우리는 CF1β에 대한 항혈청을위한 올리비에 Vallon 감사드립니다. 이 작업은 최대 플랑크 사회와 독일 Forschungsgemeinschaft (Schr 617/5-1)와 Bundesministerium 털 Bildung 숙녀 Forschung (시스템 생물 이니셔티브 FORSYS, 프로젝트 GoFORSYS)에서 보조금에 의해 지원되었다.

<ol>
	<li>Perrakis, A., Musacchio, A., Cusack, S., Petosa, C. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Investigating+a+macromolecular+complex:+the+toolkit+of+methods.">Investigating a macromolecular complex: the toolkit of methods.</a> J. Struct. Biol. 175, 106-112 (2011).</li><li>Gavin, A. C., Maeda, K., Kuhner, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Recent+advances+in+charting+protein-protein+interaction:+mass+spectrometry-based+approaches.">Recent advances in charting protein-protein interaction: mass spectrometry-based approaches.</a> Curr. Opin. Biotechnol. 22, 42-49 (2011).</li><li>Markham, K., Bai, Y., Schmitt-Ulms, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Co-immunoprecipitations+revisited:+an+update+on+experimental+concepts+and+their+implementation+for+sensitive+interactome+investigations+of+endogenous+proteins.">Co-immunoprecipitations revisited: an update on experimental concepts and their implementation for sensitive interactome investigations of endogenous proteins.</a> Anal. Bioanal. Chem. 389, 461-473 (2007).</li><li>Selbach, M., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Protein+interaction+screening+by+quantitative+immunoprecipitation+combined+with+knockdown+(QUICK.">Protein interaction screening by quantitative immunoprecipitation combined with knockdown (QUICK.</a> Nat Methods. 3, 981-983 (2006).</li><li>Ong, S. E., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+practical+recipe+for+stable+isotope+labeling+by+amino+acids+in+cell+culture+(SILAC).">A practical recipe for stable isotope labeling by amino acids in cell culture (SILAC).</a> Nat Protoc. 1, 2650-2660 (2006).</li><li>Heide, H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Application+of+quantitative+immunoprecipitation+combined+with+knockdown+and+cross-linking+to+Chlamydomonas+reveals+the+presence+of+vesicle-inducing+protein+in+plastids+1+in+a+common+complex+with+chloroplast+HSP90C.">Application of quantitative immunoprecipitation combined with knockdown and cross-linking to Chlamydomonas reveals the presence of vesicle-inducing protein in plastids 1 in a common complex with chloroplast HSP90C.</a> Proteomics. 9, 3079-3089 (2009).</li><li>Nordhues, A., Miller, S. M., Muhlhaus, T., Schroda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=New+insights+into+the+roles+of+molecular+chaperones+in+Chlamydomonas+and+Volvox.">New insights into the roles of molecular chaperones in Chlamydomonas and Volvox.</a> International review of cell and molecular biology. 285, 75-113 (2010).</li><li>Mayer, M. P., Bukau, B. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Hsp70+chaperones:+cellular+functions+and+molecular+mechanism.">Hsp70 chaperones: cellular functions and molecular mechanism.</a> Cell Mol. Life Sci. 62, 670-684 (2005).</li><li>Schroda, M., Vallon, O., Whitelegge, J. P., Beck, C. F., Wollman, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+chloroplastic+GrpE+homolog+of+Chlamydomonas:+two+isoforms+generated+by+differential+splicing.">The chloroplastic GrpE homolog of Chlamydomonas: two isoforms generated by differential splicing.</a> The Plant Cell. 13, 2823-2839 (2001).</li><li>Willmund, F., Schroda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=HEAT+SHOCK+PROTEIN+90C+is+a+bona+fide+Hsp90+that+interacts+with+plastidic+HSP70B+in+Chlamydomonas+reinhardtii.">HEAT SHOCK PROTEIN 90C is a bona fide Hsp90 that interacts with plastidic HSP70B in Chlamydomonas reinhardtii.</a> Plant Physiol. 138, 2310-2322 (2005).</li><li>Harris, E. H. <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 Chlamydomonas Sourcebook: Introduction to Chlamydomonas and Its Laboratory Use. , (2008).</li><li>Mortensen, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=MSQuant,+an+open+source+platform+for+mass+spectrometry-based+quantitative+proteomics.">MSQuant, an open source platform for mass spectrometry-based quantitative proteomics.</a> J. Proteome Res. 9, 393-403 (2010).</li><li>M?hlhaus, T., Weiss, J., Hemme, D., Sommer, F., Schroda, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Quantitative+Shotgun+Proteomics+Using+a+Uniform+15N-Labeled+Standard+to+Monitor+Proteome+Dynamics+in+Time+Course+Experiments+Reveals+New+Insights+into+the+Heat+Stress+Response+of+Chlamydomonas+reinhardtii.">Quantitative Shotgun Proteomics Using a Uniform 15N-Labeled Standard to Monitor Proteome Dynamics in Time Course Experiments Reveals New Insights into the Heat Stress Response of Chlamydomonas reinhardtii.</a> Mol. Cell Proteomics. 10, (2011).</li><li>Bukau, B., Horwich, A. L. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Hsp70+and+Hsp60+chaperone+machines.">The Hsp70 and Hsp60 chaperone machines.</a> Cell. 92, 351-366 (1998).</li><li>Wandinger, S. K., Richter, K., Buchner, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+Hsp90+chaperone+machinery.">The Hsp90 chaperone machinery.</a> J. Biol. Chem. 283, 18473-18477 (2008).</li></ol>

