저자는 Scott Haard와 Abigail Marinack에게이 작업에 대한 일반적인 지원과 조정에 대해 인정할 것입니다.

인간의 소변 샘플을 포함한 모든 연구는 Pharmasan / Neuroscience Institutional Review Board (IRB)의 승인 절차를 거쳤습니다. 1. 소변 채취 및 크레아티닌 (Cr) 측정 <ol><li> 다음 아침 소변 샘플 5 mL를 ca. 사용하기 전까지 -20 ° C에서 방부제 및 저장액으로 3 N HCl 250 μL를 함유하는 5 mL 수송 튜브 A에서 10 시간 하룻밤 동안 단식. </li><li> 해동과 소용돌이 5 mL 수송 뇨 튜브와 원심 분리기 ( 예 : Sorvall) (2297 xg, 10 분)에서 원심 분리하십시오. </li><li> 소변 1 mL를 5 mL 수송 튜브 A에서 두 번 두 번. </li><li> 비뇨기 creatinine 방법으로 크롬을 결정하십시오 26 . </li></ol> 2. 표준, IS 및 품질 관리 (QC) 샘플 준비 <ol><li> 0.01 % HCOOH 이동상 A (MA)가 함유 된 물에서 100 μg / mL의 3-NT의 스톡 용액을 준비하고 -20 ° C에서 보관하십시오. </li><li> 하다MA로 100 μg / mL 3-NT 저장 용액을 희석하여 100 ng / mL의 농도로 3-NT 작업 표준 용액을 만듭니다. </li><li> 100 ng / mL 작업 표준의 희석으로 5에서 2500 pg / mL 범위의 표준을 생성하고 0.15 M HCl 산성화 된 3-NT 공시 뇨와 공시험 시료 및 이중 여백 시료를 사용하십시오. </li><li> -20 ℃에서 13 C IS MA 물에 100 ㎍ / ㎖의에 9 -3- NT 및 저장소의 스톡 용액을 제조 하였다. </li><li> 100 ㎍ / ㎖의 13 C 9 -3- NT 희석하여 500 pg / ml이다에서 작동 용액을 제조는 MA와 원액이다. </li><li> 산성화 된 공백 소변에서 3-NT 작업 표준을 희석하여 LLOQ, 저, 중 및 고 수준 ( 즉 , 10, 25, 100, 500 및 1,250 pg / mL)을 포함하는 5 가지 수준의 QC 시료를 만듭니다. </li></ol> 3. 고체상 추출 과정 <ol><li> 해동 및 소용돌이 샘플, 표준 및 QC 샘플. </li><li> 소변 샘플, 표준 및 QC samp 추가les (각각 250 μL)을 깨끗한 2 mL 96- 웰 수집 플레이트 32 웰로 옮겼습니다. </li><li> 이중 빈 샘플 웰을 제외한 각 웰에 500 pg / mL IS 작동 용액 (50 μL)을 주입하십시오. 이중 빈 샘플 우물에 0.01 % HCOOH (50 μL)를 첨가한다. </li><li> 0.1 % HCOOH (250 μL)가 첨가 된 LC-MS / MS 물을 넣는다. </li><li> 위의 혼합물을 8 채널 피펫으로 3 번 섞는다. </li><li> SPE 로딩 될 때까지 플레이트를 덮으십시오. </li><li> MCX 96-well 추출 플레이트와 수집 저장소를 양압 프로세서에 놓습니다. </li><li> 흡착제를 통해 MeOH (200 μL)를 흐르게하여 추출 플레이트를 컨디셔닝한다. </li><li> 흡착제를 통해 2 % HCOOH (200 μL)로 물을 흐르게하여 평형을 이룬다. </li><li> 8 채널 피펫을 사용하여 미리 혼합 된 각 샘플의 전체 부피를 신중하게 사전 조건화 된 추출 플레이트에로드하십시오. </li><li> 혼합물이 흐를 수 있도록 양압 프로세서에서 낮은 양의 압력 ( 예 : 3 psi)을 설정하십시오흡착제를 천천히 가볍게 두드려서 필요하면 압력을 조절하십시오. </li><li> 흡착제를 통해 2 % HCOOH (200 μL)로 물을 흘려 우물을 씻으십시오. </li><li> 흡착제에 메탄올 (200 μL)을 흘려 우물을 씻으십시오. </li><li> 흡착제에 물 (200 μL)을 흘려 우물을 씻으십시오. </li><li> 양압 프로세서에서 높은 양압 설정 ( 예 : 40 psi)으로 웰을 완전히 말립니다. </li><li> Reservoir를 깨끗한 2 mL 96-well collection plate로 교체하십시오. </li><li> 추출 플레이트에서 보존 된 분석 물 및 IS를 용리시키기 위해 pH 9 (50 μL)에서 25 mM NH 4 OAc를 적용한다. </li><li> 용리액을 중화하기 위해 5 % HCOOH (50 μL)가 함유 된 LC-MS 물을 피펫에 넣습니다. </li><li> 8 채널 피펫으로 3 번 혼합하고 분석을 위해 LC-MS / MS 스테이션에 제출하십시오. </li></ol> 4. LC-MS / MS 분석 <ol><li> 눈금이 매겨진 실린더로 2,000 mL의 LC-MS 물을 정확하게 측정하고 2 L 병으로 옮깁니다. </li><li> 피LC - MS 물이 담긴 위의 병에 200 μL 순수 HCOOH를 넣으십시오. </li><li> 철저하게 혼합하고 이동상 A (MA)로 표시하십시오. 이니셜, 준비 날짜 및 만료 날짜를 포함하십시오. </li><li> LC-MS 메탄올 2 L 병을 가져 와서 시작 날짜와 만기 날짜가있는 이동상 B (MB)로 표시하십시오. </li><li> 자동 샘플러의 온도를 4 ° C로 설정하십시오. </li><li> 준비 샘플과 함께 수집 판을 오토 샘플러에 놓습니다. </li><li> 오븐에 PFP 컬럼 (150 mm x 2.1 mm id, 3 μm)과 보호 컬럼을 놓습니다. </li><li> 오븐 온도를 30 ° C로 설정하십시오. </li><li> 표 1에 표시된대로 LC 그라디언트 용리로 획득 방법을 사용하여 10 분간 평형을 이룬다. </li></ol><table border="1" fo:keep-together.within-page="1" fo:keep-with-next.within-page="always"><tbody><tr><td> 시간 (분) </td><td> 기준 치수 </td><td> 이벤트 </td><td> 매개 변수 </td></tr><tr><td> 0 </td><td> 슬리퍼 </td><td> 펌프 B Conc. </td><td> 5 </td></tr><tr><td> 0.5 </td><td> 슬리퍼 </td><td> 펌프 B Conc. </td><td> 20 </td></tr><tr><td> 1 </td><td> 슬리퍼 </td><td> 펌프 B Conc. </td><td> 50 </td></tr><tr><td> 삼 </td><td> 슬리퍼 </td><td> 펌프 B Conc. </td><td> 80 </td></tr><tr><td> 4 </td><td> 슬리퍼 </td><td> 펌프 B Conc. </td><td> 90 </td></tr><tr><td> 4.01 </td><td> 슬리퍼 </td><td> 펌프 B Conc. </td><td> 95 </td></tr><tr><td> 5.5 </td><td> 슬리퍼 </td><td> 펌프 B Conc. </td><td> 95 </td></tr><tr><td> 5.6 </td><td> 슬리퍼 </td><td> 펌프 B Conc. </td><td> 5 </td></tr><tr><td> 7 </td><td> 제어 장치 </td><td> 중지 </td><td></td></tr></tbody></table> 표 1 : 액체 크로마토 그라데이션 용출 조건 <ol start="10"><li> 다음을 포함하는 일괄 처리 목록 만들기표준, 품질 관리 및 소변 샘플. </li><li> 준비 샘플 (12 μL)의 주사에 의해 배치를 시작합니다. </li></ol> 5. 피크 식별, 통합 및 데이터 프로세스 <ol><li> 소프트웨어를 사용하여 데이터 수집 및 처리를 제어합니다. </li><li> 모든 샘플에 대해 3-NT 및 IS 피크를 확인하고 통합하십시오. </li><li> 1-x의 가중치 인자로 3-NT와 IS의 피크 면적비와 명목상 3-NT 농도의 선형 회귀에 의한 3-NT 정량을위한 10-2,500 pg / mL 범위의 표준 곡선을 확립하십시오. </li><li> 표준 곡선을 사용하여 모든 샘플을 정량화하십시오. </li><li> QC 샘플이 설정된 범위에 속하는지 확인하십시오. </li><li> 검출 된 소변 샘플의 농도를 nM 또는 nmol / mmol Cr 단위로 최종 결과로 변환하십시오. </li></ol>

