablation plume is intercepted by highly charged droplets to capture a fraction of the ejected sample material and convert its chemical constituents into gas-phase ions. A mass spectrometer equipped with an atmospheric-pressure ion source interface is employed to analyze and record the composition of the released ions originating from the probed area (pixel) of the sample. A systematic interrogation over an array of pixels opens a way for molecular imaging in the microprobe analysis mode. A unique aspect of LAESI mass spectrometric imaging is depth profiling that, in combination with lateral imaging, enables three-dimensional (3D) molecular imaging. With current lateral and depth resolutions of ~100 &mu;m and ~40 &mu;m, respectively, LAESI mass spectrometric imaging helps to explore the molecular structure of biological tissues. Herein, we review the major elements of a LAESI system and provide guidelines for a successful imaging experiment.

Laser ablation electrospray ionization (LAESI) is an atmospheric-pressure ion source for mass spectrometry. In the imaging mode, a mid-infrared laser probes the distributions of molecules across a tissue section or a biofilm. This technique presents a new approach for diverse bioanalytical studies carried out under native experimental conditions.

LAESI 질량 분석법에 의해 생물 학적 조직 및 Biofilms의 대기 압력 분자 이미징

Ambient ionization methods in mass spectrometry allow analytical investigations to be performed directly on a tissue or biofilm under native-like ...

Direct Analysis of Single Cells by Mass Spectrometry at Atmospheric Pressure

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell types exhibit heterogeneous biochemical makeup depending on their physiological conditions and interactions with the environment. Conventional methods of mass spectrometry (MS) used for the analysis of biomolecules in single cells rely on extensive sample preparation. Removing the cells from their natural environment and extensive sample processing could lead to changes in the cellular composition. Ambient ionization methods enable the analysis of samples in their native environment and without extensive sample preparation.1 The techniques based on the mid infrared (mid-IR) laser ablation of biological materials at 2.94 &mu;m wavelength utilize the sudden excitation of water that results in phase explosion.2 Ambient ionization techniques based on mid-IR laser radiation, such as laser ablation electrospray ionization (LAESI) and atmospheric pressure infrared matrix-assisted laser desorption ionization (AP IR-MALDI), have successfully demonstrated the ability to directly analyze water-rich tissues and biofluids at atmospheric pressure.3-11 In LAESI the mid-IR laser ablation plume that mostly consists of neutral particulate matter from the sample coalesces with highly charged electrospray droplets to produce ions. Recently, mid-IR ablation of single cells was performed by delivering the mid-IR radiation through an etched fiber. The plume generated from this ablation was postionized by an electrospray enabling the analysis of diverse metabolites in single cells by LAESI-MS.12 This article describes the detailed protocol for single cell analysis using LAESI-MS. The presented video demonstrates the analysis of a single epidermal cell from the skin of an Allium cepa bulb. The schematic of the system is shown in Figure 1. A representative example of single cell ablation and a LAESI mass spectrum from the cell are provided in Figure 2.

Single cell analysis is performed by mass spectrometry on plant and animal cells at atmospheric pressure by using a sharpened optical fiber to sample the cells for laser ablation electrospray ionization (LAESI) mass spectrometry.

대기압에서 질량 분석법에 의해 단일 ​​세포의 직접 분석

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell ...