관심 없음 충돌이 선언 없습니다.

과도 단백질 단백질 상호 작용 (QUICK-X)를 캡처하기위한 crosslinking 단계, 그리고 불평등 침전 효율 6 정상화 할 수있는 제어 강수량 : 우리는 최근 QUICK 방법 두 가지 개선을 도입했습니다. 다음은 QUICK의 두 개선이 포함 된 프로토콜 제시 : 먼저, 우리는 15 N 신진 대사 라벨링에 대한 SILAC 5 바꿉니다. 장점은 15 N은 단순 무기 소금으로 제공되는 경우 15 N 신진 대사 라벨이 SILAC보다 훨씬 저렴하다고합니다. 또한, 15 N 신진 대사와 QUICK 라벨 대부분의 식물, 곰팡이 및 박테리아처럼, 모든 아미노산에 대한 생물의 prototrophic에 적용 할 수 있습니다. 그리고 마지막으로, SILAC 5,6에 내재 아르기닌 - 투 - 프롤린 interconversion 15 N이라는 펩티드의 정량화에 대한 문제를 제시하지 않습니다. 15 N의 단백질 체학 데이터의 양적 평가에 적합한 도구 예를 들면 MSQUANT 12 I 아르OMIQS 13. 둘째, 우리는 특별히 다른 비해 한 샘플에서 주어진 목표 단백질과 상호 작용하는 단백질의 양을 감소시키기위한 수단으로 친화력 변조를 소개합니다. 이 방법의 장점은 일부 모델 시스템을 생성하기가 어렵습니다 또는 모든 필수 대상 단백질의 경우에서 생성 할 수없는 최저의 돌연변이의 건설을 circumvents한다는 점입니다. 또한, 잠재적 대상 단백질을 쓰러 뜨린에 세포의 응답으로 발생하는 차동 단백질 발현에 의한 misinterpretations을 방지 : 다른 단백질뿐만 아니라 다운 규제하고 immunoprecipitation에 사용 된 항혈청과 교차 반응하는 경우, 그들은 해석 될 대상 단백질의 진정한 상호 작용 파트너 있습니다. 마지막으로, 화성 변조는 적절한 항체 - 투 - 항원 비율을 찾는 필요성을 시킨다는. 우리는 모델 생물로 Chlamydomonas reinhardtii의 우리의 프로토콜을 적용되지만, 그것은 쉽게 세포 배양에서 재배 및 질소 소스로 암모니아 또는 질산을 사용 할 수 있습니다 할 수있는 다른 생물체에 적용 할 수 있습니다. ATP / ADP에 의한 단백질 단지의 연관성 변조는 직접 GroEL/HSP60/Cpn60 또는 HSP90 자야 시스템 14,15 같은 그의 상호 작용 기판과 일대 단백질과 ATP 상태에 따라 다른 보호자에 적용, 또는 다른 시스템에 위치 할 수 있습니다 바인딩 동질성은 ATP에 의해 조절됩니다. 화성 변조는 또한 단백질 상호 작용 사이의 동질성이 HSP90 시스템 (15)의 경우 radicicol 또는 geldanamycin 같은 특정 약물에 의해 변경 된 경우에 작동합니다. 우리 프로토콜의 명확한 제한이 필요하다는 것입니다 친화 - 정화 특정 치료 / 파트너 단백질위한 친화력을 변조 약물에 민감한 것으로 알려진 대상 단백질에 대한 항체를. 따라서 더 높은 처리량 방법이다.

