우리는 대량 spectrometric 지원 UAMS의 Proteomics 시설 감사드립니다. 이 프로젝트에 대한 자금 지원은 NIH 보조금 P20RR015569, P20RR016460 및 R01DA025755에 의해 제공되었다.

이 프로토콜은 블레어 외에서 수정됩니다. 6. 1. LSD1 Demethylation 분석 <ol><li> 20 μL의 최종 볼륨에서 demethylase 버퍼에서 0.25 μg 디 - 메틸 히스톤 H3 펩타이드 (ARTKme2QTARKSTGGKAPRKQLYK-비오틴) (50 MM 트리스-CL 산도 8.5, 50 MM KCl, 5 밀리미터 MgCl 2, 5 %의 125 NG 재조합 LSD1을 결합 글리세롤). 또한, 아니 효소와 펩티드 혼자 컨트롤을 수행합니다. 제어 섹션 3이며 reductive methylation이 필요하지 않습니다. </li><li> 37에서 2 시간 동안 품어 ° C. </li><li> 펩티드를 수집하려면 각 샘플 ~ 8 μL POROS의 R2 20 마이크론 구슬을 추가하고 실온에서 15 분 동안 선동. 메탄올 1 볼륨으로 구슬 1 볼륨을 정지하여 POROS 비즈를 준비하고 5퍼센트 개미의 산성 / 0.2 % TFA 10 볼륨을 추가합니다. </li><li> 2 급 끝에 0.1 %의 TFA 20 μL를 aspirating하고 pipettor들과 다시 한번 밀어 C 18 ZipTips. 0.1 %의 T로 두번이 작업을 수행FA, 넷 70 % acetonitrile과 회 / 0.1 %의 TFA, 그리고 0.1 %의 TFA로 다시 네 번. </li><li> ZipTips에서 C 18 수지 뒤에 비드 슬러리를 pipetting과 pipettor로를 통해 볼륨을 밀어 에어컨 C 18 ZipTips에 두 개의 샘플을 (제어 및 demethylase 반응)로드합니다. </li><li> 0.1 %의 TFA 20 μL으로 두 번 ZipTips 씻으십시오. </li><li> 70 % acetonitrile / 0.1 %의 TFA의 40 μL에 Elute. </li><li> Speed​​Vac 농축기로 완전히 각 eluant을 Lyophilize. </li></ol> 2. Reductive Methylation 프로토콜의이 부분은 ​​블레어 외에서 수정됩니다. 및 Rayment 외. 6,7. <ol><li> 50 밀리미터 인산 버퍼, 산도 7.4의 100 μL의 demethylase 반응을 다시 일시 중지합니다. </li><li> demethylase 반응으로, 15 밀리그램 / ML borane dimethylamine 8 μL를 추가합니다. Borane dimethylamine은 15 밀리그램 / ML 재고 솔루션을 제공하기 위해, 50 MM 인산 버퍼, 산도 7.4에 녹아있다.이것은 신선한하여야한다. </li><li> demethylase 반응으로, 250 MM 진정제를 맞았을 포름 알데히드의 16 μL를 추가합니다. 250 MM 진정제를 맞았을 포름 알데히드는 H 2 0에서 주식을 diluting에 의해 준비가되어 있습니다. 이것은 신선한하여야한다. </li><li> 12 시간 동안 4 ° C에서 품어. </li><li> 단계 2.2, 2.3 및 2.4 반복합니다. </li><li> 단계 2.2를 반복합니다. </li><li> 4에서 품어 ° C ~ 15 시간. </li><li> demethylase 샘플로 1M 트리스, reductive methylation 반응을 끄지하는 산도 7.4의 12.5 μL를 추가합니다. </li></ol> 3. 든 질량 분광법 <ol><li> demethylase 반응에 POROS R2 20 마이크론 구슬을 추가하고 단계 1.3-1.6에 설명된대로 ZipTips를 수집합니다. </li></ol> 참고 : 50 MM 인산 버퍼, 산도 7.4의 100 μL에 컨트롤을 다시 중지되고 다른 샘플 단계 3.1으로 시작하는으로 취급합니다. <ol start="2"><li> 33%가 포화 2 μL를 사용 든 샘플 플레이트에 ZipTips에서 제어 및 demethylase 반응 샘플을 EluteD 2,5 - dihydroxybenzoic 산. 33%에게 2,5-dihydroxybenzoic 산성을 준비하려면 70 % acetonitrile / 0.1 % TFA에 포화 33%로 희석 후 70 % acetonitrile / 0.1 % TFA에 2,5-dihydroxybenzoic 산성의 포화 용액을 만들고. eluted 샘플 건조하고 구체화하도록 허용합니다. </li><li> PerkinElmerSciex 든-prOTOF 질량 분석기 또는 가능한 고해상도 든 질량 분석계 8,9,10을 사용하여 질량 스펙트럼을 수집합니다. </li><li> 스펙트럼보기 및 질량 분광계 제조 업체에서 제공 PerkinElmerSciex TOFworks 소프트웨어 또는 소프트웨어를 사용하여 피크 영역을 추출합니다. </li><li> 탠덤 질량 spectrometric 기능을 제공하는 사용 가능한 대량 spectrometric 시스템과 석사 2로 반응 제품을 확인합니다. </li></ol> 4. 