The authors thank Epithelix Sarl and Biopredic International for providing the airway cells and hepatic cells micrographies and Neil Smith for the figure illustrating the experimental procedure.

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AB와 EM이 원고 작성 당시 브리티쉬 아메리칸 토바코의 직원이었다. 이 논문에서 설명하는 작업은 브리티쉬 아메리칸 토바코에 의해 투자되었다,이 비디오 기사에 대한 출판 비용은 브리티쉬 아메리칸 토바코 지불했다.

현재, 많은 표준화 된 독성 시험 시스템은 정상적인 인간의 신진 대사 하나의 대표하지 않습니다 엔지니어링 세균, 세포 선, 또는 배아 세포를 사용합니다. 이것은 화학 물질 또는 약의 잠재적 인 독성 활동의 부정확 한 예측으로 이어질 수 있습니다. 이러한 칩 3D 세포 배양 및 장기 등의 생체 외 모델의 혁신적인 개선 인체 조직 21, 22, 23의 일반적인 형태 및 대사 활성을 복제하기위한 시도로 개발되고있다. 대사 능력 모델이 가장 독성 약물 평가 및 연구에 적합한 생물 전환 된 해독하는데 사용될 수있는 하나 개의 기준이다. 우리는이 논문에서 설명했다 프로토콜은 살아있는 세포를 사용하여 CYP 효소의 활동을 측정하도록 설계되었습니다. 그것은 유연하고 2D 또는 3D이 배양 다른 전지 시스템에 사용할 수 있습니다ES와 luminogenic 프로브 - 또는 질량 분석 기반 접근 방식을 사용 할 수있는 옵션을 제공합니다. 두 가지 방법은 재료의 나노 그램 수량을 감지하고 만 다공 형태로 성장하는 소수의 세포를 필요로 할만큼 민감하다. 또한 회전 타원체에 적용 할 수 또는 세포는 복잡한 매트릭스에서 재배. 프로브 기판의 선택은 분명 프로토콜의 중요한 요소이다. 상기 분석의 특이성은 선택적 CYP 억제제의 사용에 의해 얻어 질 수 CYP 효소 보증 다른 층의 선택적 인 프로브에 의존한다. 기판 및이 문서에 나와 억제제는 몇 가지 예에 불과하지만, 다른 사람은 문헌에서 찾을 수 있습니다. 이 효소는 낮은 수준으로 발현되는 것에 기인 할 수있는 네거티브 CYP 활성 결과로서 새로운 세포 유형으로 작동 할 때 복수의 배양 시간을 고려하는 것이 중요하다. 양성 대조군으로 사용할 수있는 세포 유형의 추가한다는 보장하는 것이 바람직부정적인 결과는 기술적 인 문제에 어떤 문제 해결에 도움이 연결되지 않습니다. 주 간 세포가 대사 능력이 서스펜션 예를 차 간세포를 들어, 대조군으로 사용 할 수있는 것은 매우 쉽게 쓸 수 있지만 그들의 생존은 시간에 제한됩니다. 간 세포 라인이 프로토콜에 설명 된대로 사용하는 것이 상대적으로 용이 가능한 대안이지만, 일부는 신진 대사 활동 (6), (21)의 측면에서 다른 사람보다 더 낫다. 분석은 총 단백질 정량되지 않는 비 파괴적이기 때문에, 종 공부 (20)에 시간 경과 CYP 활성을 수행 할 수있다. 일부 세포는 배양 시간이 지남에 따라 변수 CYP 활동을하고 있기 때문에 이것은 중요한 포인트입니다. 음의 결과는 시험관 내에서 분화 또는 탈분화로 포화 상태의 부족, 진행과 연관 될 수 있습니다. 이 경우, 접근 방식은 반 quantitati입니다데이터는 각각의 세포 배양 물에서 단백질의 총량에 의해 정규화되지 않으므로했습니다. TCDD의 경우와 같이 조직에 대한 독성 효과를 가질 수있는 프로브, 억제제 또는 유도 물질에 대한 노출로 CYP 활성을 측정 한 후, 조직을 재사용하는 것이 불가능하거나 바람직하지 않습니다. 같은 마이크로 솜 같은 셀 분수는 주어진 세포 유형의 CYP 활동을 특성화하는 데 사용할 수 있습니다. 미크로 제제는, 그러나, 셀 재 적절한 보조 인자의 첨가 많이 필요하고, 효소 활성을 유지하도록 버퍼. 살아있는 세포를 사용하여 까다로운 마이크로 좀의 준비 단계를 제거하고 버퍼링 공동 인자가 가장 잘 작동하는 운동. 일반적인 추천 정보로서, 추가로이 효소 활성과 일치하는지 확인하는 세포 배양에서 CYPs의 발현 프로파일을 특성화하기위한 가장 좋은 방법이다. 검증이 추가 레벨은 신진 대사 프로브는 enti되지 않습니다 주어진 권장단일 CYP 특정 의존하며 측정 된 대사 활성은 해당 효소의 발현에 의해 일치 증가 된 신뢰도를 제공한다. CYPs 멤브레인 단백질이기 때문에 그러므로 그들은 정량적 RT-PCR이 효소 20 프로파일에 바람직한 접근왔다 웨스턴 블랏을 준비하기 위해 상대적으로 어렵다. 이 프로토콜은 단지 하나의 중요한이라도 대사 효소 패밀리 중 하나를 나타내는 분석 CYP 효소 활성에 대한 방법을 설명하는 것이 중요하다. 질량 분광 분석 방법의 유연성은 프로브 기판의 다른 대사 변환에 대한 취득 방법의 개발을 허용한다. 그러나, 그것들은 한번에 하나씩 개발되어야 할 현재 공지 적은 특정 프로브 및 다른 대사 효소 군에 대한 선택적 억제제가있다. 이 격차는 체외 세포 등산용의 신진 대사 능력의 종합적인 평가를 할 수 있도록 해결해야MS.

