Name: simultaneous quantification of selected kynurenines analyzed by liquid chromatography-mass spectrometry in medium collected from cancer cell cultures
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이 작품은 동유럽 2007-2013의 운영 프로그램 개발에 따라 유럽 지역 개발 기금에서 유럽 연합 (EU)에 의해 지원되었다 (POPW.01.03.00-06-003/000), 루블린의 존 폴 II 가톨릭 대학의 생명 공학 및 환경 과학 학부에 의해 (일론사사옥에 대한 젊은 과학자 보조금), 폴란드 국립 과학 센터, 폴란드 국립 과학 센터, 폴란드 국립 과학 센터, 폴란드 OPUS13 (막달레나 스타니스제프스카, 원칙 조사관에 대한 OPUS13 (2017/25/B/NZ4/01198)  저자는 산화 스트레스 연구소에서 교수 Agnieszka Şcibior 감사, 학제 간 연구 센터, 세포 배양 및 단백질 정량화를위한 장비를 공유하기위한 JPII CUL.

1. 표준 3NT, 킨, 3HKyn, 3HAA, XA 표준 재고 솔루션의 준비
<ol><li>가장 높은 정확도 (각각 0.3 mg)로 바이알에서 시약을 계량합니다. 더 나은 정확도를 위해 시약을 확장하여 1.2 단계에 따라 용매의 부피를 조정합니다.</li>
	<li>시약을 용매의 300 μL로 용해하여 물에서 1g/L의 스톡 용액을 1% (v/v) 포믹산(FA)으로 용해하는; 동전 설플옥화물 (DMSO)에서 킨, 3HAA 및 XA; 3HKyn은 염산(HCl)으로 pH 2.5로 산성화하였다. ?...

<ol>
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본 논문은 체외 배양 인간 암세포로부터 배지에서 측정되는 4개의 주요 Trp 대사산물(3HKyn, Kyn, 3HAA, XA)의 동시 정량화를 위한 상세한 LC-SQ 프로토콜을 제시합니다. 샘플 준비, 크로마토그래피/질량 분광법 절차 및 분석 에서 가장 중요한 점인 데이터 해석에 특별한 주의를 기울입니다.
일반적으로 LC-MS 분석은 높은 감도로 인해 사용되는 재료의 프로토콜 엄격성 및 순도의 ...