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 코멘트 (선택 사항) </td></tr><tr><td> ProteinA 세 파로스 </td><td> 시그마 - 알드리치 </td><td> P3391 </td><td></td></tr><tr><td> DMP (디메틸 pimelimidate) </td><td> 시그마 - 알드리치 </td><td> D8388 </td><td> -20 ° C에서 말라 저장, 사용하기 전에 직접 분해 </td></tr><tr><td> (전체, EDTA (에틸렌 다이아 민 테트라 초산) - 무료) 프로테아제 억제제 </td><td> 로슈 응용 과학 </td><td> 11873580001 </td><td></td></tr><tr><td> ATP (Adenosin-5&#39;-triphosphat) </td><td> 칼 로스 </td><td> K054 </td><td></td></tr><tr><td> 크레아틴 인산 </td><td> 시그마 - 알드리치 </td><td> 27,920 </td><td></td></tr><tr><td> 크리 phosphokinase </td><td> 시그마 - 알드리치 </td><td> C7886 </td><td>; </td></tr><tr><td> DSP Dithiobis [succinimidyl 프로 피오 네이트] </td><td> Sientific 열 </td><td> 22,585 </td><td> 4에 말라 저장 ° C </td></tr><tr><td> 15 NH 4 망할 CIA </td><td> 캠브리지 동위 원소 실험실, 앤도버, MA </td><td> NLM-467 </td><td></td></tr><tr><td> LYS-C (Endoproteinase LYS-C) </td><td> 로슈 응용 과학 </td><td> 11047825001 </td><td></td></tr><tr><td> 트립신 비즈 (Poroszyme는 트립신을 고정) </td><td> 응용 Biosystems </td><td> 2-3127-00 </td><td> 직접 사용하기 전에 힘차게 믹스 </td></tr><tr><td> Empore C 18 47mm 디스크 </td><td> Varian </td><td> 12145004 </td><td></td></tr></tbody></table>

a protocol for the identification of protein-protein interactions based on 15n metabolic labeling, immunoprecipitation, quantitative mass spectrometry and affinity modulation