데이터 분석 <ol><li> 무거운 포름 알데히드 (그림 1B)를 사용하여 만든 isotopic 중복 보상하기 위해, 피크 면적 13 C 2와 13 C 4 동위 원소 relativ의 (A)의 비율을 결정reductive methylation (그림 2A)를 겪은하지 않은 컨트롤 샘플에 대한 monoisotopic 최대 5. EQ 1 : 13C2 / 12C = R 1 EQ 2 : 13C4 / 12C = R 2 이렇게하면 실험 및 질량 분석계에 특정한 이들 isotopic 상태에서 기존 펩타이드의 비율 (R 1 &amp; R 2)를 제공할 것입니다. </li><li> demethylase 반응 시료의 각 변형 상태 (그림 2B)에 기존 펩타이드의 상대적 양을 quantifying에 대해 다음 수식에서 R 1 &amp; R이 값을 사용 EQ 3 : H3K4me2 = 1 EQ 4 : H3K4me = 2 - R 1 (1) EQ 5 : H3K4 = 3 - R 2 (1) - [R 1 (2 - R 1 (1))] </li></ol> 5. 대표 결과 LSD1를 사용하여, 우리는 quantifying 위해 MassSQUIRM의 효과를 보여라이신 demethylation. 와 같은 프로토콜 텍스트 섹션에서 설명한, 우리는 LSD1 125 NG와 0.25 μg 디 - 메틸 히스톤 H3 펩타이드 (ARTKme2QTARKSTGGKAPRKQLYK-비오틴)와 demethylase 분석을 수행. 제어 및 demethylase 반응 표본 reductive methylation 및 든 질량 분광법를 받게되었다. reductive methylation을 받다 없었 제어 반응이 펩타이드에 대한 전형적인 isotopic 봉투를 보여주와 방정식 1과 2 (그림 2A)에 피크 영역을 제공했습니다. demethylase 반응에 대한 질량 스펙트럼은 방정식 3-5 (그림 2B)의 피크 영역을 제공했습니다. 제어 및 demethylase 반응의 피크 영역을 사용하여, 우리는 LSD1는 다음과 같은 반응 제품을주는 펩타이드를 demethylated 것을 결정 : 33.7 % 디 - 메틸 Lys4, 42.3 % 모노 메틸 Lys4 및 24% 해제 methylated LYS 4. 블레어 외 참조하십시오. 전체 LSD1 demethylase 활동의 분석뿐만 아니라 억제제 연구 6. 참고 이러한 반응 시간, 효소 등 변화하는 변수농도와 기판 농도는 활동에 대한 심층 분석에있게됩니다. <img alt="그림 1" src="/files/ftp_upload/3604/3604fig1.jpg" /> 그림 1. MassSQUIRM 개요. () 히스톤 H3의 N-터미널 펩타이드가되는대로 표시됩니다 취소 (녹색), 모노 (빨간색) 또는 디 - (파란색) 라이신 4에 methylated. 각 펩타이드의 화학적 구성의 변화는 부량 복합 만들기 차등 이온화로 안내합니다. (B) Reductive methylation은 모든 펩티드가 비슷하게 이온화로 인해 디 - 메틸 상태에 대한 모든 리진 잔류물을 변환합니다. reductive methylation 반응에 무거운 포름 알데히드의 사용은 원래 methylation의 정체성 유지 수 있습니다. 닫힌 서클이 심한 methylation을 나타내는 반면 오픈 서클 밝은 methylation을 나타냅니다. 블레어 외. 2,011 6에서 수정하였습니다. <img alt="그림 2" src="/files/ftp_upload/3604/3604fig2.jpg" /> FigurE 2. MassSQUIRM는 differentially methylated 펩티드를 계량하는 데 사용할 수 있습니다. () 디 - methylated 합성 히스톤 H3 펩타이드 (ARTKme2QTARKSTGGKAPRKQLYK-비오틴)은 R 1 및 방정식 1과 2에서 R 2로 언급했다 질량 분광법과 monoisotopic에서 최대 상대적인 최대 비율을 사용하여 분석되었다. (B) 같은 합성 펩타이드는 37 ° C.에 두 시간 동안이나 demethylase 버퍼에 125 NG의 LSD1 함께 incubated되었다 샘플은 다음 분석을 MassSQUIRM를 받게되었다. 봉우리 중복의 혼합 인구는 그림 1B에서 본 세 가지 다른 methylation 상태를 나타냅니다. monoisotopic 봉우리 아래의 영역은 1, 2 및 3으로 언급되었다. 피크 영역은 방정식 3-5에 사용되었다. 닫힌 서클이 심한 methylation을 나타내는 반면 오픈 서클 밝은 methylation을 나타냅니다. 블레어 외에서 수정하였습니다. 2011 6.