Kynurenine 통로 (KP)는 인간 세포에 있는 트립토판 (Trp) 이화작용의 주요 경로입니다. 외세포에서 인돌리아민-2,3-디산소효소(IDO-1)는 KP의 최초 및 제한 효소이며 Trp를 N-formylkynurenine1로변환한다. KP 내의 추가 단계는 다른 이차 대사 산물, 즉 다양한 생물학적 특성을 나타내는 kynurenines를 생성합니다. Kynurenine (Kyn)은 아릴 탄화수소 수용체 (AhR)2에결합 한 후 독성 특성 및 조절 세포 이벤트를 보여주는 최초의 안정적인 Trp catabolite이다. 그 후, 킨은 자발적으로 또는 효소 매개 공정에서 여러 분자로 변형되어 3-하이드록시키누레닌(3HKyn), 탄트라날산(AA), 3-하이드록산(3-HAA), 키누레닉산(Kyna), xanethurnicacid(Xanethurnic)와 같은 대사산물을 생성합니다. 또 다른 하류 대사 산물, 2 아미노산-3-카복시무코닉 산-6-세미알데히드(ACMS),퀴놀린산(QA) 또는 피콜리닉산(PA)1에비효소 순환화를 겪는다. 마지막으로, QA는 니코틴아미드-아데닌 디뉴클레오티드(NAD+)3,중요한 효소 보조인인 KP 엔드포인트 대사산물로 더욱 변형된다. 일부 키누레닌은 키나와 PA와 같은 신경 보호 특성을 가지고 있으며, 다른 사람, 즉 3HAA 및 3HKyn은 독성4입니다. 3HKyn에서 형성되는 산투레닉 산은 항산화 및 혈관 완화 특성5를제시합니다. XA는 노화 렌즈에 축적되어 상피 세포의 세포멸각증으로이어진다 6. 20세기 중반에 설명된 KP는 다양한 장애에 대한 참여가 입증되었을 때 더 많은 관심을 얻었습니다. 이러한 대사 경로의 증가된 활동과 일부 키누레닌의 축적은 면역 반응을 조절하고 우울증, 정신 분열증, 뇌병증, HIV, 치매, 근위축성 측삭 경화증, 말라리아, 알츠하이머병, 헌팅턴병 및 암4,7과같은 다른 병리학적 조건과 관련이 있다. Trp 대사의 일부 변화는 종양 미세 환경 및 암세포에서관찰된다2,8. 또한, kynurenines 유망한 질병 마커로 간주됩니다9. 암 연구에서, 체외 세포 배양 모델은 잘 확립되어 널리 항암제에 대한 반응의 전임상 평가에사용된다(10). Trp 대사 산물은 배양 배지로 세포에 의해 분비되고 kynurenine 통로의 상태를 평가하기 위하여 측정될 수 있습니다. 따라서 쉽고 유연하며 신뢰할 수 있는 프로토콜을 가진 다양한 생물학적 시편에서 가능한 한 많은 KP 대사산물의 동시 검출을 위한 적절한 방법을 개발할 필요가 있다.
이 논문에서, 우리는 4개의 kynurenine 통로 대사산물의 동시 결정에 대한 프로토콜을 기술합니다: Kyn, 3HKyn, 3HAA 및 XA에 의하여 LC-SQ에 의하여 암세포에서 집합된 포스트 배양 배지에. 현대 분석 접근법에서, 액체 크로마토그래피(13,14,15,16)는 Ehrlich 시약11,12를활용한 생화학적 비특이적 분석과는 달리 개별 트립토판 이화산물의 동시 검출 및 정량화에 바람직하다. 현재, 주로 자외선 또는 형광 검출기13,17,18,19를가진 액체 크로마토그래피를 기반으로 인간 표본에서 키누레닌 결정에 사용할 수 있는 많은 방법이 있다. 질량 분석 검출기(LC-MS)와 결합된 액체 크로마토그래피는 높은 감도(검출의 하한), 선택성 및 반복성으로 인해 이러한 유형의 분석에 더 적합한 것으로 보입니다.
Trp 대사 산물은 이미 인간 혈청, 혈장 및소변(13,20,21,22,23)에서결정되었지만, 세포 배양 배지와 같은 다른 생물학적 표본에 대한 방법도 원하고 있다. 이전에는 LC-MS가 인간 신경교종 세포, 단핵구, 수지상 세포 또는 인터페론 감마(IFN-γ)24,25,26으로처리된 성상세포를 배양한 후 수집된 배지에서 Trp 유래화합물에사용되었다. 현재, 암 모델에 사용되는 다른 배양 매체, 세포 및 치료에 있는 몇몇 대사산물의 평가를 허용할 수 있는 새로운 검증된 프로토콜을 위한 필요가 있습니다.
개발된 방법의 목적은 KP에서 이상을 나타낼 수 있는 4개의 주요 키누레닌(1개의 분석 실행 내)을 정량화하는 것입니다. 여기에 제시된 시험관내 배양 인간암세포(27)로부터수집된 배지에서 하나의 내부 표준(3-니트로티로신, 3NT)을 사용하여 선택된 의미 있는 kynurenines의 정량적 LC-SQ 분석을 위한 당사의 최근 발표된 프로토콜의 중요한 단계이다. 우리의 가장 좋은 지식에, 그것은 체외 성장 세포에서 얻은 배양 배지에서 3HKyn, 3HAA, Kyn 및 XA의 동시 정량화를위한 최초의 LC-SQ 프로토콜입니다. 일부 수정시, 방법은 세포 배양 모델의 넓은 범위에서 Trp 물질 대사의 변화를 연구하기 위해 더 적용 될 수있다.