단백질 - 단백질 상호 작용은 세포의 많은 생물학적 과정에 대한 기본입니다. 따라서, 그들의 특성은 조사 1 확인할 수 있습니다 방법을 현재의 연구 및 과다에 중요한 역할을합니다. 단백질 - 단백질 상호 작용은 종종 매우 역동적이며, subcellular 지방화, 포스트 병진 수정과 지역 단백질 환경이에 따라 달라질 수 있습니다. 따라서, 그들은 공동 immunoprecipitation 접근 방식이 선택 (3)의 방법입니다있는 그들의 자연 환경에서 조사해야합니다. 공동 시켰던 상호 작용 파트너는 타겟이 불분명 한 방식으로 타겟팅 된 접근, 또는 질량 분석법 (LC-MS/MS)에 의해 immunoblotting 중 하나로 식별됩니다. 후자의 전략은 종종 부정적인 주로 자신 unspecifically precipitating 단백질의 흔적을 감지 현대적인 질량 분석기의 높은 감도에서 파생 된 잘못된 긍정적 인 발견의 많은에 의해 영향을받습니다. recen이 문제를 극복 할 수 t의 접근 방식은 아이디어에 기초 unspecifically precipitating 단백질의 양이 영향을받지 않습니다 동안 특정 상호 작용 파트너의 감소 금액은, 세포 농도 RNAi에 의해 감소​​ 주어진 대상 단백질과 공동 침전물 할 것입니다. 이 방법은, 최저 4 결합 양적 Immunoprecipitation에 대한 QUICK 칭했다 야생 유형과 노크 다운 긴장에서 immunoprecipitated 단백질의 양을 정량화하는 안정적인 동위 원소 전지 문화에 아미노산의 라벨링 (SILAC) 5 MS을 사용합니다. 1:1 비율에서 발견 된 단백질은 오염 물질, 대상 단백질의 특정 상호 작용 파트너로 야생 유형에서 침전물에 풍부한 사람들로 간주 될 수 있습니다. 혁신, QUICK 일부 제한이지지하지만 : 먼저, SILAC 비용이 많이 이상적 아르기닌 및 / 또는 리신에 대한 auxotrophic 아르 생물로 제한됩니다. addit에 또한, 무거운 아르기닌이 공급 될 때, 아르기닌 - 투 - 프롤린 interconversion 결과ional 질량이 펩타이드 각 프롤린에 대한 연속 근무 약간 정량화 더 지루하고 5,6 정확도가 떨어질 수 있습니다 빛 아르기닌과 무거운 dilutes. 둘째, QUICK는 항체 그들이 노크 다운 뮤턴트에서 추출의 대상 단백질과 포화가되지 않는 등 titrated되어 있어야합니다. 여기 15 N 신진 대사 라벨에 SILAC을 대체하여, 단백질 - 단백질 상호 작용의 연관성 변조에 대한 RNAi로 인한 똑 다운 대체하여 QUICK의 abovementioned 한계를 극복 수정 QUICK 프로토콜을 소개합니다. 우리는 단세포 녹색 alga의 Chlamydomonas이 모델 생물 및 대상 단백질 7 (그림 1)과 엽록체의 HSP70B의 보호자로 reinhardtii 사용하여이 프로토콜의 적용을 보여줍니다. HSP70s는 특정 공동 보호자 만 ADP 상태 8 기판과 상호 작용하는 것으로 알려져 있습니다. 우리는 특정을 확인하는 수단으로이 속성을 이용의 염기 교환 요소 CGE1 9 HSP70B의 상호 작용.

Watch this Scientific Journal Video about A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Aff

A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

우리는 TRUE와 FALSE 단백질 단백질 상호 작용을 구별하기 이전에 도입 된 QUICK (최저과 결합하여 정량 Immunoprecipitation) 접근 방식의 변화를 제시한다. 우리 접근 방식은 기반으로합니다<sup&gt; 15N</sup&gt; 신진 대사 라벨, ATP, immunoprecipitation, 그리고 양적 질량 분광법의 존재 / 부재에 의한 단백질 단백질 상호 작용의 동질성의 변조.

을 바탕으로 단백질 - 단백질 상호 작용의 확인에 프로토콜<sup&gt; 15</sup&gt; N 신진 대사 라벨링, Immunoprecipitation, 수량 질량 분석법 및 선호도 변조

Protein-protein interactions are fundamental for many biological processes in the cell. Therefore, their characterization plays an important role in ...