생체이 대사는 신체 외부 화학 물질들이 배출을 용이하게 한 친수성기와 복합체의 첨가에 의해 수정되는 과정이다. 일반적으로 생체 이물질의 대사 제가 하나 이상의 수산기 (1, 2)의 첨가와 함께 산화 주로 이루어지는 단계와 두 단계 프로세스이다. 예컨대 글루 쿠로 니드 또는 황산 부분 (1, 2)과 같은 친수성 복합체에 대한 수용체로서 단계 II에서, 수산기가 사용된다. 셉터 그룹이 이미 분자에 존재하는 경우, 접합 공정 독립적 I 상 대사 일어날 수있다. 각 화학 반응 기판 (3)의 특정 효소의 기, 예를 들어, 히드 록 실화를 촉매 CYPs (시토크롬 P450s) (도 1), 탈 알킬화에 의해 수행된다. ConjugatioN sulfotransferases에 의해 촉매되는, UDP-glucuronosyltransferases (도 1), 글루타티온 S 트랜스퍼 - 및 N- 아세틸 트랜스퍼 4. 각 조직 및 기관은 그 대부분의 단백질을 발현시키는 간암으로 특정 대사 효소의 발현 프로파일을 가질 것이다. <img alt="그림 1" src="/files/ftp_upload/55502/55502fig1.jpg" /> 그림 1 : 단계 I와 쿠마린을위한 단계 II 대사의 예. CYP2A6은 / 쿠마린의 7 수산화을 촉진 CYP2A13. 7- 하이드 록시 쿠마린이 UGT1A6 및 UGT1A9 가장 높은 활성을 보여주는, 단계 II 효소에 의한 글루 쿠 UGTs 잔기에 접합된다. 생체 이물질의 신진 대사를 이해하는 것은 두 가지 이유에 대한 약물 안전성 평가 및 화학 위험 평가에 중요한 : 반응 속도론은 약물이나 화학 물질은 활성 또는 비활성 형태 B의 몸에 남아 시간을 결정합니다는 efore 배설; 및 모 화합물 대사 효소에 의해 더 반응성 불안정한 독성 종으로 변형 될 수있다. 또한 &quot;생리 활성&quot;로 알려진 이러한 반응은 대부분 내가뿐만 아니라 상 II 결합 (5)에 의해 희소 한 경우에 효소를 CYP 단계에 의해 구동된다. 이를 바탕으로 정확하게 약물이나 화학 물질과 관련된 위험을 예측하는 체외 모델의 능력은 전지 시스템의 신진 대사 능력에 크게 의존한다. 병든 조직 또는 정상 세포의 변형 유래 세포주에 종종 부분 아니라면 원점 (6)의 이들 조직의 대사 효소 프로필 대표 모두 잃는다. 정상 대사 효소 프로파일의 유지는 (적어도 단기간 배양)에서 일차 세포 배양 물에 잘 나타나 조직이 그것의 3 차원 구조 (1)를 유지할 수 있도록 매트릭스 배양 할 경우 더욱 향상된다. 따라서 문자체외 전지 시스템의 신진 대사 능력의 acterization 세포 모델은 화학 물질 안전성 평가를 수행 할 적절한에 관한 결정을 안내하는 중요한 예비 단계입니다. 본 연구에서 우리는 간 세포주 7 3D 차 폐암 세포 배양 8 애플리케이션의 예는 시험관 내에서의 활성 및 단계 I CYP 효소의 발현을 프로필에 대한 프로토콜을 제시한다. CYP 특정 기질 대사 억제제 제품 및 컨트롤 질량 spectrometry- 및 luminogenic 기반 정량 방법과 함께 설명한다. 일부 CYPs을 유도하고 다른 구성적인 때문에, 예는 그 두 가지 시나리오에 대해 구체적으로 살펴 보도록한다.

