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

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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 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 ...

연구

생물학

Quantitative, Real-time Analysis of Base Excision Repair Activity in Cell Lysates Utilizing Lesion-specific Molecular Beacons

We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates using base excision repair (BER) molecular beacons. The substrate (beacon) is comprised of a deoxyoligonucleotide containing a single base lesion with a 6-Carboxyfluorescein (6-FAM) moiety conjugated to the 5'end and a Dabcyl moiety conjugated to the 3' end of the oligonucleotide. The BER molecular beacon is 43 bases in length and the sequence is designed to promote the formation of a stem-loop structure with 13 nucleotides in the loop and 15 base pairs in the stem1,2. When folded in this configuration the 6-FAM moiety is quenched by Dabcyl in a non-fluorescent manner via F&ouml;rster Resonance Energy Transfer (FRET)3,4. The lesion is positioned such that following base lesion removal and strand scission the remaining 5 base oligonucleotide containing the 6-FAM moiety is released from the stem. Release and detachment from the quencher (Dabcyl) results in an increase of fluorescence that is proportionate to the level of DNA repair. By collecting multiple reads of the fluorescence values, real-time assessment of BER activity is possible. The use of standard quantitative real-time PCR instruments allows the simultaneous analysis of numerous samples. The design of these BER molecular beacons, with a single base lesion, is amenable to kinetic analyses, BER quantification and inhibitor validation and is adaptable for quantification of DNA Repair activity in tissue and tumor cell lysates or with purified proteins. The analysis of BER activity in tumor lysates or tissue aspirates using these molecular beacons may be applicable to functional biomarker measurements. Further, the analysis of BER activity with purified proteins using this quantitative assay provides a rapid, high-throughput method for the discovery and validation of BER inhibitors.

We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates. The assay yields rates of DNA Repair activity amenable to kinetic analysis and is adaptable for quantification of DNA Repair activity in tissue and tumor lysates or with purified proteins.

병변 특정 분자 비콘을 활용 세포 Lysates의 기본 절단 복구 활동의 양적, 실시간 분석

We describe a method for the quantitative, real-time measurement of DNA glycosylase and AP endonuclease activities in cell nuclear lysates using base ...

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for using FISH to quantify the number of mRNAs in single yeast cells. Cells can be grown in any condition of interest and then fixed and made permeable. Subsequently, multiple single-stranded deoxyoligonucleotides conjugated to fluorescent dyes are used to label and visualize mRNAs. Diffraction-limited fluorescence from single mRNA molecules is quantified using a spot-detection algorithm to identify and count the number of mRNAs per cell. While the more standard quantification methods of northern blots, RT-PCR and gene expression microarrays provide information on average mRNAs in the bulk population, FISH facilitates both the counting and localization of these mRNAs in single cells at single-molecule resolution.

Visualization and Analysis of mRNA Molecules Using Fluorescence In Situ Hybridization in Saccharomyces cerevisiae

This protocol describes an experimental procedure for performing Fluorescence in situ Hybridization (FISH) for counting mRNAs in single cells at single-molecule resolution.

형광을 사용하여 mRNA의 분자의 시각화 및 분석<em&gt; 제자리에서</em에서&gt; 하이브리드<em&gt; 사카 cerevisiae의</em

The Fluorescence in situ Hybridization (FISH) method allows one to detect nucleic acids in the native cellular environment. Here we provide a protocol for ...

Cytosolic Calcium Measurements in Renal Epithelial Cells by Flow Cytometry

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial function, cell death, cell metabolism and cell migration to name but a few. Cytosolic calcium is normally maintained at low nanomolar concentrations; rather it is found in high micromolar to millimolar concentrations in the endoplasmic reticulum, mitochondrial matrix and the extracellular compartment. Upon stimulation, a transient increase in cytosolic calcium serves to signal downstream events. Detecting changes in cytosolic calcium is normally performed using a live cell imaging set up with calcium binding dyes that exhibit either an increase in fluorescence intensity or a shift in the emission wavelength upon calcium binding. However, a live cell imaging set up is not freely accessible to all researchers. Alternative detection methods have been optimized for immunological cells with flow cytometry and for non-immunological adherent cells with a fluorescence microplate reader. Here, we describe an optimized, simple method for detecting changes in epithelial cells with flow cytometry using a single wavelength calcium binding dye. Adherent renal proximal tubule epithelial cells, which are normally difficult to load with dyes, were loaded with a fluorescent cell permeable calcium binding dye in the presence of probenecid, brought into suspension and calcium signals were monitored before and after addition of thapsigargin, tunicamycin and ionomycin.