<ol>
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R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Immunoaffinity+liquid+chromatography-tandem+mass+spectrometry+detection+of+nitrotyrosine+in+biological+fluids+development+of+a+clinically+translatable+biomarker.">Immunoaffinity liquid chromatography-tandem mass spectrometry detection of nitrotyrosine in biological fluids development of a clinically translatable biomarker.</a> Anal. Biochem. 380 (1), 68-76 (2008).</li><li>Li, X. G., Li, S., Kellermann, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Tailored+96-well+&#181;Elution+solid-phase+extraction+combined+with+UFLC-MS/MS:+a+significantly+improved+approach+for+determination+of+free+3-nitrotyrosine+in+human+urine.">Tailored 96-well &#181;Elution solid-phase extraction combined with UFLC-MS/MS: a significantly improved approach for determination of free 3-nitrotyrosine in human urine.</a> Anal. Bioanal. Chem. 407 (25), 7703-7712 (2015).</li><li>. Roche Creatinine Jaffé Gen.2, package insert 2011-11, V7 Available from: <a target="_blank" href="https://usdiagnostics.roche.com/products/06407137190/PARAM2083/overlay.html">https://usdiagnostics.roche.com/products/06407137190/PARAM2083/overlay.html</a> (2011)</li><li>Li, X. G., Li, S., Kellermann, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+novel+mixed-mode+solid+phase+extraction+coupled+with+LC-MS/MS+for+the+re-evaluation+of+free+3-nitrotyrosine+in+human+plasma+as+an+oxidative+stress+biomarker.">A novel mixed-mode solid phase extraction coupled with LC-MS/MS for the re-evaluation of free 3-nitrotyrosine in human plasma as an oxidative stress biomarker.</a> Talanta. 140, 45-51 (2015).</li><li>Li, X. G., Li, S., Kellermann, G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+integrated+liquid+chromatography-tandem+mass+spectrometry+approach+for+the+ultra-sensitive+determination+of+catecholamines+in+human+peripheral+blood+mononuclear+cells+to+assess+neural-immune+communication.">An integrated liquid chromatography-tandem mass spectrometry approach for the ultra-sensitive determination of catecholamines in human peripheral blood mononuclear cells to assess neural-immune communication.</a> J. Chromatogr. A. 1449, 54-61 (2016).</li></ol>

저자는 이해 상충이 없다고 선언합니다.

인간 소변 샘플의 내인성 자유 3-NT에 대한 문헌에서 이전에보고 된 농도의 상당한 변화는 이용 가능한 분석 8 , 9 , 10 , 11 과 관련된 방법 론적 문제점을 나타낸다. 인간 소변에서 3-NT의 낮은 기본 수준을 정확하게 결정하는 것은 시료 준비 및 LC-MS / MS 분석을위한 특별한 예방 조치가 필요한 까다로운 ?...