<table border="0" cellpadding="0" cellspacing="0" style="width:1360px;" width="1359">
	<colgroup>
		<col />
		<col />
		<col />
		<col />
	</colgroup>
	<tbody>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">3-hydroksy-DL-kynurenina</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:261px;">H1771</td>
			<td style="width:513px;"></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">3-hydroxyanthranilic acid</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:261px;">148776</td>
			<td>97%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">3-nitro-L-tyrosine</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:261px;">N7389</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Acetonitrile</td>
			<td style="width:175px;">Supelco</td>
			<td>1.00029</td>
			<td>hypergrade for LC-MS LiChrosolv</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Activated charcoal</td>
			<td style="width:175px;">Supelco</td>
			<td>05105</td>
			<td>powder</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Analytical balance</td>
			<td style="width:175px;">Ohaus</td>
			<td></td>
			<td></td>
		</tr>
		<tr height="29">
			<td height="29" style="height:29px;width:411px;">Analytical column</td>
			<td style="width:175px;">Agilent Technologies</td>
			<td>959764-902</td>
			<td>Zorbax Eclipse Plus C18 rapid resolution HT (2.1 x 100 mm, 1.8 &#181;m)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ammonium formate</td>
			<td style="width:175px;">Supelco</td>
			<td style="width:261px;">7022</td>
			<td>eluent additive for LC-MS, LiChropur, &#8805;99.0%</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Bovine Serum Albumin</td>
			<td style="width:175px;">Sigma</td>
			<td>1001887398</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Caps for chromatographic vials</td>
			<td style="width:175px;">Agilent Technologies</td>
			<td>5185-5820</td>
			<td>blue screw caps,PTFE/red sil sepa</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell culture dish</td>
			<td style="width:175px;">Nest</td>
			<td>704004</td>
			<td>polystyrene, non-pyrogenic, sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell culture plate</td>
			<td style="width:175px;">Biologix</td>
			<td>07-6012</td>
			<td>12-well, non-pyrogenic, non-cytoxic, sterille</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Cell incubator</td>
			<td>Thermo Fisher Scientific</td>
			<td></td>
			<td>HERAcell 150i Cu</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge</td>
			<td style="width:175px;">Eppendorf</td>
			<td style="width:261px;"></td>
			<td>model 5415R</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge</td>
			<td style="width:175px;">Eppendorf</td>
			<td style="width:261px;"></td>
			<td>model 5428</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge tubes</td>
			<td style="width:175px;">Bionovo</td>
			<td>B-2278</td>
			<td>Eppendorf type, 1.5 mL</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Centrifuge tubes</td>
			<td style="width:175px;">Bionovo</td>
			<td>B-3693</td>
			<td>Falcon type, 50 mL, PP</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chromatographic data acqusition and analysis software</td>
			<td style="width:175px;">Agilent Technologies</td>
			<td></td>
			<td>LC/MSD ChemStation (B.04.03-S92)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chromatographic insert vials</td>
			<td style="width:175px;">Agilent Technologies</td>
			<td>9301-1387</td>
			<td>100 &#181;L</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Chromatographic vials</td>
			<td style="width:175px;">Agilent Technologies</td>
			<td>5182-0714</td>
			<td>2 mL, clear glass</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Dimethyl sulfoxide</td>
			<td style="width:175px;">Supelco</td>
			<td style="width:261px;">1.02950</td>
			<td>Uvasol</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Dubelcco&#8217;s Modified Eagle&#8217;s Medium (DMEM)</td>
			<td style="width:175px;">PAN Biotech</td>
			<td>P04-41450</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Dual meter pH/conductivity</td>
			<td style="width:175px;">Mettler Toledo</td>
			<td style="width:261px;"></td>
			<td>SevenMulti</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Evaporator</td>
			<td style="width:175px;">Genevac</td>
			<td style="width:261px;"></td>
			<td>model EZ-2.3 Elite</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Fetal bovine serum</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td>F9665</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Formic acid</td>
			<td style="width:175px;">Supelco</td>
			<td style="width:261px;">5.33002</td>
			<td>for LC-MS LiChropur</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Glass bottle for reagents storage</td>
			<td style="width:175px;">Bionovo</td>
			<td>S-2070</td>
			<td>50 mL, clear glass</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Guard column</td>
			<td style="width:175px;">Agilent Technologies</td>