Calcium is involved in numerous physiological and pathophysiological signaling pathways. Live cell imaging requires specialized equipment and can be time consuming. A quick, simple method using a flow cytometer to determine relative changes in cytosolic calcium in adherent epithelial cells brought into suspension was optimized.

유동 세포 계측법에 의한 신장 상피 세포에서 세포 내 칼슘 측정

A variety of cellular processes, both physiological and pathophysiological, require or are governed by calcium, including exocytosis, mitochondrial ...

Specificity Analysis of Protein Lysine Methyltransferases Using SPOT Peptide Arrays

Lysine methylation is an emerging post-translation modification and it has been identified on several histone and non-histone proteins, where it plays crucial roles in cell development and many diseases. Approximately 5,000 lysine methylation sites were identified on different proteins, which are set by few dozens of protein lysine methyltransferases. This suggests that each PKMT methylates multiple proteins, however till now only one or two substrates have been identified for several of these enzymes. To approach this problem, we have introduced peptide array based substrate specificity analyses of PKMTs. Peptide arrays are powerful tools to characterize the specificity of PKMTs because methylation of several substrates with different sequences can be tested on one array. We synthesized peptide arrays on cellulose membrane using an Intavis SPOT synthesizer and analyzed the specificity of various PKMTs. Based on the results, for several of these enzymes, novel substrates could be identified. For example, for NSD1 by employing peptide arrays, we showed that it methylates K44 of H4 instead of the reported H4K20 and in addition H1.5K168 is the highly preferred substrate over the previously known H3K36. Hence, peptide arrays are powerful tools to biochemically characterize the PKMTs.

Peptide arrays synthesized by the SPOT method can be used to analyze the substrate specificity of Protein lysine methyltransferases (PKMTs) and to define the substrate spectrum of PKMTs to understand their biological role. This protocol describes how to synthesize peptide arrays, methylate them with PKMTs, and analyze the results.

SPOT 펩타이드 배열을 사용하여 단백질 리신 Methyltransferases의 특이성 분석

Lysine methylation is an emerging post-translation modification and it has been identified on several histone and non-histone proteins, where it plays ...

Functional Reconstitution and Channel Activity Measurements of Purified Wildtype and Mutant CFTR Protein

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of transporters. The phosphorylation and nucleotide dependent chloride channel activity of CFTR has been frequently studied in whole cell systems and as single channels in excised membrane patches. Many Cystic Fibrosis-causing mutations have been shown to alter this activity. While a small number of purification protocols have been published, a fast reconstitution method that retains channel activity and a suitable method for studying population channel activity in a purified system have been lacking. Here rapid methods are described for purification and functional reconstitution of the full-length CFTR protein into proteoliposomes of defined lipid composition that retains activity as a regulated halide channel. This reconstitution method together with a novel flux-based assay of channel activity is a suitable system for studying the population channel properties of wild type CFTR and the disease-causing mutants F508del- and G551D-CFTR. Specifically, the method has utility in studying the direct effects of phosphorylation, nucleotides and small molecules such as potentiators and inhibitors on CFTR channel activity. The methods are also amenable to the study of other membrane channels/transporters for anionic substrates.

Described here is a rapid and effective procedure for functional reconstitution of purified wild-type and mutant CFTR protein that preserves activity for this chloride channel, which is defective in Cystic Fibrosis. Iodide efflux from reconstituted proteoliposomes mediated by CFTR allows studies of channel activity and the effects of small molecules.

정제 된 야생형과 돌연변이 체 CFTR 단백질의 기능의 재구성 및 채널 활동 측정

The Cystic Fibrosis Transmembrane Conductance Regulator (CFTR) is a unique channel-forming member of the ATP Binding Cassette (ABC) superfamily of ...