최근 임상 연구에서 산화 스트레스의 영향이 최전방으로 치닫고 있습니다 1 . 연구되는 바이오 마커 중 하나는 반응성 질소 종 (RNS)이 티로신 (catecholamine neurotransmitter precursor)과 상호 작용할 때 형성된 안정한 제품인 3- 니트로 티로신 (3-NT)입니다. 3-NT는 생체 내 RNS의 바이오 마커로서 임상 적 가치를 가질 수 있지만 , 티로신의 성질과 기능의 실질적인 변화는 해당 단백질과 세포 기능에 악영향을 미칠 수 있습니다 1 , 2 . 신생 연구는 3-NT가 염증성 질환 3 , 신경 퇴행성 질환 4 , 5 , 심혈관 질환 6 및 당뇨병 7 뿐만 아니라 산화 스트레스와 관련된 상태에서 중요한 역할을 할 수 있다고 제안했다. 그러나, 이러한 obse민감도 및 / 또는 선택성이 결여 된 방법론 8 , 9 , 10 , 11의 결과에 근거합니다. 이전에 문헌에보고 된 생물학적 시료에 대한 거대한 3-NT 농도 범위는 심각한 분석 문제가 이러한 분석법과 관련되어 있으며 3-NT 수준을 정확하게 계량화하고 이러한 병리 현상에서의 역할을 확인하기위한 기술적 개선이 필요함을 보여줍니다 . 생물학적 기질에서 유리 3-NT의 정량은 사람과 도구 8 , 9 , 10 , 11에 특별한 도전이됩니다. 첫째, 내인성 3-NT의 추적 수준은 매우 민감한 탐지를 요구합니다. 둘째로 구조적으로 유사한 많은 유사체, 특히 티로신이 존재한다.광대 한 과잉은, 높은 선택성을 요구한다; 셋째, 유비쿼터스 질산염과 아질산염으로 티로신 니트로 화에 의한 3-NT의 인공 생성물은 3-NT의 잘못된 과대 평가를 피하기 위해 시료 준비 중에 특별한 고려가 필요하다. 3-NT를 측정하기 위해 사용 된 다양한 방법론 중에서 MS / MS는 탁월한 감도 및 선택성 11 , 12 , 13 , 14 로 인해 금 표준 방법으로 간주되었습니다. 가스 크로마토 그래피 (GC) 결합 MS / MS가 가장 우수한 감도를 제공하지만 필수적인 유도체 화 단계는 임상 유틸리티 15 , 16에 비해 너무 지루하고 시간 소모적입니다. LC-MS / MS는 복잡한 표본 유도체 화를 필요로하지 않으므로 더 유망한 옵션입니다. 그럼에도 불구하고 se와 같은 극복해야 할 몇 가지 장애물이 있습니다.낮은 농도의 3-NT 7 , 17 , 18 의 측정을 위해서는 문헌에보고 된 LC-MS / MS 방법의 사용성이 향상되어야하고 고 처리량 응용 12 , 13 , 17 , 19에 대해서는 상대적으로 긴 처리 시간을 단축해야합니다 . 또한, 임상 적용을 고려할 때, 사용 된 생물학적 기질이 중요한 역할을한다. 가능한 경우 쉽고 저렴해야하며 가능한 경우 비 침습적이어야합니다 20 , 21 , 22 . 문헌에서 전통적으로 사용 된 혈장은 임상 적으로 바람직한 매트릭스가 아니므로, 비 침습적이고 비용 효과가있는 소변을 이용하는 방법론이 필요하다. rel 개발을위한 몇 가지 시도iable 및 특정 LC-MS / MS 방법은 소변 9 , 10 , 11을 사용하여 이루어졌습니다. 그러나, 그들은 모두 임상 적 사용을 위해 선택적이고, 신뢰할 만하 며, 효율적이지 못하다. 3 NT 분석을위한 샘플 정리 등과 같은 전통적인 역상 카트리지 (C18 타입)를 사용하여 지배적 인 SPE의 효과는 의문을 제기하고 강력한 양이온 교환의 순차적 인 SPE (SCX)와 역상 C18-OH 6 제안되었다되었습니다 7 , 19 . 한 최근 개발 된 LC-MS / MS 방법 3- NT (23)의 분석을위한 설명서 C18 SPE, 예비 고압 액체 크로마토 그래피 (HPLC), 온라인 SPE의 다단계 정제 공정을 이용했다. 이 방법은 0.041 nM의 LLOQ로 임상 목적에 충분히 민감했지만, 정화 과정은 집중적이고 지루하고 requi적색 3 mL의 소변. 높은 처리량에 대한 가능성을 제한합니다. 분자 임프린트 폴리머 정리 프로세스 (14)의 효율을 개선하기 위해 SPE 흡착제로서 사용되었지만, 생성 된 LLOQ (0.7 ㎍ / mL)의 임상 검체 충분히 낮은 아니었다. 또 다른 방법은 0.022 nM 24 의 검출 한계 (LOD)를 달성하기 위해 샘플 정화를 위해 2 차원 (2D) LC-MS / MS 및 면역 친화 크로마토 그래피가 필요했습니다. 이러한 모든 방법들이 3-NT의 평가에서 진전을 보였지만 임상 적용에 필요한 민감성, 신뢰성 및 효율성을 달성 한 것은 없습니다. 무료 3-NT의 병리 및 임상 설정에서 산화 스트레스의 바이오 마커의 역할을 조사하기 위해, 우리는 높은 처리량 임상 적용 25 수 있도록, 간단하고 효율적 정확하고 정밀한하는 방법을 개발했다. 소형화 된 혼합 모드 양이온 엑시드유도체 화, 증발 및 2D-LC를 필요로하는 기존 방법에서 나타나는 단점을 우회하여 단일 추출에서 3-NT의 간단하고 효과적인 시료 정화 및 농축을 달성하기 위해 hange (MCX) 96-well 추출 마이크로 플레이트가 구현되었습니다. 0.01 % HCOOH가 첨가 된 액체 크로마토 그래피는 신속한 사이클 시간으로 향상된 신호 응답을 제공합니다. 선택성은 3-NT의 선택적 용리 및 3-NT 및 내부 표준 (IS) 모두에 대한 MRM 전이의 사용을위한 약한 NH 4 OAc 용출 용액의 적용을 통해 더욱 향상되었다. 기질 효과는 정량화를 위해 선호되는 13 C- 동위 원소 IS의 감소 된 양을 사용함으로써 보상되었다. 이 방법론의 출현과 함께 연구원 및 임상의는 임상 적 상태에서 3-NT의 역할을 검증하고 산화 스트레스의 영향을 더 탐구 할 수있을 것이다.