<table border="0" cellpadding="0" cellspacing="0" width="697">
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Reagent &#38; Cells</td>
			<td style="width:156px;"></td>
			<td style="width:203px;"></td>
			<td style="width:63px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">2 &#956;m syringe filter</td>
			<td>Whatman</td>
			<td>UN203NPEORG</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">24 well plate</td>
			<td>Corning</td>
			<td>3524</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">4-methylumbelliferone</td>
			<td>Sigma Aldrich</td>
			<td>M1381</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">5-phenyl pentyne</td>
			<td>Sigma Aldrich</td>
			<td>CD5001437</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6-hydroxybupropion</td>
			<td>Sigma Aldrich</td>
			<td>H3167</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">6-hydroxychlorzoxazone</td>
			<td>Sigma Aldrich</td>
			<td>UC148</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">7-ethoxycoumarin</td>
			<td>Sigma Aldrich</td>
			<td>195642</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">7-hydroxycoumarin</td>
			<td>Sigma Aldrich</td>
			<td>H24003</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">8-methoxypsoralen</td>
			<td>Sigma Aldrich</td>
			<td>M3501</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">acetic acid</td>
			<td>Sigma Aldrich</td>
			<td>695092</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">acetonitrile</td>
			<td>Fisher</td>
			<td>A/0626/17</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">&#945;-naphthoflavone</td>
			<td>Sigma Aldrich</td>
			<td>N5757</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">BCA kit</td>
			<td>Thermo Scientific</td>
			<td>23227</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">b-glucuronidase/arylsulfatase</td>
			<td>Sigma Aldrich</td>
			<td>G0876</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bupropion</td>
			<td>Sigma Aldrich</td>
			<td>B102</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Carbamazepine</td>
			<td>Sigma Aldrich</td>
			<td>C4024</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">chlorzoxasone</td>
			<td>Sigma Aldrich</td>
			<td>C4397</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">collagen</td>
			<td>Cell Systems</td>
			<td>5005-B</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">coumarin</td>
			<td>Sigma Aldrich</td>
			<td>C4261</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">disulfiram</td>
			<td>Sigma Aldrich</td>
			<td>86720</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">fluvoxamine</td>
			<td>Sigma Aldrich</td>
			<td>F2802</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Glutamax (Glutamine supplement)</td>
			<td>Fisher</td>
			<td>35050061</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HepaRG metabolism supplement</td>
			<td>Merck</td>
			<td>MMAD621</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HepaRG thaw media supplement</td>
			<td>Merck</td>
			<td>MMADD671</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">HepaRG</td>
			<td>Merck</td>
			<td>MMHPR116</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Luciferin-CEE</td>
			<td>Promega</td>
			<td>V8751</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">methanol</td>
			<td>Fisher</td>
			<td>M/4056/17</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">MucilAir airway cells</td>
			<td>Epithelix</td>
			<td>EP01</td>
			<td></td>
		</tr>
		<tr height="30">
			<td height="30" style="height:30px;">MucilAir airway cells maintenance media</td>
			<td>Epithelix</td>
			<td>EP04MM</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:276px;">Phenomenex Kinetex 2.6&#956;m, PFP 100A</td>
			<td>Phenomenex</td>
			<td>00B-4477-AN</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">TCDD</td>
			<td>Sigma Aldrich</td>
			<td>48599</td>
			<td></td>
		</tr>
		<tr height="36">
			<td height="36" style="height:36px;">thioTEPA</td>
			<td>Sigma Aldrich</td>
			<td>T6069</td>
			<td></td>
		</tr>
		<tr height="41">
			<td height="41" style="height:41px;width:276px;">Waters Acquity UPLC HSS T3 1.8&#181;m 2.1 x 50mm</td>
			<td>Waters</td>
			<td>86003538</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Williams&#8217; E media</td>
			<td>Fisher</td>
			<td>17704-024</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Name</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">UPLC</td>
			<td>Waters</td>
			<td>Acquity</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QTRAP MS</td>
			<td>Sciex&#160;</td>
			<td>ABI Sciex 4000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">QTRAP MS software</td>
			<td>Sciex&#160;</td>
			<td>Analyst 1.4.2</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">luminometer</td>
			<td>Molecular Devices</td>
			<td>SpectraMax M3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">spectrophotometer</td>
			<td>Molecular Devices</td>
			<td>SpectraMax M3</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">cell counter</td>
			<td>Beckman Coulter</td>
			<td>Vi-Cell XR</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;">rotary evaporator</td>
			<td>Eppendorf</td>
			<td>Eppendorf-5301</td>
			<td></td>
		</tr>
	</tbody>
</table>