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우리는 공개 할게 없다.

MassSQUIRM은 모노와 DI-methylation에 관여 라이신 demethylases의 활동의 종합적인 분석을위한 저렴하고 양적 방법입니다. MassSQUIRM는 반응의 제품뿐만 아니라 중간체를위한뿐만 아니라 quantitation를 제공합니다. 이 분석은 LSD1 및 기타 히스톤 demethylases의 메커니즘을 연구의 강력한 도구로 사용될 수 있습니다. 그것은 또한 PHF8 많은 새로 발견된 라이신 demethylase 효소를 분류하고 특정 methyltransferase 효소에 사...

<table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td></tr><tr><td> LSD1 </td><td> BPS Biosciences </td><td> 50,100 </td></tr><tr><td> H3K4me2-비오틴 펩타이드 </td><td> 자체 준비 </td><td> 없음 </td></tr><tr><td> POROS R2 20 마이크론 구슬 </td><td> 응용 Biosystems </td><td> 1-1129-06 </td></tr><tr><td> C 18 ZipTip </td><td> Millipore </td><td> ZTC18M </td></tr><tr><td> Trifluoroacetic 산성 (TFA) </td><td> 써모 </td><td> 28,904 </td></tr><tr><td> acetonitrile </td><td> 어부 </td><td> A996 </td></tr><tr><td> 2,5-dihydroxybenzoic 산 </td><td> 시그마 </td><td> 85,707 </td></tr><tr><td> Borane dimethylamine </td><td> 시그마 </td><td> 180,238 </td></tr><tr><td> isotopically 무거운 D 2 - 포름 알데히드 </td><td> 캠브리지 동위 원소 연구소 </tD&gt; <td> DLM - 805-20 </td></tr><tr><td> 트리스 </td><td> 어부 </td><td> BP154 </td></tr><tr><td> KCl </td><td> 어부 </td><td> BP366 </td></tr><tr><td> MgCl 2 </td><td> 어부 </td><td> BP214 </td></tr><tr><td> 글리세린 </td><td> 어부 </td><td> G33 </td></tr><tr><td> 개미의 산 </td><td> Fluka </td><td> 06,440 </td></tr><tr><td> 메탄올 </td><td> 어부 </td><td> A452 </td></tr><tr><td> 나 인산 </td><td> 어부 </td><td> BP329 </td></tr><tr><td> Speed​​Vac 농축기 </td><td> 학자 </td><td> DNA110 </td></tr><tr><td> 든 - prOTOF 질량 분석계 및 TOFworks 소프트웨어 </td><td> PerkinElmerSciex </td><td> 없음 </td></tr></tbody></table>

application of masssquirm for quantitative measurements of lysine demethylase activity