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A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

20.4K 조회수.  Max Planck Institute of Molecular Plant Physiology. 우리는 TRUE와 FALSE 단백질 단백질 상호 작용을 구별하기 이전에 도입 된 QUICK (최저과 결합하여 정량 Immunoprecipitation) 접근 방식의 변화를 제시한다. 우리 접근 방식은 기반으로합니다 15N 신진 대사 라벨, ATP, immunoprecipitation, 그리고 양적 질량 분광법의 존재 / 부재에 의한 단백질 단백질 상호 작용의 동질성의 변조.

비디오: A Protocol for the Identification of Protein-protein Interactions Based on 15N Metabolic Labeling, Immunoprecipitation, Quantitative Mass Spectrometry and Affinity Modulation

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 ...

연구

생물학

A Quantitative Assessment of The Yeast Lipidome using Electrospray Ionization Mass Spectrometry

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and very simple lipidome, is a valuable model for studying biological functions of various lipid species in multicellular eukaryotes2,3,5. S. cerevisiae has 10 major classes of lipids with chain lengths mainly of 16 or 18 carbon atoms and either zero or one degree of unsaturation6,7. Existing methods for lipid identification and quantification - such as high performance liquid chromatography, thin-layer chromatography, fluorescence microscopy, and gas chromatography followed by MS - are well established but have low sensitivity, insufficiently separate various molecular forms of lipids, require lipid derivitization prior to analysis, or can be quite time consuming. Here we present a detailed description of our experimental approach to solve these inherent limitations by using survey-scan ESI/MS for the identification and quantification of the entire complement of lipids in yeast cells. The described method does not require chromatographic separation of complex lipid mixtures recovered from yeast cells, thereby greatly accelerating the process of data acquisition. This method enables lipid identification and quantification at the concentrations as low as g/ml and has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. Lipids extraction from whole yeast cells for using this method of lipid analysis takes two to three hours. It takes only five to ten minutes to run each sample of extracted and dried lipids on a Q-TOF mass spectrometer equipped with a nano-electrospray source.

We describe a new quantitative lipidomics method for identifying numerous lipid species in yeast using survey-scan electrospray ionization mass spectrometry (ESI/MS). This method exceeds currently available methods for lipid identification and quantification in the ability to resolve various molecular forms of lipids, sensitivity, and speed.

전기 분무 이온화 질량 분광법을 사용하여 효모 Lipidome의 양적 평가

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

의 단백질 단지의 식별<em&gt; 대장균</em&gt; 직렬 질량 분석법과 함께 순차적 펩타이드의 동질 정화를 사용하여

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex composition under different conditions. In this protocol the approach is demonstrated by comparing the composition of the protein complex responsible for cyclic electron flow (CEF) in Chlamydomonas reinhardtii, isolated from genetically different strains. The procedure comprises the isolation of thylakoid membranes, followed by their separation into multiprotein complexes by sucrose density gradient centrifugation, SDS-PAGE, immunodetection and comparative, quantitative mass spectrometry (MS) based on differential metabolic labeling (14N/15N) of the analyzed strains. Detergent solubilized thylakoid membranes are loaded on sucrose density gradients at equal chlorophyll concentration. After ultracentrifugation, the gradients are separated into fractions, which are analyzed by mass-spectrometry based on equal volume. This approach allows the investigation of the composition within the gradient fractions and moreover to analyze the migration behavior of different proteins, especially focusing on ANR1, CAS, and PGRL1. Furthermore, this method is demonstrated by confirming the results with immunoblotting and additionally by supporting the findings from previous studies (the identification and PSI-dependent migration of proteins that were previously described to be part of the CEF-supercomplex such as PGRL1, FNR, and cyt f). Notably, this approach is applicable to address a broad range of questions for which this protocol can be adopted and e.g. used for comparative analyses of multiprotein complex composition isolated from distinct environmental conditions.