mass spectrometry and luminogenic-based approaches to characterize phase i metabolic competency of in vitro cell cultures

생체이 대사 효소는 용해도를 증가 및 배설을 촉진 작용기를 첨가하여 약제의 독성 물질 및 생물 전환에 중요한 기능을 담당한다. 어떤 경우에 그 구조 변경은 새로운 독성 제품의 형성으로 이어질. 동물 실험을 줄이기 위해, 화학적 위험은 대사 능력이 세포를 이용하여 평가 될 수있다. 대사 효소의 발현은, 그러나, 체외 차 문화 시스템에서 많은 시간 이상 안정되지 종종 세포 라인의 일부 또는 존재하지 않는다. 따라서, 생체 외 의약품, 첨가제, 환경 오염 물질 대사의 연구는 이상적으로 신진 대사 활동이 특징되었습니다 전지 시스템에서 수행되어야한다. 우리는 UPLC 질량 분석에 의한 정량적 화학 프로브 및 대사 제품을 사용하여 2D 세포주 및 기본 3D의 문화 대사 효소의 클래스의 활동 (인간 단계 I)를 측정하기 위해 여기에 방법을 설명하고luminometry. 방법 세포주 및 다양한 조직으로부터 유도 된 세포의 주 대사 활성을 테스트하도록 구현 될 수있다.