최근 epigenetic 규제는 1-3 여러 가지 질병의 주요 선수로 발견되었습니다. 결과적으로 이러한 효소는 작은 분자 연구 및 약물 개발 4 주요 목표입니다. 많은 epigenetic 규제는 최근에 발견하고 분류하는 과정이 아직 없습니다. 이들 효소 중 histones 및 다른 단백질에 lysines에서 메틸 그룹을 제거 라이신 demethylases 있습니다. 효소의이 클래스의 소설 특성으로 인해 몇 assays들은 활동을 공부하기 위해 개발되었습니다. 이 분류와 히스톤 demethylases의 높은 처리량 연구 양쪽에 도로를 차단했습니다. 현재 거의 demethylase의 assays가 존재합니다. 존재하지 그것들은 자연에서 질적 경향 동시에 다른 라이신 methylation 상태 사이에 분별없는 수 있습니다 (취소, 모노 -, 디 - 및 트라이 -). 질량 분석법은 일반적으로 demethylase 활동을 결정하는 데 사용하지만 현재 대량 spectrometric assays 할있다differentially methylated 펩티드 다르게 이온화 여부를 해결할 수 없습니다. methylated 펩티드의 차동 이온화는 methylation 상태가 어렵고 (그림 1A) 확실히 양적되지 비교 있습니다. 따라서 가능한 assays는 demethylase 활동의 포괄적인 분석을 위해 최적화되지 않습니다. 여기 따라서 본질적으로 동일한 화학 종족들을 만들고, 모든 lysines는 디 - methylated로 강제로 진정제를 맞았을 포름 알데히드와 아민 그룹의 reductive methylation을 기반으로 MassSQUIRM라는 방법을 (isotopic reductive methylation을 사용하여 질량 spectrometric quantitation) 설명하고 있으므로이 이온화 동일한 (그림 1B). reductive methylation에 따라 유일한 화학 변화는 수소와 든 이온화 효율에 영향을주지 않습니다 중수소입니다. MassSQUIRM 분석이 해제, 모노 또는 디 - methylated lysines와 demethylase 반응 제품에 대한 구체적인 것입니다. 분석은 또한 SA주는 라이신 methyltransferases에 적용나에게 반응 제품보기. 여기서는 reductive methylation 화학 및 든 질량 분광법 LSD1의 활동을 측정, 합성 펩티드 기질 5, 디 - 및 모노 - 메틸 그룹을 제거하는 능력이 라이신 demethylase.의 조합을 사용 이 분석은 간단하고 실험실이나 proteomics 시설을 통해 든 질량 분석기에 대한 액세스 권한이있는 모든 실험실로 쉽게받을 수있는 것입니다. 검정은 ~ 8 배 다이나믹 레인지를 가지고 있으며, 플레이트 형식 5 ~ 쉽게 확장 가능합니다.

Watch this Scientific Journal Video about Application of MassSQUIRM for Quantitative Measurements of Lysine Demethylase Activity at JoVE.com

Application of MassSQUIRM for Quantitative Measurements of Lysine Demethylase Activity

우리는 라이신 methylation의 변화를 계량하는 데 든 질량 분광법과 reductive methylation 화학을 사용하는 방법을 제시한다.

라이신 Demethylase 활동의 양적 측정을위한 MassSQUIRM의 응용

Recently, epigenetic regulators have been discovered as key players in many different diseases 1-3. As a result, these enzymes are prime targets for small ...

application-masssquirm-for-quantitative-measurements-lysine

Universidad de Cartagena UDEC

Research

JoVE Journal

Biology

13.3K 조회수.  University of Arkansas for Medical Sciences. 우리는 라이신 methylation의 변화를 계량하는 데 든 질량 분광법과 reductive methylation 화학을 사용하는 방법을 제시한다.