A New Approach for the Comparative Analysis of Multiprotein Complexes Based on 15N Metabolic Labeling and Quantitative Mass Spectrometry

The described comparative, quantitative proteomic approach aims at obtaining insights into the composition of multiprotein complexes&#160;under different conditions and is demonstrated by comparing genetically different strains. For quantitative analysis equal volumes of different fractions from a sucrose density gradient are mixed and analyzed by mass spectrometry.

multiprotein의 단지의 비교 분석을위한 새로운 접근 방식을 기반으로<sup&gt; 15</sup&gt; N 대사 라벨링 및 정량 질량 분석

The introduced protocol provides a tool for the analysis of multiprotein complexes in the thylakoid membrane, by revealing insights into complex ...

Investigating Protein-protein Interactions in Live Cells Using Bioluminescence Resonance Energy Transfer

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live cells. BRET is the non-radiative transfer of energy from a 'donor' luciferase enzyme to an 'acceptor' fluorescent protein. In the most common configuration of this assay, the donor is Renilla reniformis luciferase and the acceptor is Yellow Fluorescent Protein (YFP). Because the efficiency of energy transfer is strongly distance-dependent, observation of the BRET phenomenon requires that the donor and acceptor be in close proximity. To test for an interaction between two proteins of interest in cultured mammalian cells, one protein is expressed as a fusion with luciferase and the second as a fusion with YFP. An interaction between the two proteins of interest may bring the donor and acceptor sufficiently close for energy transfer to occur. Compared to other techniques for investigating protein-protein interactions, the BRET assay is sensitive, requires little hands-on time and few reagents, and is able to detect interactions which are weak, transient, or dependent on the biochemical environment found within a live cell. It is therefore an ideal approach for confirming putative interactions suggested by yeast two-hybrid or mass spectrometry proteomics studies, and in addition it is well-suited for mapping interacting regions, assessing the effect of post-translational modifications on protein-protein interactions, and evaluating the impact of mutations identified in patient DNA.

Interactions between proteins are fundamental to all cellular processes. Using Bioluminescence Resonance Energy Transfer, the interaction between a pair of proteins can be monitored in live cells and in real time. Furthermore, the effects of potentially pathogenic mutations can be assessed.

생물 발광 공명 에너지 전달을 이용하여 살아있는 세포에서 단백질 - 단백질 상호 작용을 조사

Assays based on Bioluminescence Resonance Energy Transfer (BRET) provide a sensitive and reliable means to monitor protein-protein interactions in live ...

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and silencing of genes mediate the response of plants to environmental signals and developmental cues. Therefore, insights into the mechanisms that control plant gene expression are essential to gain a deep understanding of how biological processes are regulated in plants. The chromatin immunoprecipitation (ChIP) technique described here is a procedure to identify the DNA-binding sites of proteins in genes or genomic regions of the model species Arabidopsis thaliana. The interactions with DNA of proteins of interest such as transcription factors, chromatin proteins or posttranslationally modified versions of histones can be efficiently analyzed with the ChIP protocol. This method is based on the fixation of protein-DNA interactions in vivo, random fragmentation of chromatin, immunoprecipitation of protein-DNA complexes with specific antibodies, and quantification of the DNA associated with the protein of interest by PCR techniques. The use of this methodology in Arabidopsis has contributed significantly to unveil transcriptional regulatory mechanisms that control a variety of plant biological processes. This approach allowed the identification of the binding sites of the Arabidopsis chromatin protein EBS to regulatory regions of the master gene of flowering FT. The impact of this protein in the accumulation of particular histone marks in the genomic region of FT was also revealed through ChIP analysis.

Chromatin Immunoprecipitation Assay for the Identification of Arabidopsis Protein-DNA Interactions In Vivo

Chromatin immunoprecipitation is a powerful technique for the identification of DNA binding sites of Arabidopsis proteins in vivo. This procedure includes chromatin cross-linking and fragmentation, immunoprecipitation with selective antibodies against the protein of interest, and qPCR analysis of bound DNA. We describe a simple ChIP assay optimized for Arabidopsis plants.