세 가지 공여체 (기도 M360, M370, M417) 및 간 세포주 HEP (선)에서기도 세포 배양 두 CYPs 쌍 (CYP1A1 / 1B1 및 CYP2A6 / 2A13) 측정 대사 활성의 예는도 6에 나타낸다 (20). CYP 활성 측정 luminometry 도 6a는 호흡기 세포에서 룩 - CEE (루시페린 6&#39;- 클로로 에틸 에테르) (3 도너)와 함께 및 TCDD 유도없이 간 세포의 CYP1A1 / 1B1 O-탈 알킬화 활동에 의해 생성 된 상대 발광을 나타낸다. CYP1A1 / 1B1 높게 유도 효소이며, 통상적으로 대부분의 조직의 구성 적 수준에서 발현되지 않는다. 세포가 유도되지 않는 경우도 6a에서 알 수있는 바와 같이, 더 luminogenic 신호가 검출되지 않는다. 두 세포 유형은 TCDD 강한 CYP1A1 / 1B1 유발제 마크로 사전 배양하면ED luminogenic 신호는 제어부 (도 6a)와 비교하여 검출된다. 이는 룩 - CEE 프로브 이후 정량화 루시페린 부분의 박리로 이어지는 대사되었음을 나타낸다. 전체적으로 TCDD 처리 간 세포주에서 활성 심지어 총 단백질 정규화 후에,기도 세포보다 낮다. 세포주는 일차 전지보다 대사 활성화하는 경향이 있기 때문에 이것은 드문 일이 아니다. α-naphthoflavone 첨가 CEE 상기 룩 - 대사 CYP 의존성을 확인 모든 세포 유형에서 CYP1A1 / 1B1 활성에 명확한 억제 효과를 갖는다. UPLC-MS를 사용 CYP450 대사 산물의 정량 도 6b는 INHI의 존재와 부재 및 쿠마린과 배양 후 3 도너 (기도 M343, M345, M347) 및 간 세포 (간염 라인)에서기도 세포 CYP2A6 / 2A13 활성의 정량화를 도시BITOR 8 methoxypsoralen. 데이터는 전체 단백질 정규화된다. 0 분 쿠마린 배양 컨트롤 어떠한 하이드 록시 쿠마린 (7)는 (도 6b) 검출되지 않는다. CYP2A6 / 2A13 특정 조직에서 구성 적으로 발현되는 7 하이드 록시 쿠마린의 형성 쿠마린 따라서 후 16 시간 배양에도 유도하지 않고, 테스트를 다른 유형의 세포에서 발견된다. 8-methoxypsoralen 때 CYP2A 억제제 (도 6B)는, 7 하이드 록시 쿠마린의 형성이 상당히 감소된다 첨가한다. 억제가 거의 완료되는 다른 CYPs 또한 기판을 인식하지만, 훨씬 덜 효율적으로 처리 할 수 ​​있습니다 이후 CYP 억제제의 존재, 여전히 잔존 활동을 감지하는 것이 정상입니다 주목해야한다. 또한 그것이기도 세포 CYP2A6 볼 같이 일차 전지를 사용하는 경우, 기록 대사 활성은 공여체와 상이 할 수 있음을 관찰 할 수있다(도 6b). 이것은 CYP 활동이 각 기증자의 유전자형과 유전자 다형성에 대한 책임이로 예상된다. <img alt="그림 6" src="/files/ftp_upload/55502/55502fig6.jpg" /> 도 6 : 활성 분석법은 CYP1A1에 대한 결과 / 1B1 활성 (A) 및 간 세포주 세기도 세포 기증자 (M360, M370, M417)에 CYP2A6 / 2A13 활성 (B). (A) - 루크 CEE 탈 알킬화 활성와 TCDD 의한 CYP1A1 / 1B1의 유발없이 나타낸다. α-naphthoflavone은 CYP1A1 / 1B1 억제제로서 사용 하였다. 활성 분 (최소)으로 배양 시간에 의해 정규화 된 상대 발광 단위 (RLU) 총 단백질 (㎎)으로 표현된다. 들은 항시 적으로 발현 될 때 (B) CYP2A6 / 2A13 유도를 필요로하지 않는다. pmol의 / 분 / mg의 단백질의 쿠마린 -7- 히드 록 실화 활성 억제제와 8 methoxypsor없이 다양한 시점에서 도시ALEN. 모든 결과는 평균의 표준 편차와 3 개 개의 독립적 인 측정의 평균 값으로 표시하고 있습니다. 이 수치는 박스터 (20)에서 수정되었습니다. 보충 표 1 : UPLC 및 질량 분석기 설정. <a href="http://ecsource.jove.com/files/ftp_upload/55502/Supplemental_Table_1.xlsx" target="_blank">이 파일을 다운로드하려면 여기를 클릭하십시오.</a>

Watch this Scientific Journal Video about Mass Spectrometry and Luminogenic-based Approaches to Characterize Phase I Metabolic Competency of In Vitro Cell Cultures at JoVE.com

Mass Spectrometry and Luminogenic-based Approaches to Characterize Phase I Metabolic Competency of In Vitro Cell Cultures

체외 시스템의 신진 대사 능력은 약물 및 독성 물질의 생체 내 변화 및 처분을위한 핵심 요구 사항입니다. 이러한 프로토콜에서는, I는 세포 배양 대사 위상을 평가하기위한 기준 대사 프로브의 적용을 설명한다.

질량 분석 및 Luminogenic 기반의 접근 방법은 단계 I 대사 역량의의 특성을<em&gt; 체외</em&gt; 세포 배양

Xenobiotic metabolizing enzymes play a key function in the biotransformation of medicines and toxicants by adding functional groups that increase ...

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Biochemistry

Mass Spectrometry and Luminogenic-based Approaches to Characterize Phase I Metabolic Competency of In Vitro Cell Cultures

9.7K 조회수.  British American Tobacco.  체외 시스템의 신진 대사 능력은 약물 및 독성 물질의 생체 내 변화 및 처분을위한 핵심 요구 사항입니다. 이러한 프로토콜에서는, I는 세포 배양 대사 위상을 평가하기위한 기준 대사 프로브의 적용을 설명한다. 