<table><tbody><tr><td>3-Nitro-L-tyrosine</td><td>Sigma</td><td>N7389-5g</td><td></td></tr><tr><td>3-Nitro-L-tyrosine-13C&lt;sub&gt;9&lt;/sub&gt;</td><td>Sigma</td><td>652296-5.0mg</td><td></td></tr><tr><td>Mass Spec Gold Urine</td><td>Golden West Biologicals</td><td>MSG 5000-1L</td><td></td></tr><tr><td>Oasis MCX 96-well &amp;micro;Elution plate</td><td>Waters</td><td>186001830BA</td><td></td></tr><tr><td>2 mL 96 well collection plate</td><td>Phenomenex&amp;nbsp;&amp;nbsp;</td><td>AH0-7194</td><td></td></tr><tr><td>96 positive processor</td><td>Waters&amp;nbsp;</td><td>186005521</td><td></td></tr><tr><td>LC-MS Ultra CHROMASOLV methanol&amp;nbsp;&amp;nbsp;</td><td>Sigma</td><td>14262-2L</td><td></td></tr><tr><td>LC-MS Ultra CHROMASOLV water</td><td>Sigma</td><td>14263-2L</td><td></td></tr><tr><td>Formic acid for mass spectrometry</td><td>Sigma</td><td>94318-50ML-F</td><td></td></tr><tr><td>Ammonium hydroxide solution</td><td>Sigma</td><td>338818-1L</td><td></td></tr><tr><td>Ultra PFP propyl columns</td><td>Restek</td><td>9179362</td><td></td></tr><tr><td>5500 Triple quad</td><td>AB Sciex&amp;nbsp;</td><td>/</td><td>Contact manufacture for more detail</td></tr><tr><td>UFLC-XR</td><td>Shimadzu&amp;nbsp;</td><td>/</td><td>Contact manufacture for more detail</td></tr><tr><td>Integra 400 Plus&amp;nbsp;</td><td>Roche</td><td>/</td><td>Urinary Creatinine Jaff&amp;eacute; Gen 2 method</td></tr><tr><td>LCMS certified 12 x 32 mm screw neck vial</td><td>Waters</td><td>600000751CV</td><td></td></tr><tr><td>LCGC certified 12 x 32 mm screw neck total recovery vial</td><td>Waters</td><td>186000384C</td><td></td></tr><tr><td>5 mL transport tube</td><td>Phenix</td><td>TT-3205</td><td></td></tr><tr><td>50 mL Centrifuge tube</td><td>Crystalgen&amp;nbsp;</td><td>23-2263</td><td></td></tr><tr><td>15 mL Centrifuge tube</td><td>Crystalgen&amp;nbsp;</td><td>23-2266</td><td></td></tr><tr><td>eLine electronic pipette</td><td>Sartorius</td><td>730391</td><td></td></tr><tr><td>Microfuge centrifuge&amp;nbsp;</td><td>Beckman Coulter</td><td>A46474</td><td></td></tr><tr><td>OHAUS balance&amp;nbsp;&amp;nbsp;</td><td>Kennedy Scales, inc.</td><td>735</td><td></td></tr><tr><td>Vortex mixer&amp;nbsp;</td><td>Bernstead Thermolyne</td><td>M16715</td><td></td></tr></tbody></table>

integration of miniaturized solid phase extraction and lc-ms/ms detection of 3-nitrotyrosine in human urine for clinical applications

Free 3- nitrotyrosine (3-NT)은 산화 스트레스에 대한 가능한 바이오 마커로 광범위하게 사용되어 왔습니다. 3-NT의 증가 된 수준은 다양한 병리학 적 조건에서보고되었다. 그러나 기존의 방법은 3-NT의 낮은 내인성 수준을 안정적으로 측정하는 데 필요한 충분한 감도 및 / 또는 특이성이 결여되어 있으며 임상 적용에 너무 복잡합니다. 따라서, 3-NT의 수준을 정확히 정량화하고 병리학 적 조건에서 3-NT의 역할을 검증하기 위해서는 분석적 개선이 시급히 필요하다. 이 프로토콜은 비 침습성 바이오 마커로서 인간의 소변에서 3-NT의 신속하고 정확한 측정을위한 소형 고체상 추출 (SPE)과 결합 된 새로운 액체 크로마토 그래피 탠덤 질량 분석 (LC-MS / MS) 검출의 개발을 제시합니다 산화 스트레스. SPE는 96- 웰 플레이트를 사용하여 지루한 유도체 화 및 증발 단계없이 샘플 클린업 및 분석 물 농축을 결합함으로써 공정을 현저히 단순화시켰다, 용매 소비, 폐기물 처리, 오염 위험 및 전반적인 처리 시간 감소. SPE 용출 용액으로서 pH 9에서 25 mM 암모늄 아세테이트 (NH 4 OAc)를 사용함으로써 선택성이 실질적으로 향상되었다. 질량 분석법 신호 응답은 다중 반응 모니터링 (MRM) 전환의 조정을 통해 향상되었습니다. 펜타 플루오로 페닐 (PFP) 컬럼 (150 mm x 2.1 mm, 3 μm)에서 첨가제로 0.01 % HCOOH를 사용하면 신호 응답이 2.5 배 향상되었고 전체 실행 시간이 7 분으로 단축되었습니다. 10 pg / mL (0.044 nM)의 정량 하한 (LLOQ)이 달성되어보고 된 분석법에 비해 현저한 민감도 향상을 나타냅니다. 이 간단하고 신속한 선택적이고 민감한 방법으로 24 시간 동안 2 장의 소변 샘플 (n = 192)을 처리 할 수 ​​있습니다. 현저하게 개선 된 분석 성능과 비 침습성 및 저렴한 소변 샘플링을 고려하면, 제안 된 분석법은 전임상 및 임상연구.