비디오: Application of MassSQUIRM for Quantitative Measurements of Lysine Demethylase Activity

An Efficient Method for the Synthesis of Peptoids with Mixed Lysine-type/Arginine-type Monomers and Evaluation of Their Anti-leishmanial Activity

This protocol describes the manual solid-phase synthesis of linear peptoids that contain two differently functionalized cationic monomers. In this procedure amino functionalized 'lysine' and guanido functionalized 'arginine' peptoid monomers can be included within the same peptoid sequence. This procedure uses on-resin (N-(1-(4,4-dimethyl-2,6-dioxocyclohexylidene)ethyl) or Dde protection, orthogonal conditions to the Boc protection of lysine monomers. Subsequent deprotection allows an efficient on-resin guanidinylation reaction to form the arginine residues. The procedure is compatible with the commonly used submonomer method of peptoid synthesis, allowing simple peptoids to be made using common laboratory equipment and commercially available reagents. The representative synthesis, purification and characterization of two mixed peptoids is described. The evaluation of these compounds as potential anti-infectives in screening assays against Leishmania mexicana is also described. The protozoan parasite L. mexicana is a causative agent of cutaneous leishmaniasis, a neglected tropical disease that affects up to 12 million people worldwide.

A protocol to synthesize peptoids with mixed cationic functionality in the same sequence is presented (lysine- and arginine-type monomers). Subsequent testing of these compounds against Leishmania mexicana, the protozoan parasites that cause cutaneous leishmaniasis, is also described.

이들 안티 leishmanial 역가의 혼합 리신 형 / 아르기닌 형 단량체 및 평가를 펩 토이 드의 합성을위한 효과적인 방법

This protocol describes the manual solid-phase synthesis of linear peptoids that contain two differently functionalized cationic monomers. In this ...

연구

생화학

Methods to Identify the NMR Resonances of the 13C-Dimethyl N-terminal Amine on Reductively Methylated Proteins

Nuclear magnetic resonance (NMR) spectroscopy is a proven technique for protein structure and dynamic studies. To study proteins with NMR, stable magnetic isotopes are typically incorporated metabolically to improve the sensitivity and allow for sequential resonance assignment. Reductive 13C-methylation is an alternative labeling method for proteins that are not amenable to bacterial host over-expression, the most common method of isotope incorporation. Reductive 13C-methylation is a chemical reaction performed under mild conditions that modifies a protein&#39;s primary amino groups (lysine &#949;-amino groups and the N-terminal &#945;-amino group) to 13C-dimethylamino groups. The structure and function of most proteins are not altered by the modification, making it a viable alternative to metabolic labeling. Because reductive 13C-methylation adds sparse, isotopic labels, traditional methods of assigning the NMR signals are not applicable. An alternative assignment method using mass spectrometry (MS) to aid in the assignment of protein 13C-dimethylamine NMR signals has been developed. The method relies on partial and different amounts of 13C-labeling at each primary amino group. One limitation of the method arises when the protein&#39;s N-terminal residue is a lysine because the &#945;- and &#949;-dimethylamino groups of Lys1 cannot be individually measured with MS. To circumvent this limitation, two methods are described to identify the NMR resonance of the 13C-dimethylamines associated with both the N-terminal &#945;-amine and the side chain &#949;-amine. The NMR signals of the N-terminal &#945;-dimethylamine and the side chain &#949;-dimethylamine of hen egg white lysozyme, Lys1, are identified in 1H-13C heteronuclear single-quantum coherence spectra.

Methods to Identify the NMR Resonances of the 13C-Dimethyl N-terminal Amine on Reductively Methylated Proteins

Two methods for assigning the &#945;- and &#949;-dimethylamine nuclear magnetic resonance signals of a reductively 13C-methylated N-terminal lysine are described. One method utilizes the pH-induced selectivity of the reductive methylation reaction, and the other uses aminopeptidase to selectively remove the N-terminal lysine.