비디오: Mass Spectrometry and Luminogenic-based Approaches to Characterize Phase I Metabolic Competency of In Vitro Cell Cultures

Sheathless Capillary Electrophoresis&#8211;Mass Spectrometry for Metabolic Profiling of Biological Samples

In metabolomics, a wide range of analytical techniques is used for the global profiling of (endogenous) metabolites in complex samples. In this paper, a protocol is presented for the analysis of anionic and cationic metabolites in biological samples by capillary electrophoresis&#8211;mass spectrometry (CE-MS). CE is well-suited for the analysis of highly polar and charged metabolites as compounds are separated on the basis of their charge-to-size ratio. A recently developed sheathless interfacing design, i.e., a porous tip interface, is used for coupling CE to electrospray ionization (ESI) MS. This interfacing approach allows the effective use of the intrinsically low-flow property of CE in combination with MS, resulting in nanomolar detection limits for a broad range of polar metabolite classes. The protocol presented here is based on employing a bare fused-silica capillary with a porous tip emitter at low-pH separation conditions for the analysis of a broad array of metabolite classes in biological samples. It is demonstrated that the same sheathless CE-MS method can be used for the profiling of cationic metabolites, including amino acids, nucleosides and small peptides, or anionic metabolites, including sugar phosphates, nucleotides and organic acids, by only switching the MS detection and separation voltage polarity. Highly information-rich metabolic profiles in various biological samples, such as urine, cerebrospinal fluid and extracts of the glioblastoma cell line, can be obtained by this protocol in less than 1 hr of CE-MS analysis.

A protocol for metabolic profiling of biological samples by capillary electrophoresis&#8211;mass spectrometry using a sheathless porous tip interface design is presented.

생물 시료의 대사 프로파일 링에 대한 쉬스 모세관 전기 영동 - 질량 분석

In metabolomics, a wide range of analytical techniques is used for the global profiling of (endogenous) metabolites in complex samples. In this paper, a ...

연구

생화학

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

시간 해결 전기 분무 이온화 수소 - 중수소 교환 질량 분석 단백질 구조와 역학 연구를위한

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

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 ...

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most cases, the action of noncoding RNAs is mediated by proteins whose functions are in turn regulated by these interactions with noncoding RNAs. Consistent with this, a growing number of proteins involved in nuclear functions have been reported to bind RNA and in a few cases the RNA-binding regions of these proteins have been mapped, often through laborious, candidate-based methods.
Here, we report a detailed protocol to perform a high-throughput, proteome-wide unbiased identification of RNA-binding proteins and their RNA-binding regions. The methodology relies on the incorporation of a photoreactive uridine analog in the cellular RNA, followed by UV-mediated protein-RNA crosslinking, and mass spectrometry analyses to reveal RNA-crosslinked peptides within the proteome. Although we describe the procedure for mouse embryonic stem cells, the protocol should be easily adapted to a variety of cultured cells.

We describe a protocol to identify RNA-binding proteins and map their RNA-binding regions in live cells using UV-mediated photocrosslinking and mass spectrometry.

질량 분석 기반 식별 RNA 바인딩 영역에 대 한 샘플 준비

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

The Determination of Protease Specificity in Mouse Tissue Extracts by MALDI-TOF Mass Spectrometry: Manipulating PH to Cause Specificity Changes

Proteases have several biological functions, including protein activation/inactivation and food digestion. Identifying protease specificity is important for revealing protease function. The method proposed in this study determines protease specificity by measuring the molecular weight of unique substrates using Matrix-assisted Laser Desorption/Ionization Time-of-Flight (MALDI-TOF) mass spectrometry. The substrates contain iminobiotin, while the cleaved site consists of amino acids, and the spacer consists of polyethylene glycol. The cleaved substrate will generate a unique molecular weight using a cleaved amino acid. One of the merits of this method is that it may be carried out in one pot using crude samples, and it is also suitable for assessing multiple samples. In this article, we describe a simple experimental method optimized with samples extracted from mouse lung tissue, including tissue extraction, placement of digestive substrates into samples, purification of digestive substrates under different pH conditions, and measurement of the substrates&#39; molecular weight using MALDI-TOF mass spectrometry. In summary, this technique allows for the identification of protease specificity in crude samples derived from tissue extracts using MALDI-TOF mass spectrometry, which may easily be scaled up for multiple sample processing.

Here, we present a protocol to determine protease specificity in crude mouse tissue extracts using MALDI-TOF spectrometry.

마우스 조직에서 효소 특이성 결정 TOF MALDI 질량 분석에 의해 추출: PH 특이성 변경를 조작

Proteases have several biological functions, including protein activation/inactivation and food digestion. Identifying protease specificity is important ...