애기 장대 단백질 DNA 상호 작용의 식별을위한 염색질 면역 침전 분석<em&gt; 생체</em

Intricate gene regulatory networks orchestrate biological processes and developmental transitions in plants. Selective transcriptional activation and ...

Identification of MyoD Interactome Using Tandem Affinity Purification Coupled to Mass Spectrometry

Skeletal muscle terminal differentiation starts with the commitment of pluripotent mesodermal precursor cells to myoblasts. These cells have still the ability to proliferate or they can differentiate and fuse into multinucleated myotubes, which maturate further to form myofibers. Skeletal muscle terminal differentiation is orchestrated by the coordinated action of various transcription factors, in particular the members of the Muscle Regulatory Factors or MRFs (MyoD, Myogenin, Myf5, and MRF4), also called the myogenic bHLH transcription factors family. These factors cooperate with chromatin-remodeling complexes within elaborate transcriptional regulatory network to achieve skeletal myogenesis. In this, MyoD is considered the master myogenic transcription factor in triggering muscle terminal differentiation. This notion is strengthened by the ability of MyoD to convert non-muscle cells into skeletal muscle cells. Here we describe an approach used to identify MyoD protein partners in an exhaustive manner in order to elucidate the different factors involved in skeletal muscle terminal differentiation. The long-term aim is to understand the epigenetic mechanisms involved in the regulation of skeletal muscle genes, i.e., MyoD targets. MyoD partners are identified by using Tandem Affinity Purification (TAP-Tag) from a heterologous system coupled to mass spectrometry (MS) characterization, followed by validation of the role of relevant partners during skeletal muscle terminal differentiation. Aberrant forms of myogenic factors, or their aberrant regulation, are associated with a number of muscle disorders: congenital myasthenia, myotonic dystrophy, rhabdomyosarcoma and defects in muscle regeneration. As such, myogenic factors provide a pool of potential therapeutic targets in muscle disorders, both with regard to mechanisms that cause disease itself and regenerative mechanisms that can improve disease treatment. Thus, the detailed understanding of the intermolecular interactions and the genetic programs controlled by the myogenic factors is essential for the rational design of efficient therapies.

MyoD is a myogenic transcription factor with a strong capacity to induce myogenic transdifferentiation of many fully differentiated non-muscle cell lines. The epigenetic mechanisms involved in this transdifferentiation are largely unknown. Here we describe a double-affinity purification method followed by mass spectrometry to exhaustively characterize MyoD partners.

탠덤 선호도 정화를 사용하여 MyoD 상호 작용 체의 식별 질량 분석에 결합

Skeletal muscle terminal differentiation starts with the commitment of pluripotent mesodermal precursor cells to myoblasts. These cells have still the ...

Study of Protein-protein Interactions in Autophagy Research

Protein-protein interactions are important for understanding cellular signaling cascades and identifying novel pathway components and protein dynamics. The majority of cellular activities require physical interactions between proteins. To analyze and map these interactions, various experimental techniques as well as bioinformatics tools were developed. Autophagy is a cellular recycling mechanism that allows the cells to cope with different stressors, including nutrient deprivation, chemicals, and hypoxia. In order to better understand autophagy-related signaling events and to discover novel factors that regulate protein complexes in autophagy, we performed protein-protein interaction screens. Validation of these screening results requires the use of immunofluorescence and immunoprecipitation techniques. In this system, specific autophagy-related protein-protein interactions that we discovered were tested in Neuro2A (N2A) and HEK293T cell lines. Details of the technical procedures used are explained in this visualized experiment paper.

Presented here are two antibody-based protein-protein interaction research techniques: immunofluorescence and immunoprecipitation. These techniques are suitable for studying physical interactions between proteins for the discovery of novel components of cellular signaling pathways and for understanding protein dynamics.