그림 1 은 3-NT가 최적화 된 LC 조건 하에서 구조적으로 유사한 다른 티로신 유사체로부터 완전히 크로마토 그래피 적으로 분리되어있어 이러한 과도한 화합물로 인한 용출 간섭을 제거하여 결과적으로 분석 선택도를 향상 시킨다는 것을 보여줍니다. 또한 MA 및 메탄올의 첨가제 인 0.01 % HCOOH를 유속 0.45 mL / min로 사용하는 구배 용출은 3-NT ( 즉 , ...

Watch this Scientific Journal Video about Integration of Miniaturized Solid Phase Extraction and LC-MS/MS Detection of 3-Nitrotyrosine in Human Urine for Clinical Applications at JoVE.com

Integration of Miniaturized Solid Phase Extraction and LC-MS/MS Detection of 3-Nitrotyrosine in Human Urine for Clinical Applications

혼합 모드 양이온 교환 (MCX) 96- 웰 마이크로 플레이트상에서 효율적인 고체상 추출과 결합 된 선택적이고 민감한 액체 크로마토 그래피 탠덤 질량 분석법 (LC-MS / MS)이 유리 3- 니트로 티로신의 측정을 위해 개발되었다 3-NT)을 임상 적으로 사용하기에 적합합니다.

임상 응용을위한 인간의 소변에서 3- 니트로 타이로신의 소형 고체 상 추출 및 LC-MS / MS 검출의 통합

Free 3-nitrotyrosine (3-NT) has been extensively used as a possible biomarker for oxidative stress. Increased levels of 3-NT have been reported in a wide ...

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Osmoregulation and Excretion

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9.2K 조회수.  Pharmasan Labs, Inc..  혼합 모드 양이온 교환 (MCX) 96- 웰 마이크로 플레이트상에서 효율적인 고체상 추출과 결합 된 선택적이고 민감한 액체 크로마토 그래피 탠덤 질량 분석법 (LC-MS / MS)이 유리 3- 니트로 티로신의 측정을 위해 개발되었다 3-NT)을 임상 적으로 사용하기에 적합합니다. 

비디오: Integration of Miniaturized Solid Phase Extraction and LC-MS/MS Detection of 3-Nitrotyrosine in Human Urine for Clinical Applications

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry (UPLC-HRMS)

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first separated into polar and non-polar fractions by a liquid-liquid phase extraction, and partially purified to facilitate downstream analysis. Both aqueous (polar metabolites) and organic (non-polar metabolites) phases of the initial extraction are processed to survey a broad range of metabolites. Metabolites are separated by different liquid chromatography methods based upon their partition properties. In this method, we present microflow ultra-performance (UP)LC methods, but the protocol is scalable to higher flows and lower pressures. Introduction into the mass spectrometer can be through either general or compound optimized source conditions. Detection of a broad range of ions is carried out in full scan mode in both positive and negative mode over a broad m/z range using high resolution on a recently calibrated instrument. Label-free differential analysis is carried out on bioinformatics platforms. Applications of this approach include metabolic pathway screening, biomarker discovery, and drug development.

Untargeted Metabolomics from Biological Sources Using Ultraperformance Liquid Chromatography-High Resolution Mass Spectrometry UPLC-HRMS

Untargeted metabolomics provides a hypothesis generating snapshot of a metabolic profile. This protocol will demonstrate the extraction and analysis of metabolites from cells, serum, or tissue. A range of metabolites are surveyed using liquid-liquid phase extraction, microflow ultraperformance liquid chromatography/high-resolution mass spectrometry (UPLC-HRMS) coupled to differential analysis software.

Ultraperformance 액체 크로마토 그래피 고해상도 질량 분석 (UPLC-HRMS)를 사용하여 생물 소스에서 타겟이 불분명 한 대사

Here we present a workflow to analyze the metabolic profiles for biological samples of interest including; cells, serum, or tissue. The sample is first ...

연구

화학

A General Method for Detecting Nitrosamide Formation in the In Vitro Metabolism of Nitrosamines by Cytochrome P450s

N-nitrosamines are a well-established group of environmental carcinogens, which require cytochrome P450 oxidation to exhibit activity. The accepted mechanism of metabolic activation involves formation of &#945;-hydroxynitrosamines that spontaneously decompose to DNA alkylating agents. Accumulation of DNA damage and the resulting mutations can ultimately lead to cancer. New evidence indicates that &#945;-hydroxynitrosamines can be further oxidized to nitrosamides processively by cytochrome P450s. Because nitrosamides are generally more stable than &#945;-hydroxynitrosamines and can also alkylate DNA, nitrosamides may play a role in carcinogenesis. In this report, we describe a general protocol for evaluating nitrosamide production from in vitro cytochrome P450-catalyzed metabolism of nitrosamines. This protocol utilizes a general approach to the synthesis of the relevant nitrosamides and an in vitro cytochrome P450 metabolism assay using liquid chromatography-nanospray ionization-high resolution tandem mass spectrometry for detection. This method detected N&#8242;-nitrosonorcotinine as a minor metabolite of N&#8242;-nitrosonornicotine in the example study. The method has high sensitivity and selectively due to accurate mass detection. Application of this method to a wide variety of nitrosamine-cytochrome P450 systems will help determine the generality of this transformation. Because cytochrome P450s are polymorphic and vary in activity, a better understanding of nitrosamide formation could aid in individual cancer risk assessment.

A General Method for Detecting Nitrosamide Formation in the In Vitro Metabolism of Nitrosamines by Cytochrome P450s

&#945;-hydroxylation of carcinogenic nitrosamines by cytochrome P450s is the accepted metabolic pathway that produces DNA-damaging intermediates, which cause mutations. However, new data indicates further oxidation to nitrosamides can occur. We describe a general method for detecting nitrosamides produced from in vitro cytochrome P450-catalyzed metabolism of nitrosamines.