의 NMR 공명 확인하는 방법<sup&gt; 13</sup환원 메틸을 단백질에&gt; C-디메틸 N-말단 아민

Nuclear magnetic resonance (NMR) spectroscopy is a proven technique for protein structure and dynamic studies. To study proteins with NMR, stable magnetic ...

화학

Direct Measurement of KDM1A Target Engagement Using Chemoprobe-based Immunoassays

The assessment of the target engagement, defined as the interaction of a drug with the protein it was designed for, is a basic requirement for the interpretation of the biological activity of any compound in drug development or in basic research projects. In epigenetics, target engagement is most often assessed by the analysis of proxy markers instead of measuring the union of the compound to the target. Downstream biological readouts that have been analyzed include the histone mark modulation or gene expression changes. KDM1A is a lysine demethylase that removes methyl groups from mono- and dimethylated H3K4, a modification associated with the silencing of gene expression. Modulation of the proxy markers is dependent on the cell type and function of the genetic make-up of the cells investigated, which can make interpretation and cross-case comparison quite difficult. To circumvent these problems, a versatile protocol is presented to assess the dose effects and dynamics of the direct KDM1A target engagement. The assay described makes use of a KDM1A chemoprobe to capture and quantify uninhibited enzyme, can be broadly applied to cells or tissue samples without the need for genetic modification, has an excellent window of detection, and can be used both for basic research and analysis of clinical samples.

Here, we present a protocol to measure KDM1A target engagement in a human or animal cell, tissue or blood samples treated with KDM1A inhibitors. The protocol employs chemoprobe tagging of the free KDM1A enzyme and direct quantification of the target occupation using chemoprobe-based immunoassays and can be used in preclinical and clinical studies.

화학 프로브 기반 면역 분석제사용 KDM1A 표적 결합의 직접 측정

The assessment of the target engagement, defined as the interaction of a drug with the protein it was designed for, is a basic requirement for the ...

Exploring the Arginine Methylome by Nuclear Magnetic Resonance Spectroscopy

Protein-bound arginine is commonly methylated in many proteins and regulates their function by altering the physicochemical properties, their interaction with other molecules, including other proteins or nucleic acids. This work presents an easily implementable protocol for quantifying arginine and its derivatives, including asymmetric and symmetric dimethylarginine (ADMA and SDMA, respectively) and monomethyl arginine (MMA). Following protein isolation from biological body fluids, tissues, or cell lysates, a simple method for homogenization, precipitation of proteins, and protein hydrolysis is described. Since the hydrolysates contain many other components, such as other amino acids, lipids, and nucleic acids, a purification step using solid-phase extraction (SPE) is essential. SPE can either be performed manually using centrifuges or a pipetting robot. The sensitivity for ADMA using the current protocol is about 100 nmol/L. The upper limit of detection for arginine is 3 mmol/L due to SPE saturation. In summary, this protocol describes a robust method, which spans from biological sample preparation to NMR-based detection, providing valuable hints and pitfalls for successful work when studying the arginine methylome.

The present protocol describes the preparation and quantitative measurement of free and protein-bound arginine and methyl-arginines by 1H-NMR spectroscopy.

핵자기공명분광법에 의한 아르기닌 메틸롬 탐구

Protein-bound arginine is commonly methylated in many proteins and regulates their function by altering the physicochemical properties, their interaction ...

암 연구학

A Mass Spectrometry-Based Proteomics Approach for Global and High-Confidence Protein R-Methylation Analysis

Protein Arginine (R)-methylation is a widespread protein post-translational modification (PTM) involved in the regulation of several cellular pathways, including RNA processing, signal transduction, DNA damage response, miRNA biogenesis, and translation.
In recent years, thanks to biochemical and analytical developments, mass spectrometry (MS)-based proteomics has emerged as the most effective strategy to characterize the cellular methyl-proteome with single-site resolution. However, identifying and profiling in vivo protein R-methylation by MS remains challenging and error-prone, mainly due to the substoichiometric nature of this modification and the presence of various amino acid substitutions and chemical methyl-esterification of acidic residues that are isobaric to methylation. Thus, enrichment methods to enhance the identification of R-methyl-peptides and orthogonal validation strategies to reduce False Discovery Rates (FDR) in methyl-proteomics studies are required.
Here, a protocol specifically designed for high-confidence R-methyl-peptides identification and quantitation from cellular samples is described, which couples metabolic labeling of cells with heavy isotope-encoded Methionine (hmSILAC) and dual protease in-solution digestion of whole cell extract, followed by off-line High-pH Reversed Phase (HpH-RP) chromatography fractionation and affinity enrichment of R-methyl-peptides using anti-pan-R-methyl antibodies. Upon high-resolution MS analysis, raw data are first processed with the MaxQuant software package and the results are then analyzed by hmSEEKER, a software designed for the in-depth search of MS peak pairs corresponding to light and heavy methyl-peptide within the MaxQuant output files.