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and exhibit a pro-inflammatory phenotype. Although the development of microfluidic devices has been embraced by engineers over two decades, their biological applications remain limited. A more physiologically relevant in vitro microvessel model validated by biological applications is important to advance the field and bridge the gaps between in vivo and in vitro studies. Here, we present detailed procedures for the development of cultured microvessel network using a microfluidic device with a long-term perfusion capability. We also demonstrate its applications for quantitative measurements of agonist-induced changes in EC [Ca2+]i and nitric oxide (NO) production in real time using confocal and conventional fluorescence microscopy. The formed microvessel network with continuous perfusion showed well-developed junctions between ECs. VE-cadherin distribution was closer to that observed in intact microvessels than statically cultured EC monolayers. ATP-induced transient increases in EC [Ca2+]i and NO production were quantitatively measured at individual cell levels, which validated the functionality of the cultured microvessels. This microfluidic device allows ECs to grow under a well-controlled, physiologically relevant flow, which makes the cell culture environment closer to in vivo than that in the conventional, static 2D cultures. The microchannel network design is highly versatile, and the fabrication process is simple and repeatable. The device can be easily integrated to the confocal or conventional microscopic system enabling high resolution imaging. Most importantly, because the cultured microvessel network can be formed by primary human ECs, this approach will serve as a useful tool to investigate how pathologically altered blood components from patient samples affect human ECs and provide insight into clinical issues. It also can be developed as a platform for drug screening.

Development and Characterization of In Vitro Microvessel Network and Quantitative Measurements of Endothelial [Ca2+]i and Nitric Oxide Production

Primary human umbilical vein endothelial cells (HUVECs) were grown to confluence within a microfluidic network device. The endothelial cell junction and F-actin distributions were illustrated and the changes in intracellular calcium concentration and nitric oxide production in response to adenosine triphosphate (ATP) were quantified in real-time at individual cell levels.

개발 및 특성화의<em&gt; 체외</em&gt; 미세 혈관 네트워크 및 내피의 정량적 측정 [칼슘<sup&gt; 2 +</sup&gt;]<sub&gt; 전</sub&gt; 및 질소 산화물 생산

Endothelial cells (ECs) lining the blood vessel walls in vivo are constantly exposed to flow, but cultured ECs are often grown under static conditions and ...

Application of Electrophysiology Measurement to Study the Activity of Electro-Neutral Transporters

The transport of ions through cell membranes ensures the fine control of ion content within and outside the cell that is indispensable for cell survival. These transport mechanisms are mediated by the activities of specialized transporter proteins. Specifically,pH dynamics are finely controlled by plasma membrane proton (H+) extrusion systems, such as the Na+/H+ exchanger (NHE) protein family. Despite extensive efforts to study the mechanisms underlying NHE regulation, our current understanding of the biophysical and molecular properties of the NHE family is inadequate because of the limited availability of methods to effectively measure NHE activity. In this manuscript, we used H+-selective electrodes during whole-cell patch clamping recording to measure NHE-induced H+ flux. We proposed this approach to overcome some limitations of typically used methods to measure NHE activity, such as radioactive uptake and fluorescent membrane permeants. Measurement of NHE activity using the described method enables high sensitivity and time resolution and more efficient control of intracellular H+ concentrations. H+-selective electrodes are based on the fact that transporter activity creates an ion gradient in close proximity to the cell membrane. An H+-selective electrode moving up to and away from the cell membrane in a repetitive, oscillatory fashion records a voltage difference that is dependent on H+ flux. While H+-selective electrodes are used to detect H+ flux moving out of the cell, the patch clamp method in the whole-cell configuration is used to control the intracellular ion composition. Moreover, application of the giant patch clamp technique allows modification of the intracellular composition of not only ions but also lipids. The transporter activity of NHE isoform 3 (NHE3) was measured using this technical approach to study the molecular basis of NHE3 regulation by phosphoinositides.

This manuscript describes the applications of proton-selective electrodes and patch clamping methods to measure the activity of proton transport systems. These methods overcome some limitations of techniques commonly used to study proton transport activity, such as moderate sensitivity, time resolution and insufficient intracellular milieu control.

전기 중립 전송기의 활동을 연구 전기 생리학 측정의 응용

The transport of ions through cell membranes ensures the fine control of ion content within and outside the cell that is indispensable for cell survival. ...

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 ...

Particle Templated Emulsification enables Microfluidic-Free Droplet Assays

Reactions performed in monodispersed droplets afford enhanced accuracy and sensitivity compared to equivalent ones performed in bulk. However, the requirement of microfluidics to form controlled droplets imposes a barrier to non-experts, limiting their use. Here, we describe particle templated emulsification, an approach to generate monodisperse droplets without microfluidics. Using templating hydrogel spheres, we encapsulate samples in monodispersed droplets by simple vortexing. We demonstrate the approach by using it to perform microfluidic-free digital PCR.

Water-in-oil droplet assays are useful for analytical chemistry, enzyme evolution, and single cell analysis, but typically require microfluidics to form the droplets. Here, we describe particle templated emulsification, a microfluidic-free approach to perform droplet assays.