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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Biology

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

Protein Complex Affinity Capture from Cryomilled Mammalian Cells

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this technique is also frequently referred to as immunoprecipitation. Affinity capture can be applied in a bench-scale and in a high-throughput context. When coupled with protein mass spectrometry, affinity capture has proven to be a workhorse of interactome analysis. Although there are potentially many ways to execute the numerous steps involved, the following protocols implement our favored methods. Two features are distinctive: the use of cryomilled cell powder to produce cell extracts, and antibody-coupled paramagnetic beads as the affinity medium. In many cases, we have obtained superior results to those obtained with more conventional affinity capture practices. Cryomilling avoids numerous problems associated with other forms of cell breakage. It provides efficient breakage of the material, while avoiding denaturation issues associated with heating or foaming. It retains the native protein concentration up to the point of extraction, mitigating macromolecular dissociation. It reduces the time extracted proteins spend in solution, limiting deleterious enzymatic activities, and it may reduce the non-specific adsorption of proteins by the affinity medium. Micron-scale magnetic affinity media have become more commonplace over the last several years, increasingly replacing the traditional agarose- and Sepharose-based media. Primary benefits of magnetic media include typically lower non-specific protein adsorption; no size exclusion limit because protein complex binding occurs on the bead surface rather than within pores; and ease of manipulation and handling using magnets.

Here we describe protocols to disrupt mammalian cells by solid-state milling at a cryogenic temperature, produce a cell extract from the resulting cell powder, and isolate protein complexes of interest by affinity capture upon antibody-coupled micron-scale paramagnetic beads.

Cryomilled 포유 동물 세포에서 단백질 단지 선호도 캡처

Affinity capture is an effective technique for isolating endogenous protein complexes for further study. When used in conjunction with an antibody, this ...

연구

생화학

A Protein Preparation Method for the High-throughput Identification of Proteins Interacting with a Nuclear Cofactor Using LC-MS/MS Analysis

Transcriptional coregulators are vital to the efficient transcriptional regulation of nuclear chromatin structure. Coregulators play a variety of roles in regulating transcription. These include the direct interaction with transcription factors, the covalent modification of histones and other proteins, and the occasional chromatin conformation alteration. Accordingly, establishing relatively quick methods for identifying proteins that interact within this network is crucial to enhancing our understanding of the underlying regulatory mechanisms. LC-MS/MS-mediated protein binding partner identification is a validated technique used to analyze protein-protein interactions. By immunoprecipitating a previously-identified member of a protein complex with an antibody (occasionally with an antibody for a tagged protein), it is possible to identify its unknown protein interactions via mass spectrometry analysis. Here, we present a method of protein preparation for the LC-MS/MS-mediated high-throughput identification of protein interactions involving nuclear cofactors and their binding partners. This method allows for a better understanding of the transcriptional regulatory mechanisms of the targeted nuclear factors.

We have established a method for the purification of coregulatory interaction proteins using the LC-MS/MS system.

LC-MS / MS 분석을 이용하여 핵 공동 인자와 상호 작용 단백질의 높은 처리량 식별 단백질 제조 방법

Transcriptional coregulators are vital to the efficient transcriptional regulation of nuclear chromatin structure. Coregulators play a variety of roles in ...

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

블루 기본 페이지와 단백질 상관 관계 프로파일 링에 의한 선호도 정제 단백질 복합체 해결

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

Deep Proteome Profiling by Isobaric Labeling, Extensive Liquid Chromatography, Mass Spectrometry, and Software-assisted Quantification

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) coupled to tandem mass spectrometry (LC-MS/MS) and isobaric labeling multiplexing capacity. Here, we introduce a deep-proteomics profiling protocol that combines 10-plex tandem mass tag (TMT) labeling with an extensive LC/LC-MS/MS platform, and post-MS computational interference correction to accurately quantitate whole proteomes. This protocol includes the following main steps: protein extraction and digestion, TMT labeling, 2-dimensional (2D) LC, high-resolution mass spectrometry, and computational data processing. Quality control steps are included for troubleshooting and evaluating experimental variation. More than 10,000 proteins in mammalian samples can be confidently quantitated with this protocol. This protocol can also be applied to the quantitation of post translational modifications with minor changes. This multiplexed, robust method provides a powerful tool for proteomic analysis in a variety of complex samples, including cell culture, animal tissues, and human clinical specimens.

We present a protocol to accurately quantitate proteins with isobaric labelling, extensive fractionation, bioinformatics tools, and quality control steps in combination with liquid chromatography interfaced to a high-resolution mass spectrometer.

깊은 프로테옴 Isobaric 라벨, 광범위 한 액체 크로마토그래피, 질량 분석 및 소프트웨어 기반 정량화 하 여 프로 파일링

Many exceptional advances have been made in mass spectrometry (MS)-based proteomics, with particular technical progress in liquid chromatography (LC) ...