Protein Arginine (R)-methylation is a wide-spread post-translational modification regulating multiple biological pathways. Mass spectrometry is the best technology to globally profile the R-methyl-proteome, when coupled to biochemical approaches for modified peptide enrichment. The workflow designed for the high confidence identification of global R-methylation in human cells is described here.

글로벌 및 신뢰도가 높은 단백질 R-메틸화 분석을 위한 질량분석법 기반 단백질체학 접근법

Protein Arginine (R)-methylation is a widespread protein post-translational modification (PTM) involved in the regulation of several cellular pathways, ...

Qualitative and Quantitative Assays for Detection and Characterization of Protein Antimicrobials

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods to screen for target enzymes prior to investing greater research effort and resources. The goal of this protocol is to demonstrate two complementary assays for conducting these initial evaluations. The microslide diffusion assay provides an initial or simple detection screen to enable the qualitative and rapid assessment of proteolytic activity against an array of both viable and heat-killed bacterial target substrates. As a counterpart, the increased sensitivity and reproducibility of the dye-release assay provides a quantitative platform for evaluating and comparing environmental influences affecting the hydrolytic activity of protein antimicrobials. The ability to label specific heat-killed cell culture substrates with Remazol brilliant blue R dye expands this capability to tailor the dye-release assay to characterize enzymatic activity of interest.

Here, we present protocols to rapidly screen and characterize enzymes for antimicrobial activity. The microslide diffusion assay and the dye-release assay utilize target bacterial substrates for qualitative and quantitative enzymatic activity evaluation.

정성 및 정량 검출을위한 시험 법 및 단백질 항생제 특성

Initial evaluations of large microbial libraries for potential producers of novel antimicrobial proteins require both qualitative and quantitative methods ...

면역학 및 감염병학

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme acid stresses including passage through the mammalian stomach where the pH can fall to as low as pH 1 - 22. To enable such a broad range of acidic pH survival, E. coli possesses four different inducible amino acid decarboxylases that decarboxylate their substrate amino acids in a proton-dependent manner thus raising the internal pH. The decarboxylases include the glutamic acid decarboxylases GadA and GadB3, the arginine decarboxylase AdiA4, the lysine decarboxylase LdcI5, 6 and the ornithine decarboxylase SpeF7. All of these enzymes utilize pyridoxal-5'-phospate as a co-factor8 and function together with inner-membrane substrate-product antiporters that remove decarboxylation products to the external medium in exchange for fresh substrate2. In the case of LdcI, the lysine-cadaverine antiporter is called CadB. Recently, we determined the X-ray crystal structure of LdcI to 2.0 &#197;, and we discovered a novel small-molecule bound to LdcI the stringent response regulator guanosine 5'-diphosphate,3'-diphosphate (ppGpp) 14. The stringent response occurs when exponentially growing cells experience nutrient deprivation or one of a number of other stresses9. As a result, cells produce ppGpp which leads to a signaling cascade culminating in the shift from exponential growth to stationary phase growth10. We have demonstrated that ppGpp is a specific inhibitor of LdcI 14. Here we describe the lysine decarboxylase assay, modified from the assay developed by Phan et al.11, that we have used to determine the activity of LdcI and the effect of pppGpp/ppGpp on that activity. The LdcI decarboxylation reaction removes the &alpha;-carboxy group of L-lysine and produces carbon dioxide and the polyamine cadaverine (1,5-diaminopentane)5. L-lysine and cadaverine can be reacted with 2,4,6-trinitrobenzensulfonic acid (TNBS) at high pH to generate N,N'-bistrinitrophenylcadaverine (TNP-cadaverine) and N,N&#8242;-bistrinitrophenyllysine (TNP-lysine), respectively11. The TNP-cadaverine can be separated from the TNP-lysine as the former is soluble in organic solvents such as toluene while the latter is not (See Figure 1). The linear range of the assay was determined empirically using purified cadaverine.