단백질 단백질 상호 작용 Autophagy 연구에서의 연구

Protein-protein interactions are important for understanding cellular signaling cascades and identifying novel pathway components and protein dynamics. ...

Quantification of Site-specific Protein Lysine Acetylation and Succinylation Stoichiometry Using Data-independent Acquisition Mass Spectrometry

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein functions, for example, by changing enzymatic activity or by mediating interactions. Precise quantification of site-specific protein acylation occupancy, or stoichiometry, is essential for understanding the functional consequences of both global low-level stoichiometry and individual high-level acylation stoichiometry of specific lysine residues. Other groups have reported measurement of lysine acetylation stoichiometry by comparing the ratio of peptide precursor isotopes from endogenous, natural abundance acylation and exogenous, heavy isotope-labeled acylation introduced after quantitative chemical acetylation of proteins using stable isotope-labeled acetic anhydride. This protocol describes an optimized approach featuring several improvements, including: (1) increased chemical acylation efficiency, (2) the ability to measure protein succinylation in addition to acetylation, and (3) improved quantitative accuracy due to reduced interferences using fragment ion quantification from data-independent acquisitions (DIA) instead of precursor ion signal from data-dependent acquisition (DDA). The use of extracted peak areas from fragment ions for quantification also uniquely enables differentiation of site-level acylation stoichiometry from proteolytic peptides containing more than one lysine residue, which is not possible using precursor ion signals for quantification. Data visualization in Skyline, an open source quantitative proteomics environment, allows for convenient data inspection and review. Together, this workflow offers unbiased, precise, and accurate quantification of site-specific lysine acetylation and succinylation occupancy of an entire proteome, which may reveal and prioritize biologically relevant acylation sites.

Here, we present unbiased quantification of site-specific protein acetylation and/or succinylation occupancy (stoichiometry) of an entire proteome through a ratiometric analysis of endogenous modifications to modifications introduced after quantitative chemical acylation using stable isotope-labeled anhydrides. In combination with sensitive data-independent acquisition mass spectrometry, accurate site occupancy measurements are obtained.

사이트별 단백질 Lysine Acetylation 및 Succinylation 산출할 데이터 독립적인 수집 질량 분석을 사용 하 여 정량화

Post-translational modification (PTM) of protein lysine residues by N&#400;-acylation induces structural changes that can dynamically regulate protein ...

SA-&#946;-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell culture, termed senolytics, can reduce the senescent cell burden in vivo and extend healthspan. Multiple classes of senolytics have been identified to date including HSP90 inhibitors, Bcl-2 family inhibitors, piperlongumine, a FOXO4 inhibitory peptide and the combination of Dasatinib/Quercetin. Detection of SA-&#946;-Gal at an increased lysosomal pH is one of the best characterized markers for the detection of senescent cells. Live cell measurements of senescence-associated &#946;-galactosidase (SA-&#946;-Gal) activity using the fluorescent substrate C12FDG in combination with the determination of the total cell number using a DNA intercalating Hoechst dye opens the possibility to screen for senotherapeutic drugs that either reduce overall SA-&#946;-Gal activity by killing of senescent cells (senolytics) or by suppressing SA-&#946;-Gal and other phenotypes of senescent cells (senomorphics). Use of a high content fluorescent image acquisition and analysis platform allows for the rapid, high throughput screening of drug libraries for effects on SA-&#946;-Gal, cell morphology and cell number.

SA-&amp;#946;-Galactosidase-Based Screening Assay for the Identification of Senotherapeutic Drugs

Cellular senescence is the key factor in the development of chronic age-related pathologies. Identification of therapeutics that target senescent cells show promise for extending healthy aging. Here, we present a novel assay to screen for the identification of senotherapeutics based on measurement of senescence associated &#946;-Galactosidase activity in single cells.

SA-β-갈락토시다아제 계 선별 분석법

Cell senescence is one of the hallmarks of aging known to negatively influence a healthy lifespan. Drugs able to kill senescent cells specifically in cell ...