			<td style="width:261px;">959757-902</td>
			<td>Zorbax Eclipse Plus-C18 Narrow Bore Guard Column (2.1 x 12.5 mm, 5 &#181;m)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Hydrochloric acid</td>
			<td style="width:175px;">Merck</td>
			<td>1.00317.1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">L-kynurenine</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:261px;">K8625</td>
			<td>&#8805;98% (HPLC)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Magnetic stirrer</td>
			<td style="width:175px;">Wigo</td>
			<td style="width:261px;"></td>
			<td>ES 21 H</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Microbalance</td>
			<td style="width:175px;">Mettler Toledo</td>
			<td></td>
			<td>model XP6</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Milli-Q system</td>
			<td style="width:175px;">Millipore</td>
			<td>ZRQSVPO30</td>
			<td>Direct-Q 3 UV with Pump</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Quadrupole mass spectrometer</td>
			<td style="width:175px;">Agilent Technologies</td>
			<td>G1948B</td>
			<td>model 6120</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Penicillin-Streptomycin</td>
			<td style="width:175px;">Sigma-Adrich</td>
			<td>048M4874V</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Plate reader</td>
			<td style="width:175px;">Bio Tek</td>
			<td></td>
			<td>Synergy 2 operated by Gen5</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium chloride</td>
			<td style="width:175px;">Merck</td>
			<td>1.04936.1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Potassium dihydrogen phosphate</td>
			<td style="width:175px;">Merck</td>
			<td>1.05108.0500</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Protein Assay Dye Reagent Concentrate</td>
			<td style="width:175px;">BioRad</td>
			<td>500-0006</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">See-saw rocker</td>
			<td style="width:175px;">Stuart</td>
			<td>SSL4</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Serological pipette</td>
			<td style="width:175px;">Nest</td>
			<td>326001</td>
			<td>5 mL, polystyrene, non-pyrogenic, sterile</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium chloride</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:261px;">7647-14-5</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Sodium phosphate dibasic</td>
			<td style="width:175px;">Merck</td>
			<td>1.06346.1000</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Solvent inlet filter</td>
			<td style="width:175px;">Agilent Technologies</td>
			<td>5041-2168</td>
			<td>glass filter, 20 &#956;m pore size</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Solvent reservoir (for LC-MS)</td>
			<td style="width:175px;">Agilent Technologies</td>
			<td>9301-6524</td>
			<td>1 L, clear glass</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Solvent reservoir (for LC-MS)</td>
			<td style="width:175px;">Agilent Technologies</td>
			<td>9301-6526</td>
			<td>1 L, amber glass</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Spreadsheet program</td>
			<td style="width:175px;">Microsoft Office</td>
			<td></td>
			<td>Microsoft Office Excel</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Stir bar</td>
			<td style="width:175px;">Bionovo</td>
			<td>6-2003</td>
			<td>teflon coated</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Syringe filters for culture medium filtration</td>
			<td style="width:175px;">Bionovo</td>
			<td>7-8803</td>
			<td>regenerated cellulose, &#216; 30 mm, 0,45 &#181;m</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Syringe filters for mobile phase components filtration</td>
			<td style="width:175px;">Bionovo</td>
			<td>6-0018</td>
			<td>nylon, &#216; 30 mm, 0,22 &#181;m</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Tissue culture plates</td>
			<td style="width:175px;">VWR</td>
			<td>10062-900</td>
			<td>96-wells, sterille</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Trypsin-EDTA Solution</td>
			<td style="width:175px;">Sigma-Aldrih</td>
			<td>T4049-100 mL</td>
			<td></td>
		</tr>
		<tr height="50">
			<td height="50" style="height:50px;width:411px;">Ultra-High Performance Liquid Chromatography system</td>
			<td style="width:175px;">Agilent Technologies</td>
			<td style="width:261px;">G1367D, G1379B, G1312B, G1316C</td>
			<td style="width:513px;">1200 Infinity system consisted of autosampler (G1367D), degasser (G1379B), binary pump (G1312B), column thermostat (G1316C)</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Ultrasound bath</td>
			<td>Polsonic</td>
			<td>104533</td>
			<td>model 6D</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;">Vortex</td>
			<td>Biosan</td>
			<td></td>
			<td>model V-1 plus</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:411px;">Xanthurenic acid</td>
			<td style="width:175px;">Sigma-Aldrich</td>
			<td style="width:261px;">D120804</td>
			<td>96%</td>
		</tr>
	</tbody>
</table>