Sample Preparation for Probe Electrospray Ionization Mass Spectrometry

Mass spectrometry (MS) is a powerful tool in analytical chemistry because it provides very accurate information about molecules, such as mass-to-charge ratios (m/z), which are useful to deduce molecular weights and structures. While it is essentially a destructive analytical method, recent advancements in the ambient ionization technique have enabled us to acquire data while leaving tissue in a relatively intact state in terms of integrity. Probe electrospray ionization (PESI) is a so-called direct method because it does not require complex and time-consuming pretreatment of samples. A fine needle serves as a sample picker, as well as an ionization emitter. Based on the very sharp and fine property of the probe tip, destruction of the samples is minimal, allowing us to acquire the real-time molecular information from living things in situ. Herein, we introduce three applications of PESI-MS technique that will be useful for biomedical research and development. One involves the application to solid tissue, which is the basic application of this technique for the medical diagnosis. As this technique requires only 10 mg of the sample, it may be very useful in the routine clinical settings. The second application is for in vitro medical diagnostics where human blood serum is measured. The ability to measure fluid samples is also valuable in various biological experiments where a sufficient volume of sample for conventional analytical techniques cannot be provided. The third application leans toward the direct application of probe needles in living animals, where we can obtain real-time dynamics of metabolites or drugs in specific organs. In each application, we can infer the molecules that have been detected by MS or use artificial intelligence to obtain a medical diagnosis.

This article introduces sample preparation methods for a unique real-time analytical method based on the ambient mass spectrometry. This method lets us perform real-time analysis of the biological molecules in vivo without any special pretreatments.

Mass spectrometry (MS) is a powerful tool in analytical chemistry because it provides very accurate information about molecules, such as mass-to-charge ...

Absolute Quantification of Cell-Free Protein Synthesis Metabolism by Reversed-Phase Liquid Chromatography-Mass Spectrometry

Cell-free protein synthesis (CFPS) is an emerging technology in systems and synthetic biology for the in vitro production of proteins. However, if CFPS is going to move beyond the laboratory and become a widespread and standard just in time manufacturing technology, we must understand the performance limits of these systems. Toward this question, we developed a robust protocol to quantify 40 compounds involved in glycolysis, the pentose phosphate pathway, the tricarboxylic acid cycle, energy metabolism and cofactor regeneration in CFPS reactions. The method uses internal standards tagged with 13C-aniline, while compounds in the sample are derivatized with 12C-aniline. The internal standards and sample were mixed and analyzed by reversed-phase liquid chromatography-mass spectrometry (LC/MS). The co-elution of compounds eliminated ion suppression, allowing the accurate quantification of metabolite concentrations over 2-3 orders of magnitude where the average correlation coefficient was 0.988. Five of the forty compounds were untagged with aniline, however, they were still detected in the CFPS sample and quantified with a standard curve method. The chromatographic run takes approximately 10 min to complete. Taken together, we developed a fast, robust method to separate and accurately quantify 40 compounds involved in CFPS in a single LC/MS run. The method is a comprehensive and accurate approach to characterize cell-free metabolism, so that ultimately, we can understand and improve the yield, productivity and energy efficiency of cell-free systems.

Here, we present a robust protocol to quantify 40 compounds involved in central carbon and energy metabolism in cell-free protein synthesis reactions. The cell-free synthesis mixture is derivatized with aniline for effective separation using reversed-phase liquid chromatography and then quantified by mass spectrometry using isotopically labelled internal standards.

역상 액체 크로마토그래피-질량 분광법에 의한 무세포 단백질 합성 대사의 절대 정량화

Cell-free protein synthesis (CFPS) is an emerging technology in systems and synthetic biology for the in vitro production of proteins. However, if CFPS is ...

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of biological membranes, energy storage and production, cellular signaling, vesicular transport of proteins, organelle biogenesis, and regulated cell death. Because the budding yeast Saccharomyces cerevisiae is a unicellular eukaryote amenable to thorough molecular analyses, its use as a model organism helped uncover mechanisms linking lipid metabolism and intracellular transport to complex biological processes within eukaryotic cells. The availability of a versatile analytical method for the robust, sensitive, and accurate quantitative assessment of major classes of lipids within a yeast cell is crucial for getting deep insights into these mechanisms. Here we present a protocol to use liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of major cellular lipids of S. cerevisiae. The LC-MS/MS method described is versatile and robust. It enables the identification and quantification of numerous species (including different isobaric or isomeric forms) within each of the 10 lipid classes. This method is sensitive and allows identification and quantitation of some lipid species at concentrations as low as 0.2 pmol/&#181;L. The method has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. The use of alternative mobile phase additives for electrospray ionization mass spectrometry in this method can increase the efficiency of ionization for some lipid species and can be therefore used to improve their identification and quantitation.