체 외에 대사 시 토 크롬 P450s에 의해 니트로의 Nitrosamide 형성을 탐지 하기 위한 일반적인 방법

N-nitrosamines are a well-established group of environmental carcinogens, which require cytochrome P450 oxidation to exhibit activity. The accepted ...

암 연구학

Sampling Human Indigenous Saliva Peptidome Using a Lollipop-Like Ultrafiltration Probe: Simplify and Enhance Peptide Detection for Clinical Mass Spectrometry

Although human saliva proteome and peptidome have been revealed 1-2 they were majorly identified from tryptic digests of saliva proteins. Identification of indigenous peptidome of human saliva without prior digestion with exogenous enzymes becomes imperative, since native peptides in human saliva provide potential values for diagnosing disease, predicting disease progression, and monitoring therapeutic efficacy. Appropriate sampling is a critical step for enhancement of identification of human indigenous saliva peptidome. Traditional methods of sampling human saliva involving centrifugation to remove debris 3-4 may be too time-consuming to be applicable for clinical use. Furthermore, debris removal by centrifugation may be unable to clean most of the infected pathogens and remove the high abundance proteins that often hinder the identification of low abundance peptidome.
Conventional proteomic approaches that primarily utilize two-dimensional gel electrophoresis (2-DE) gels in conjugation with in-gel digestion are capable of identifying many saliva proteins 5-6. However, this approach is generally not sufficiently sensitive to detect low abundance peptides/proteins. Liquid chromatography-Mass spectrometry (LC-MS) based proteomics is an alternative that can identify proteins without prior 2-DE separation. Although this approach provides higher sensitivity, it generally needs prior sample pre-fractionation 7 and pre-digestion with trypsin, which makes it difficult for clinical use. 
To circumvent the hindrance in mass spectrometry due to sample preparation, we have developed a technique called capillary ultrafiltration (CUF) probes 8-11. Data from our laboratory demonstrated that the CUF probes are capable of capturing proteins in vivo from various microenvironments in animals in a dynamic and minimally invasive manner 8-11. No centrifugation is needed since a negative pressure is created by simply syringe withdrawing during sample collection. The CUF probes combined with LC-MS have successfully identified tryptic-digested proteins 8-11. In this study, we upgraded the ultrafiltration sampling technique by creating a lollipop-like ultrafiltration (LLUF) probe that can easily fit in the human oral cavity. The direct analysis by LC-MS without trypsin digestion showed that human saliva indigenously contains many peptide fragments derived from various proteins. Sampling saliva with LLUF probes avoided centrifugation but effectively removed many larger and high abundance proteins. Our mass spectrometric results illustrated that many low abundance peptides became detectable after filtering out larger proteins with LLUF probes. Detection of low abundance saliva peptides was independent of multiple-step sample separation with chromatography. For clinical application, the LLUF probes incorporated with LC-MS could potentially be used in the future to monitor disease progression from saliva.

Considering saliva sampling for future clinical application, a lollipop-like ultrafiltration (LLUF) probe was fabricated to fit in the human oral cavity. Direct analysis of undigested saliva by NanoLC-LTQ mass spectrometry demonstrated the ability of LLUF probes to remove large proteins and high abundance proteins, and make low-abundant peptides more detectable.

롤리팝 - 같은 Ultrafiltration 프로브를 사용하여 인간의 원주민 타액 Peptidome를 샘플링 : 임상 질량 분광법을위한 펩타이드 감지를 단순화하고 향상

Although human saliva proteome and peptidome have been revealed 1-2 they were majorly identified from tryptic digests of saliva proteins. Identification ...

의학

Detection of 3-Nitrotyrosine in Atmospheric Environments via a High-performance Liquid Chromatography-electrochemical Detector System

3-nitrotyrosine (3-NT) is generated from the tyrosine residue in atmospheric bio-aerosol proteins via a reaction with ozone (O3) and nitrogen dioxide (NO2). Stable 3-NT is a specific marker for oxidative damage and is reported to have a promotive effect to elicit allergies. In the present study, we report the development of a highly sensitive assay to quantify 3-NT in air sampler filters to collect &#60; 2.5 &#181;m of particulate matter (PM2.5) from urban environmental air, including bio-aerosol. In this method, a 6 mm-diameter round hole was cut from the filters of air samplers and mixed with a nonspecific protease cocktail in order to hydrolyze proteins. Protein samples digested to the amino acid level were tested for 3-NT using a high-performance liquid chromatography-electrochemical detector (HPLC-ECD). The maximum 3-NT content was detected in a prefilter for PM of sizes from 4.5 to 7.3 &#956;m, with a detection limit of 1.13 pg/m3.

Detection of 3-Nitrotyrosine in Atmospheric Environments via a High-performance Liquid Chromatography-electrochemical Detector System

We present a method to detect 3-nitrotyrosine chemical modifications of atmospheric proteins with 6 mm-diameter rounds cut from air sampler prefilters using a high-performance liquid chromatography-electrochemical detector (HPLC-ECD).

3-Nitrotyrosine 대기 환경을 통해 고성능 액체 착 색 인쇄기-전기 화학 검출기 시스템에서의 탐지

3-nitrotyrosine (3-NT) is generated from the tyrosine residue in atmospheric bio-aerosol proteins via a reaction with ozone (O3) and nitrogen dioxide ...