simultaneous quantification of selected kynurenines analyzed by liquid chromatography-mass spectrometry in medium collected from cancer cell cultures

kynurenine 통로 및 kynurenines에게 불린 트립토판 이환산물은 면역 조절 및 암 생물학에 그들의 연루를 위해 증가한 관심을 수신했습니다. 체외 세포 배양 분석은 종종 질병 메커니즘에서 다른 트립토판 이환산물의 기여에 대해 배우고 치료 전략을 테스트하는 데 사용됩니다. 분비대사산물과 신호 분자가 풍부한 세포 배양 배지는 트립토판 대사 및 기타 세포 사건의 상태를 반영한다. 복합 세포 배양 배지에서 다중 키누레닌의 신뢰할 수 있는 정량화를 위한 새로운 프로토콜은 여러 시료의 안정적이고 빠른 분석을 가능하게 하고자 한다. 이것은 질량 분석과 결합된 액체 크로마토그래피로 달성될 수 있습니다. 이 강력한 기술은 대사 산물의 정량화를 위한 많은 임상 및 연구 실험실에서 채택되고 kynurenines를 측정하는 데 사용될 수 있습니다.
여기에 제시된 액체 크로마토그래피는 4개의 키누레닌, 즉 키누레닌, 3-하이드록시키누레닌, 3-하이드록시키누레닌, 3-하이드록시스라놀릭 및 산의 동시 측정을 위한 단일 쿼드러폴 질량 분광법(LC-SQ)과 결합된 액체 크로마토그래피를 사용한다. SQ 검출기는 사용하기 쉽고 다른 질량 분광계에 비해 저렴합니다. SQ-MS 분석에서, 샘플로부터의 다중 이온은 특정 질량대전비율(m/z)에따라 생성되고 분리되며, 그 다음에는 단일 이온 모니터링(SIM) 모드를 이용하여 검출한다.
이 백서는 보고된 방법의 장점에 주목하고 몇 가지 약점을 나타냅니다. 크로마토그래피 및 질량 분광분석과 함께 샘플 준비를 포함한 LC-SQ 분석의 중요한 요소에 초점을 맞추고 있습니다. 품질 관리, 방법 교정 조건 및 매트릭스 효과 문제도 논의됩니다. 우리는 모든 표적 마취에 대한 하나의 아날로그 표준으로 3 니트로티로신의 간단한 응용 프로그램을 설명했다. 인간 난소 및 유방암 세포를 이용한 실험에 의해 확인된 바와 같이, 제안된 LC-SQ 방법은 신뢰할 수 있는 결과를 생성하고 다른 체외 세포 모델에 더 적용될 수 있다.