An Assay for Measuring the Activity of Escherichia coli Inducible Lysine Decarboxyase

The activity of the inducible lysine decarboxylase is monitored by reacting the substrate L-lysine and the product cadaverine with 2,4,6-trinitrobenzensulfonic acid to form adducts that have differential solubility in toluene.

의 활동을 측정하는 어세이 대장균은 라이신 Decarboxyase을 Inducible

Escherichia coli is an enteric bacterium that is capable of growing over a wide range of pH values (pH 5 - 9)1 and, incredibly, is able to survive extreme ...

생물학

Limosilactobacillus reuteri DSM20016의 리포터 단백질 측정

Methods for Electroporation and Transformation Confirmation in Limosilactobacillus reuteri DSM20016

Lactobacillus were an incredibly large, diverse genus of bacteria comprising 261 species, several of which were commensal strains with the potential for use as a chassis for synthetic biological endeavors within the gastrointestinal tract. The wide phenotypic and genotypic variation observed within the genus led to a recent reclassification and the introduction of 23 novel genera.
Due to the breadth of variations within the old genera, protocols demonstrated in one member may not work as advertised with other members. A lack of centralized information on how exactly to manipulate specific strains has led to a range of ad hoc approaches, often adapted from other bacterial families. This can complicate matters for researchers starting in the field, who may not know which information does or does not apply to their chosen strain.
In this paper, we aim to centralize a set of protocols with demonstrated success, specifically in&#160;the Limosilactobacillus reuteri strain designation F275 (other collection numbers: DSM20016, ATCC23272, CIP109823), along with troubleshooting advice and common issues one may encounter. These protocols should enable a researcher with little to no experience working with L. reuteri DSM20016 to transform a plasmid, confirm transformation, and measure system feedback in a plate reader via a reporter protein.

저자 스포트라이트: Methods for Electroporation and Transformation Confirmation in Limosilactobacillus reuteri DSM20016  

Here, we present protocols for working with Limosilactobacillus reuteri DSM20016, detailing growth, plasmid transformation, colony PCR, fluorescent reporter protein measurement, and limited plasmid mini-prep, as well as common issues and troubleshooting. These protocols allow the measurement of reporter proteins in DSM20016, or confirmation via colony PCR if no reporter is involved.

Limosilactobacillus reuteri DSM20016의 전기천공 및 변형 확인 방법

Lactobacillus were an incredibly large, diverse genus of bacteria comprising 261 species, several of which were commensal strains with the potential for ...

라이신 Demethylase 활동의 양적 측정을위한 MassSQUIRM의 응용

요약

더 많은 비디오 탐색

이 비디오의 챕터

이들 안티 leishmanial 역가의 혼합 리신 형 / 아르기닌 형 단량체 및 평가를 펩 토이 드의 합성을위한 효과적인 방법

의 NMR 공명 확인하는 방법

화학 프로브 기반 면역 분석제사용 KDM1A 표적 결합의 직접 측정

핵자기공명분광법에 의한 아르기닌 메틸롬 탐구

글로벌 및 신뢰도가 높은 단백질 R-메틸화 분석을 위한 질량분석법 기반 단백질체학 접근법

정성 및 정량 검출을위한 시험 법 및 단백질 항생제 특성

의 활동을 측정하는 어세이 대장균은 라이신 Decarboxyase을 Inducible

Limosilactobacillus reuteri DSM20016의 전기천공 및 변형 확인 방법

Limosilactobacillus reuteri DSM20016의 전기천공 및 변형 확인 방법

Limosilactobacillus reuteri DSM20016의 전기천공 및 변형 확인 방법