2 in 1: One-step Affinity Purification for the Parallel Analysis of Protein-Protein and Protein-Metabolite Complexes

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of protein-protein interactions (PPI) is no novelty, experimental approaches aiming to characterize endogenous protein-metabolite interactions (PMI) constitute a rather recent development. Herein, we present a protocol that allows simultaneous characterization of the PPI and PMI of a protein of choice, referred to as bait. Our protocol was optimized for Arabidopsis cell cultures and combines affinity purification (AP) with mass spectrometry (MS)-based protein and metabolite detection. In short, transgenic Arabidopsis lines, expressing bait protein fused to an affinity tag, are first lysed to obtain a native cellular extract. Anti-tag antibodies are used to pull down protein and metabolite partners of the bait protein. The affinity-purified complexes are extracted using a one-step methyl tert-butyl ether (MTBE)/methanol/water method. Whilst metabolites separate into either the polar or the hydrophobic phase, proteins can be found in the pellet. Both metabolites and proteins are then analyzed by ultra-performance liquid chromatography-mass spectrometry (UPLC-MS or UPLC-MS/MS). Empty-vector (EV) control lines are used to exclude false positives. The major advantage of our protocol is that it enables identification of protein and metabolite partners of a target protein in parallel in near-physiological conditions (cellular lysate). The presented method is straightforward, fast, and can be easily adapted to biological systems other than plant cell cultures.

Protein-protein and protein-metabolite interactions are crucial for all cellular functions. Herein, we describe a protocol that allows parallel analysis of these interactions with a protein of choice. Our protocol was optimized for plant cell cultures and combines affinity purification with mass spectrometry-based protein and metabolite detection.

2 1: 원스텝 단백질 단백질과 단백질 대사 산물 단지의 병렬 분석에 대 한 친 화력 정화

Cellular processes are regulated by interactions between biological molecules such as proteins, metabolites, and nucleic acids. While the investigation of ...

Quantification of Protein Interaction Network Dynamics using Multiplexed Co-Immunoprecipitation

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic interactions among multiple proteins in a protein interaction network is technically difficult. Here, we present a protocol for Quantitative Multiplex Immunoprecipitation (QMI), which allows quantitative assessment of fold changes in protein interactions based on relative fluorescence measurements of Proteins in Shared Complexes detected by Exposed Surface epitopes (PiSCES). In QMI, protein complexes from cell lysates are immunoprecipitated onto microspheres, and then probed with a labeled antibody for a different protein in order to quantify the abundance of PiSCES. Immunoprecipitation antibodies are conjugated to different MagBead spectral regions, which allows a flow cytometer to differentiate multiple parallel immunoprecipitations and simultaneously quantify the amount of probe antibody associated with each. QMI does not require genetic tagging and can be performed using minimal biomaterial compared to other immunoprecipitation methods. QMI can be adapted for any defined group of interacting proteins, and has thus far been used to characterize signaling networks in T cells and neuronal glutamate synapses. Results have led to new hypothesis generation with potential diagnostic and therapeutic applications. This protocol includes instructions to perform QMI, from the initial antibody panel selection through to running assays and analyzing data. The initial assembly of a QMI assay involves screening antibodies to generate a panel, and empirically determining an appropriate lysis buffer. The subsequent reagent preparation includes covalently coupling immunoprecipitation antibodies to MagBeads, and biotinylating probe antibodies so they can be labeled by a streptavidin-conjugated fluorophore. To run the assay, lysate is mixed with MagBeads overnight, and then beads are divided and incubated with different probe antibodies, and then a fluorophore label, and read by flow cytometry. Two statistical tests are performed to identify PiSCES that differ significantly between experimental conditions, and results are visualized using heatmaps or node-edge diagrams.

Quantitative Multiplex Immunoprecipitation (QMI) uses flow cytometry for sensitive detection of differences in the abundance of targeted protein-protein interactions between two samples. QMI can be performed using a small amount of biomaterial, does not require genetically engineered tags, and can be adapted for any previously defined protein interaction network.

멀티플렉스 동시 면역 침전을 이용한 단백질 상호 작용 네트워크 역학의 정량화

Dynamic protein-protein interactions control cellular behavior, from motility to DNA replication to signal transduction. However, monitoring dynamic ...