Quantitative Analysis of the Cellular Lipidome of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

We present a protocol using liquid chromatography coupled with tandem mass spectrometry to identify and quantify major cellular lipids in Saccharomyces cerevisiae. The described method for a quantitative assessment of major lipid classes within a yeast cell is versatile, robust, and sensitive.

탠덤 질량 분석과 결합 된 액체 크로마토그래피를 사용하여 사카로 미세화세세비시아의 세포 지질의 정량분석

Lipids are structurally diverse amphipathic molecules that are insoluble in water. Lipids are essential contributors to the organization and function of ...

Semi-Quantitative Analysis of Peptidoglycan by Liquid Chromatography Mass Spectrometry and Bioinformatics

Peptidoglycan is an important component of bacterial cell walls and a common cellular target for antimicrobials. Although aspects of peptidoglycan structure are fairly conserved across all bacteria, there is also considerable variation between Gram-positives/negatives and between species. In addition, there are numerous known variations, modifications, or adaptations to the peptidoglycan that can occur within a bacterial species in response to growth phase and/or environmental stimuli. These variations produce a highly dynamic structure that is known to participate in many cellular functions, including growth/division, antibiotic resistance, and host defense avoidance. To understand the variation within peptidoglycan, the overall structure must be broken down into its constitutive parts (known as muropeptides) and assessed for overall cellular composition. Peptidoglycomics uses advanced mass spectrometry combined with high-powered bioinformatic data analysis to examine peptidoglycan composition in fine detail. The following protocol describes the purification of peptidoglycan from bacterial cultures, the acquisition of muropeptide intensity data through a liquid chromatograph&#8212;mass spectrometer, and the differential analysis of peptidoglycan composition using bioinformatics.

This protocol covers a detailed analysis of peptidoglycan composition using liquid chromatography mass spectrometry coupled with advanced feature extraction and bioinformatic analysis software.

액체 크로마토그래피, 질량 분석법 및 생물정보학에 의한 펩티도글리칸의 반정량 분석

Peptidoglycan is an important component of bacterial cell walls and a common cellular target for antimicrobials. Although aspects of peptidoglycan ...

Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

Metabolomics is a methodology used for the identification and quantification of many low-molecular-weight intermediates and products of metabolism within a cell, tissue, organ, biological fluid, or organism. Metabolomics traditionally focuses on water-soluble metabolites. The water-soluble metabolome is the final product of a complex cellular network that integrates various genomic, epigenomic, transcriptomic, proteomic, and environmental factors. Hence, the metabolomic analysis directly assesses the outcome of the action for all these factors in a plethora of biological processes within various organisms. One of these organisms is the budding yeast Saccharomyces cerevisiae, a unicellular eukaryote with the fully sequenced genome. Because S. cerevisiae is amenable to comprehensive molecular analyses, it is used as a model for dissecting mechanisms underlying many biological processes within the eukaryotic cell. A versatile analytical method for the robust, sensitive, and accurate quantitative assessment of the water-soluble metabolome would provide the essential methodology for dissecting these mechanisms. Here we present a protocol for the optimized conditions of metabolic activity quenching in and water-soluble metabolite extraction from S. cerevisiae cells. The protocol also describes the use of liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) for the quantitative analysis of the extracted water-soluble metabolites. The LC-MS/MS method of non-targeted metabolomics described here is versatile and robust. It enables the identification and quantification of more than 370 water-soluble metabolites with diverse structural, physical, and chemical properties, including different structural isomers and stereoisomeric forms of these metabolites. These metabolites include various energy carrier molecules, nucleotides, amino acids, monosaccharides, intermediates of glycolysis, and tricarboxylic cycle intermediates. The LC-MS/MS method of non-targeted metabolomics is sensitive and allows the identification and quantitation of some water-soluble metabolites at concentrations as low as 0.05 pmol/&#181;L. The method has been successfully used for assessing water-soluble metabolomes of wild-type and mutant yeast cells cultured under different conditions.

Quantitative Metabolomics of Saccharomyces Cerevisiae Using Liquid Chromatography Coupled with Tandem Mass Spectrometry

We present a protocol for the identification and quantitation of major classes of water-soluble metabolites in the yeast Saccharomyces cerevisiae. The described method is versatile, robust, and sensitive. It allows the separation of structural isomers and stereoisomeric forms of water-soluble metabolites from each other.

탠덤 질량 분광과 결합 된 액체 크로마토그래피를 사용하여 사카로미세스 Cerevisiae의 정량적 Metabolomics

Metabolomics is a methodology used for the identification and quantification of many low-molecular-weight intermediates and products of metabolism within ...