LC-MS는 복잡한 표본의 일부 구성 요소가 소위 매트릭스 효과를 일으키고 분석물의 이온화를 손상시키더라도 생물학적 활성 분자의 정량화에서 명백한 이점을 제시합니다. 이온 억제 또는 이온 향상으로 이어지며 정확도를 강력하게 감소시키고 LC-MS의 검출/정량화 한계에 영향을 미치며, 이는 이 방법의 '약한" 지점으로 간주됩니다. 우리의 프로토콜에서, 이온은 또한 Trp 대사 산물 결정 에 대한 다...

Watch this Scientific Journal Video about Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures at JoVE.

Simultaneous Quantification of Selected Kynurenines Analyzed by Liquid Chromatography-Mass Spectrometry in Medium Collected from Cancer Cell Cultures

여기에 설명된 항암세포 배양으로부터 수집된 배지에서 키누레닌 통로(kynurenine, 3-하이드록시키누레닌, 크산, 3-하이드록산, 3-하이드록산산)에서 생성된 4개의 상이한 트립토판 대사산물의 결정 프로토콜이 단일 사각폴 황색술과 결합된 액체 염색체 배양으로부터 수집된다.

암 세포 배양에서 수집된 매체에서 액체 크로마토그래피 질량 분광법에 의해 분석된 선택된 Kynurenines의 동시 정량화

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and ...