Label-Free Immunoprecipitation Mass Spectrometry Workflow for Large-scale Nuclear Interactome Profiling

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This publication presents a complete interaction proteomics workflow designed for identifying low abundance protein-protein interactions from the nucleus that could also be applied to other subcellular compartments. This workflow includes subcellular fractionation, immunoprecipitation, sample preparation, offline cleanup, single-shot label-free mass spectrometry, and downstream computational analysis and data visualization. Our protocol is optimized for detecting compartmentalized, low abundance interactions that are difficult to identify from whole cell lysates (e.g., transcription factor interactions in the nucleus) by immunoprecipitation of endogenous proteins from fractionated subcellular compartments. The sample preparation pipeline outlined here provides detailed instructions for the preparation of HeLa cell nuclear extract, immunoaffinity purification of endogenous bait protein, and quantitative mass spectrometry analysis. We also discuss methodological considerations for performing large-scale immunoprecipitation in mass spectrometry-based interaction profiling experiments and provide guidelines for evaluating data quality to distinguish true positive protein interactions from nonspecific interactions. This approach is demonstrated here by investigating the nuclear interactome of the CMGC kinase, DYRK1A, a low abundance protein kinase with poorly defined interactions within the nucleus.

Described is a proteomics workflow for identifying protein interaction partners from a nuclear subcellular fraction using immunoaffinity enrichment of a given protein of interest and label-free mass spectrometry. The workflow includes subcellular fractionation, immunoprecipitation, filter aided sample preparation, offline cleanup, mass spectrometry, and a downstream bioinformatics pipeline.

대규모 핵 상호 작용 프로파일링을 위한 라벨 없는 면역 침전 질량 분광 워크플로우

Immunoaffinity purification mass spectrometry (IP-MS) has emerged as a robust quantitative method of identifying protein-protein interactions. This ...

TurboID-Based Proximity Labeling for In Planta Identification of Protein-Protein Interaction Networks

Proximity labeling (PL) techniques using engineered ascorbate peroxidase (APEX) or Escherichia coli biotin ligase BirA (known as BioID) have been successfully used for identification of protein-protein interactions (PPIs) in mammalian cells. However, requirements of toxic hydrogen peroxide (H2O2) in APEX-based PL, longer incubation time with biotin (16&#8211;24 h), and higher incubation temperature (37 &#176;C) in BioID-based PL severely limit their applications in plants. The recently described TurboID-based PL addresses many limitations of BioID and APEX. TurboID allows rapid proximity labeling of proteins in just 10&#8201;min under room temperature (RT) conditions. Although the utility of TurboID has been demonstrated in animal models, we recently showed that TurboID-based PL performs better in plants compared to BioID for labeling of proteins that are proximal to a protein of interest. Provided here is a step-by-step protocol for the identification of protein interaction partners using the N-terminal Toll/interleukin-1 receptor (TIR) domain of the nucleotide-binding leucine-rich repeat (NLR) protein family as a model. The method describes vector construction, agroinfiltration of protein expression constructs, biotin treatment, protein extraction and desalting, quantification, and enrichment of the biotinylated proteins by affinity purification. The protocol described here can be easily adapted to study other proteins of interest in Nicotiana and other plant species.

Described here is a proximity labeling method for identification of interaction partners of the TIR domain of the NLR immune receptor in Nicotiana benthamiana leaf tissue. Also provided is a detailed protocol for the identification of interactions between other proteins of interest using this technique in Nicotiana and other plant species.

단백질-단백질 상호 작용 네트워크의 질라 식별을 위한 터보ID 기반 근접 라벨링

Proximity labeling (PL) techniques using engineered ascorbate peroxidase (APEX) or Escherichia coli biotin ligase BirA (known as BioID) have been ...

면역학 및 감염병학

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 ...

생물학

Identification of Protein Interaction Partners in Mammalian Cells Using SILAC-immunoprecipitation Quantitative Proteomics

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants. Low affinity interactions can be preserved through the use of less-stringent buffer conditions and remain readily identifiable. This protocol discusses the labeling of tissue culture cells with stable isotope labeled amino acids, transfection and immunoprecipitation of an affinity tagged protein of interest, followed by the preparation for submission to a mass spectrometry facility. This protocol then discusses how to analyze and interpret the data returned from the mass spectrometer in order to identify cellular partners interacting with a protein of interest. As an example this technique is applied to identify proteins binding to the eukaryotic translation initiation factors: eIF4AI and eIF4AII.

SILAC immunoprecipitation experiments represent a powerful means for discovering novel protein:protein interactions. By allowing the accurate relative quantification of protein abundance in both control and test samples, true interactions may be easily distinguished from experimental contaminants, and low affinity interactions preserved through use of less-stringent buffer conditions.

SILAC - 면역 정량 프로테오믹스를 사용하여 포유 동물 세포에있는 단백질 상호 작용 파트너의 식별

Quantitative proteomics combined with immuno-affinity purification, SILAC immunoprecipitation, represent a powerful means for the discovery of novel ...

을 바탕으로 단백질 - 단백질 상호 작용의 확인에 프로토콜

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