환경과학

A Convenient Method for Extraction and Analysis with High-Pressure Liquid Chromatography of Catecholamine Neurotransmitters and Their Metabolites

The extraction and analysis of catecholamine neurotransmitters in biological fluids is of great importance in assessing nervous system function and related diseases, but their precise measurement is still a challenge. Many protocols have been described for neurotransmitter measurement by a variety of instruments, including high-pressure liquid chromatography (HPLC). However, there are shortcomings, such as complicated operation or hard-to-detect multiple targets, which cannot be avoided, and presently, the dominant analysis technique is still HPLC coupled with sensitive electrochemical or fluorimetric detection, due to its high sensitivity and good selectivity. Here, a detailed protocol is described for the pretreatment and detection of catecholamines with high pressure liquid chromatography with electrochemical detection (HPLC-ECD) in real urine samples of infants, using electrospun composite nanofibers composed of polymeric crown ether with polystyrene as adsorbent, also known as the packed-fiber solid phase extraction (PFSPE) method. We show how urine samples can be easily precleaned by a nanofiber-packed solid phase column, and how the analytes in the sample can be rapidly enriched, desorbed, and detected on an ECD system. PFSPE greatly simplifies the pretreatment procedures for biological samples, allowing for decreased time, expense, and reduction of the loss of targets. 
Overall, this work illustrates a simple and convenient protocol for solid-phase extraction coupled to an HPLC-ECD system for simultaneous determination of three monoamine neurotransmitters (norepinephrine (NE), epinephrine (E), dopamine (DA)) and two of their metabolites (3-methoxy-4-hydroxyphenylglycol (MHPG) and 3,4-dihydroxy-phenylacetic acid (DOPAC)) in infants' urine. The established protocol was applied to assess the differences of urinary catecholamines and their metabolites between high-risk infants with perinatal brain damage and healthy controls. Comparative analysis revealed a significant difference in urinary MHPG between the two groups, indicating that the catecholamine metabolites may be an important candidate marker for early diagnosis of cases at risk for brain damage in infants.

We present a convenient solid-phase extraction coupled to high-pressure liquid chromatography (HPLC) with electrochemical detection (ECD) for simultaneous determination of three monoamine neurotransmitters and two of their metabolites in infants' urine. We also identify the metabolite MHPG as a potential biomarker for the early diagnosis of brain damage for infants.

추출 및 카 테 콜 아민 신경 전달 물질과 그들의 대사 산물의 고압 액체 크로마토그래피로 분석 하는 편리한 방법

The extraction and analysis of catecholamine neurotransmitters in biological fluids is of great importance in assessing nervous system function and ...

Nitropeptide Profiling and Identification Illustrated by Angiotensin II

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of reactive oxygen species (ROS) and reactive nitrogen species (RNS) in eukaryotic cells. Precise identification of nitration sites on proteins is crucial for understanding the physiological and pathological processes related to protein nitration, such as inflammation, aging, and cancer. Since the nitrated proteins are of low abundance in cells even under induced conditions, no universal and efficient methods have been developed for the profiling and identification of protein nitration sites. Here we describe a protocol for nitropeptide enrichment by using a chemical reduction reaction and biotin labeling, followed by high resolution mass spectrometry. In our method, nitropeptide derivatives can be identified with high accuracy. Our method exhibits two advantages compared to the previously reported methods. First, dimethyl labeling is used to block the primary amine on nitropeptides, which can be used to generate quantitative results. Second, a disulfide bond containing NHS-biotin reagent is used for the enrichment, which can be further reduced and alkylated to enhance the detection signal on a mass spectrometer. This protocol has been successfully applied to the model peptide Angiotensin II in the current paper.

Proteomic profiling of tyrosine-nitrated proteins has been a challenging technique due to the low abundance of the 3-nitrotyrosine modification. Here we describe a novel approach for nitropeptide enrichment and profiling by using Angiotensin II as the model. This method can be extended for other in vitro or in vivo systems.

안지오텐신 II에 의해 설명 니트로 펩티드 프로파일링 및 식별

Protein nitration is one of the most important post-translational modifications (PTM) on tyrosine residues and it can be induced by chemical actions of ...

생화학

High-throughput and Comprehensive Drug Surveillance Using Multisegment Injection-Capillary Electrophoresis-Mass Spectrometry

New analytical methods are urgently needed to enable high-throughput, yet comprehensive drug screening, given an alarming opioid and prescription drug crisis in public health. Conventional urine drug testing based on a two-tier immunoassay screen followed by a gas chromatography-tandem mass spectrometry (GC-MS/MS) or liquid chromatography-tandem mass spectrometry (LC-MS/MS) method are expensive and prone to bias while being limited to targeted panels of known drugs of abuse (DoA). Herein, we outline an improved method for drug surveillance that allows for the resolution and detection of an expanded panel of DoA and their metabolites when using multisegment injection-capillary electrophoresis-mass spectrometry (MSI-CE-MS). Multiplexed separations of ten urine samples with a quality control by CE (&lt; 3 min/sample) in conjunction with full-scan data acquisition using a time-of-flight mass spectrometer (TOF-MS) under positive ion mode detection allows for the identification and quantification of DoA above recommended cut-off levels. An excellent resolution of drug isomers and isobars, including background interferences, are achieved when using MSI-CE-MS with an electrokinetic spacer between sample segments, where accurate mass/molecular formula together with the comigration of a matching deuterated internal standard and the detection of one or more bio-transformed metabolites facilitate DoA identification over a wider detection window. Additionally, urine samples can be analyzed directly without enzyme deconjugation for the rapid screening without complicated sample workup. MSI-CE-MS enables the surveillance of a broad spectrum of DoA that is required for the treatment monitoring of high-risk patients, including confirming prescribed drug adherence, revealing illicit drug use/substitution, and evaluating optimal dosage regimes as required for new advances in precision medicine.

Here we describe a high-throughput method for comprehensive drug surveillance that allows for the improved resolution and detection of large panels of drugs of abuse and their metabolites, with quality control based on multisegment injection-capillary electrophoresis-mass spectrometry.

멀티 주입-모 세관 전기 이동 법 질량 분석을 사용 하 여 높은 처리량 및 포괄적인 약물 감시

New analytical methods are urgently needed to enable high-throughput, yet comprehensive drug screening, given an alarming opioid and prescription drug ...