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in quantitation, are associated with a high cost, long lead time, and are fraught with drawbacks (e.g. heterophilic antibodies, autoantibody interference, 'hook-effect').1 An alternative technique is affinity enrichment of peptides coupled with quantitative mass spectrometry, commonly referred to as SISCAPA (Stable Isotope Standards and Capture by Anti-Peptide Antibodies).2 In this technique, affinity enrichment of peptides with stable isotope dilution and detection by selected/multiple reaction monitoring mass spectrometry (SRM/MRM-MS) provides quantitative measurement of peptides as surrogates for their respective proteins. SRM/MRM-MS is well established for accurate quantitation of small molecules 3, 4 and more recently has been adapted to measure the concentrations of proteins in plasma and cell lysates.5-7 To achieve quantitation of proteins, these larger molecules are digested to component peptides using an enzyme such as trypsin. One or more selected peptides whose sequence is unique to the target protein in that species (i.e. "proteotypic" peptides) are then enriched from the sample using anti-peptide antibodies and measured as quantitative stoichiometric surrogates for protein concentration in the sample. Hence, coupled to stable isotope dilution (SID) methods (i.e. a spiked-in stable isotope labeled peptide standard), SRM/MRM can be used to measure concentrations of proteotypic peptides as surrogates for quantification of proteins in complex biological matrices. The assays have several advantages compared to traditional immunoassays. The reagents are relatively less expensive to generate, the specificity for the analyte is excellent, the assays can be highly multiplexed, enrichment can be performed from neat plasma (no depletion required), and the technique is amenable to a wide array of proteins or modifications of interest.8-13 In this video we demonstrate the basic protocol as adapted to a magnetic bead platform.

Stable Isotope Standards and Capture by Anti-Peptide Antibodies (SISCAPA) couples affinity enrichment of peptides with stable isotope dilution mass spectrometry (MRM-MS) to provide quantitative measurement of peptides as surrogates for their respective proteins. Here we describe the protocol using magnetic particles in a partially automated format.

펩타이드 면역 친 화성 농축을 사용하여 단백질의 양을 정함은 질량 분광법과 함께

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in ...

Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on Arabidopsis thaliana, a genetically tractable host plant1,2. The accumulation of reactive oxygen species (ROS) in cotyledons inoculated with DC3000 indicates a role of ROS in modulating necrotic cell death during bacterial speck disease of tomato3. Hydrogen peroxide, a component of ROS, is produced after inoculation of tomato plants with Pseudomonas3. Hydrogen peroxide can be detected using a histochemical stain 3'-3' diaminobenzidine (DAB)4. DAB staining reacts with hydrogen peroxide to produce a brown stain on the leaf tissue4. ROS has a regulatory role of the cellular redox environment, which can change the redox status of certain proteins5. Cysteine is an important amino acid sensitive to redox changes. Under mild oxidation, reversible oxidation of cysteine sulfhydryl groups serves as redox sensors and signal transducers that regulate a variety of physiological processes6,7. Tandem mass tag (TMT) reagents enable concurrent identification and multiplexed quantitation of proteins in different samples using tandem mass spectrometry8,9. The cysteine-reactive TMT (cysTMT) reagents enable selective labeling and relative quantitation of cysteine-containing peptides from up to six biological samples. Each isobaric cysTMT tag has the same nominal parent mass and is composed of a sulfhydryl-reactive group, a MS-neutral spacer arm and an MS/MS reporter10. After labeling, the samples were subject to protease digestion. The cysteine-labeled peptides were enriched using a resin containing anti-TMT antibody. During MS/MS analysis, a series of reporter ions (i.e., 126-131 Da) emerge in the low mass region, providing information on relative quantitation. The workflow is effective for reducing sample complexity, improving dynamic range and studying cysteine modifications. Here we present redox proteomic analysis of the Pst DC3000 treated tomato (Rio Grande) leaves using cysTMT technology. This high-throughput method has the potential to be applied to studying other redox-regulated physiological processes.

Reactive oxygen species level is elevated when cells encounter stress conditions. Here we show the example of 3'-3' diaminobenzidine staining as well as cysTMT labeling and mass spectrometry to profile the redox proteome in Pseudomonas syringae treated tomato leaves.

동위 원소 태그 질량 분광법을 사용하여 Thiol 산화 환원 프로테옴 프로 파일링

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on ...

Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis (diguanylate cyclases) and degradation (phosphodiesterases) of the second messenger c-di-GMP. In contrast, little information is available regarding the molecular mechanisms and cellular components through which this signaling molecule regulates a diverse range of cellular processes. Most of the known effector proteins belong to the PilZ family or are degenerated diguanylate cyclases or phosphodiesterases that have given up on catalysis and have adopted effector function. Thus, to better define the cellular c-di-GMP network in a wide range of bacteria experimental methods are required to identify and validate novel effectors for which reliable in silico predictions fail.
We have recently developed a novel Capture Compound Mass Spectrometry (CCMS) based technology as a powerful tool to biochemically identify and characterize c-di-GMP binding proteins. This technique has previously been reported to be applicable to a wide range of organisms1. Here we give a detailed description of the protocol that we utilize to probe such signaling components. As an example, we use Pseudomonas aeruginosa, an opportunistic pathogen in which c-di-GMP plays a critical role in virulence and biofilm control. CCMS identified 74% (38/51) of the known or predicted components of the c-di-GMP network. This study explains the CCMS procedure in detail, and establishes it as a powerful and versatile tool to identify novel components involved in small molecule signaling.

The ubiquitous second messenger c-di-GMP controls growth and behavior of many bacteria. We have developed a novel Capture Compound Mass Spectrometry based technology to biochemically identify and characterize c-di-GMP binding proteins in virtually any bacterial species.

캡처 복합 질량 분석 - 소설의 C 디 GMP 이펙터 단백질을 식별 할 수있는 강력한 도구

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis ...

Sequence-specific Labeling of Nucleic Acids and Proteins with Methyltransferases and Cofactor Analogues

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific positions in DNA, RNA, proteins and small biomolecules. This natural methylation reaction can be expanded to a wide variety of alkylation reactions using synthetic cofactor analogues. Replacement of the reactive sulfonium center of AdoMet with an aziridine ring leads to cofactors which can be coupled with DNA by various DNA MTases. These aziridine cofactors can be equipped with reporter groups at different positions of the adenine moiety and used for Sequence-specific Methyltransferase-Induced Labeling of DNA (SMILing DNA). As a typical example we give a protocol for biotinylation of pBR322 plasmid DNA at the 5&#8217;-ATCGAT-3&#8217; sequence with the DNA MTase M.BseCI and the aziridine cofactor 6BAz in one step. Extension of the activated methyl group with unsaturated alkyl groups results in another class of AdoMet analogues which are used for methyltransferase-directed Transfer of Activated Groups (mTAG). Since the extended side chains are activated by the sulfonium center and the unsaturated bond, these cofactors are called double-activated AdoMet analogues. These analogues not only function as cofactors for DNA MTases, like the aziridine cofactors, but also for RNA, protein and small molecule MTases. They are typically used for enzymatic modification of MTase substrates with unique functional groups which are labeled with reporter groups in a second chemical step. This is exemplified in a protocol for fluorescence labeling of histone H3 protein. A small propargyl group is transferred from the cofactor analogue SeAdoYn to the protein by the histone H3 lysine 4 (H3K4) MTase Set7/9 followed by click labeling of the alkynylated histone H3 with TAMRA azide. MTase-mediated labeling with cofactor analogues is an enabling technology for many exciting applications including identification and functional study of MTase substrates as well as DNA genotyping and methylation detection.

DNA and proteins are sequence-specifically labeled with affinity or fluorescent reporter groups using DNA or protein methyltransferases and synthetic cofactor analogues. Depending on the cofactor specificity of the enzymes, aziridine or double activated cofactor analogues are employed for one- or two-step labeling.

Methyltransferases과 공동 인자 유사체와 핵산과 단백질의 순서 특정 레이블

S-Adenosyl-l-methionine (AdoMet or SAM)-dependent methyltransferases (MTase) catalyze the transfer of the activated methyl group from AdoMet to specific ...

Methyltransferases의 프로 파일링 및 기타 S - adenosyl - L - 호모 시스테인 결합 단백질

Methyltransferases의 프로 파일링 및 기타 S - adenosyl -_{L - 호모 시스테인 결합 단백질}