Kynurenines are associated with immune response regulation in several diseases, including cancer. Reliable and validated methods for identifying multiple kynurenines aid in the development of more effective therapies. Our protocol uses liquid chromatography coupled with mass spectrometry to identify the different tryptophan metabolites generated by the kynurenine pathway in medium collected from cancer cell cultures.Unexperienced users may have some difficulty during the sample preparation or data acquisition and interpretation steps. By following our protocol and recommendations, one can avoid mistakes and obtain reliable data. To prepare stock solutions of the reagents, weigh out 0.3 milligrams of each reagent in a vial to the highest accuracy and dissolve each reagent in 300 microliters of the appropriate solvent to obtain one gram per liter stock solutions of each reagent.To prepare charcoal-treated culture medium, weigh out 280 milligrams of activated charcoal in a conical tube and add five milliliters of the liquid medium prepared for culturing the cells of interest. Shake the tube with the medium and charcoal on a seesaw rocker for two hours at room temperature and 50 oscillations per minute. At the end of the incubation, sediment the charcoal by centrifugation and carefully collect the supernatant without disturbing the charcoal, then filter the medium using a 0.45 micrometer syringe filter.To prepare the calibration solution, spike the charcoal-treated culture medium with 0.75 microliters of 0.1 gram per liter 3NT solution and add 3-H kynurenine, 3-HAA kynurenine, and xanthurenic acid to at least six different concentrations to cover all of the calibration ranges to a final volume of 150 microliters per sample. After vortexing, add 150 microliters of minus 20 degrees Celsius cold methanol supplemented with 1%formic acid to each tube for sample deproteinization and tightly cap and vortex the tubes again. After a 40-minute incubation at minus 20 degrees Celsius, centrifuge the samples and use an automatic pipette to transfer 270 microliters of each supernatant into individual flat bottom glass vials.Place the vials into an evaporator and use the appropriate program for water methanol fractions to gently evaporate the volatile components for about 30 minutes. When the tubes are dry, reconstitute each sample in 60 microliters of 0.1%formic acid in water and vortex the samples before transferring each solution into individual 1.5 milliliter tubes for centrifugation. Without disturbing the sediment, transfer the supernatants into chromatographic vials with conical glass inserts and place the vials into an LC-MS auto-sampler.Before running the samples, purge the LC system with the mobile phase to remove bubbles and to prime all of the solvent channels. Next, flush the guard and analytical column with 100%acetonitrile for about 30 minutes before flushing the system with 100%solvent A until a stable pressure is observed in the column, then set the appropriate LC parameters in the data acquisition software and construct a work list for running the samples on the LC system. To construct the calibration curve, in the data acquisition software, add the calibration standards into the work list and run the standards in triplicates, then integrate and measure the peak area corresponding to 3-hydroxykynurenine, kynurenine, 3-hydroxyanthranilic acid, 3-nitrotyrosine, and xanthurenic acid at the retention time of about 4.4, 10, 16, 21, and 30 minutes respectively.To collect supernatant samples from an in vitro cell culture, after 48 hours of standard cell culture of the cells of interest, transfer 500 microliters of supernatant from each well into individual 1.5 milliliter tubes and remove the cell debris by centrifugation, then transfer the medium supernatants into new tubes for minus 80 degrees Celsius storage until the analysis and maintain the cell pellets at minus 20 degrees Celsius until protein estimation analysis. To prepare samples for LC-MS analysis, transfer 149.25 microliters of each thawed culture medium sample into a new 1.5 microliter tube and add 0.75 microliters of 0.1 gram per liter 3NT solution to each tube. After preparing the samples as demonstrated, add 150 microliters of minus 20 degrees Celsius methanol supplemented with 1%formic acid to each tube and prepare the sample for LC-MS analysis as demonstrated.When the work list is complete, measure the peak area of each analyte and use an individual linear calibration equation dedicated for each analyte to calculate concentrations of the analytes present within each experimental sample. To assess cellular protein content corresponding to the sample of culture medium, resuspend each cell pellet in 100 microliters of PBS and freeze the samples at minus 20 degrees Celsius for one hour before thawing them on ice. After freeze-thawing the samples three times, collect the cell lysates by centrifugation and transfer the supernatants into new tubes without disturbing the pellets.Dilute the sample 10 times with fresh PBS and add the appropriate volumes of bovine serum albumin standard solution in duplicate to the appropriate wells of a 96-well plate. Load 10 to 15 microliters of each cell lysate sample supernatant to the appropriate wells of the 96-well plate and fill both the standard and sample wells to a final volume of 50 microliters of PBS per well. Next, add 200 microliters of a Bradford reagent diluted five times with ultra pure water to each well and incubate the plate for at least five minutes at room temperature.At the end of the incubation, load the plate onto a microplate reader and measure the absorbance at 595 nanometers, then construct a calibration curve to allow calculation of the amount of protein in each sample and divide the concentration of each kynurenine by the total protein content to normalize the kynurenine amount in each sample per one microgram of protein. Here are single quadrupole mass spectrometry results obtained during the analysis of medium containing 4.5 grams per liter of D-glucose and 10%fetal bovine serum. Note the small peak indicating the presence of kynurenine within the sample.Run a quality control sample before requiring the actual sample data to confirm the appropriate retention times of the analytes as a shift in LC signals or mismatches can be observed. Co-eluting matrix components can generate ions with the master charge ratio selected for the target analytes. For example, in this analysis, the peak from the unknown compound appeared in the scan period during which the ion of master charge ratio 206 was monitored.Due to the incompatibility of the retention time, the signal did not correspond to the analyte. Although the tryptophan isotope shared the same ion as master charge ratio 206, chromatographic analysis revealed that the tryptophan signal was well separated from the xanthurenic acid signal, indicating that the level of tryptophan present in the tested cancer cell medium did not impact xanthurenic acid quantification. This analysis of the culture medium from two types of cancer cell lines demonstrates that the evaluated human epithelial breast cancer cells released different amounts of tryptophan metabolites while the tested human ovarian cancer cells produced significantly higher levels of kynurenine.Using an internal standard during the sample preparation helps with reliable data acquisition. Normalization of the data to the protein content corrects for cell number variabilities within individual wells. Our protocol can be further expanded to determine additional analytes, but may accumulate in the culture medium under different experimental conditions.

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