Fast Enzymatic Processing of Proteins for MS Detection with a Flow-through Microreactor

The vast majority of mass spectrometry (MS)-based protein analysis methods involve an enzymatic digestion step prior to detection, typically with trypsin. This step is necessary for the generation of small molecular weight peptides, generally with MW &#60; 3,000-4,000 Da, that fall within the effective scan range of mass spectrometry instrumentation. Conventional protocols involve O/N enzymatic digestion at 37 &#186;C. Recent advances have led to the development of a variety of strategies, typically involving the use of a microreactor with immobilized enzymes or of a range of complementary physical processes that reduce the time necessary for proteolytic digestion to a few minutes (e.g., microwave or high-pressure). In this work, we describe a simple and cost-effective approach that can be implemented in any laboratory for achieving fast enzymatic digestion of a protein. The protein (or protein mixture) is adsorbed on C18-bonded reversed-phase high performance liquid chromatography (HPLC) silica particles preloaded in a capillary column, and trypsin in aqueous buffer is infused over the particles for a short period of time. To enable on-line MS detection, the tryptic peptides are eluted with a solvent system with increased organic content directly in the MS ion source. This approach avoids the use of high-priced immobilized enzyme particles and does not necessitate any aid for completing the process. Protein digestion and complete sample analysis can be accomplished in less than ~3 min and ~30 min, respectively.

A quick protocol for proteolytic digestion with an in-house built flow-through tryptic microreactor coupled to an electrospray ionization (ESI) mass spectrometer is presented. The fabrication of the microreactor, the experimental setup and the data acquisition process are described.

플로우를 통해 마이크로 반응기와 MS 검출을위한 단백질의 빠른 효소 처리

The vast majority of mass spectrometry (MS)-based protein analysis methods involve an enzymatic digestion step prior to detection, typically with trypsin. ...

생체공학

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

The kynurenine pathway and the tryptophan catabolites called kynurenines have received increased attention for their involvement in immune regulation and cancer biology. An in vitro cell culture assay is often used to learn about the contribution of different tryptophan catabolites in a disease mechanism and for testing therapeutic strategies. Cell culture medium that is rich in secreted metabolites and signaling molecules reflects the status of tryptophan metabolism and other cellular events. New protocols for the reliable quantification of multiple kynurenines in the complex cell culture medium are desired to allow for a reliable and quick analysis of multiple samples. This can be accomplished with liquid chromatography coupled with mass spectrometry. This powerful technique is employed in many clinical and research laboratories for the quantification of metabolites and can be used for measuring kynurenines.
Presented here is the use of liquid chromatography coupled with single quadrupole mass spectrometry (LC-SQ) for the simultaneous determination of four kynurenines, i.e., kynurenine, 3-hydroxykynurenine, 3-hydroxyanthranilic, and xanthurenic acid in the medium collected from in vitro cultured cancer cells. SQ detector is simple to use and less expensive compared to other mass spectrometers. In the SQ-MS analysis, multiple ions from the sample are generated and separated according to their specific mass-to-charge ratio (m/z), followed by the detection using a Single Ion Monitoring (SIM) mode.
This paper draws the attention on the advantages of the reported method and indicates some weak points. It is focused on critical elements of LC-SQ analysis including sample preparation along with chromatography and mass spectrometry analysis. The quality control, method calibration conditions and matrix effect issues are also discussed. We described a simple application of 3-nitrotyrosine as one analog standard for all target analytes. As confirmed by experiments with human ovary and breast cancer cells, the proposed LC-SQ method&#160;generates reliable results and can be further applied to other in vitro cellular models.

Described here is a protocol for the determination of four different tryptophan metabolites generated in the kynurenine pathway (kynurenine, 3-hydroxykynurenine, xanthurenic acid, 3-hydroxyanthranilic acid) in the medium collected from cancer cell cultures analyzed by liquid chromatography coupled with a single quadrupole mass spectrometry.

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 ...

Liquid Chromatography Coupled to Refractive Index or Mass Spectrometric Detection for Metabolite Profiling in Lysate-based Cell-free Systems

Engineering cellular metabolism for targeted biosynthesis can require extensive design-build-test-learn (DBTL) cycles as the engineer works around the cell&#39;s survival requirements. Alternatively, carrying out DBTL cycles in cell-free environments can accelerate this process and alleviate concerns with host compatibility. A promising approach to cell-free metabolic engineering (CFME) leverages metabolically active crude cell extracts as platforms for biomanufacturing and for rapidly discovering and prototyping modified proteins and pathways. Realizing these capabilities and optimizing CFME performance requires methods to characterize the metabolome of lysate-based cell-free platforms. That is, analytical tools are necessary for monitoring improvements in targeted metabolite conversions and in elucidating alterations to metabolite flux when manipulating lysate metabolism. Here, metabolite analyses using high-performance liquid chromatography (HPLC) coupled with either optical or mass spectrometric detection were applied to characterize metabolite production and flux in E. coli S30 lysates. Specifically, this report describes the preparation of samples from CFME lysates for HPLC analyses using refractive index detection (RID) to quantify the generation of central metabolic intermediates and by-products in the conversion of low-cost substrates (i.e., glucose) to various high-value products. The analysis of metabolite conversion in CFME reactions fed with 13C-labeled glucose through reversed-phase liquid chromatography coupled to tandem mass spectrometry (MS/MS), a powerful tool for characterizing specific metabolite yields and lysate metabolic flux from starting materials, is also presented. Altogether, applying these analytical methods to CFME lysate metabolism enables the advancement of these systems as alternative platforms for executing faster or novel metabolic engineering tasks.

The protocols describe high-performance liquid chromatography methods coupled to refractive index or mass spectrometric detection for studying metabolic reactions in complex lysate-based cell-free systems.

리자테 기반 세포 없는 시스템에서 대사산물 프로파일링을 위한 굴절률 또는 질량 분광 검출에 결합된 액체 크로마토그래피

Engineering cellular metabolism for targeted biosynthesis can require extensive design-build-test-learn (DBTL) cycles as the engineer works around the ...