We thank SP Gygi and GM Church for help and guidance. This work was supported by a CNRS chaire d'excellence to ACT.

참고 :이 방법은 이전에 12를 설명했다. 1. 단백질 분리 바람직하게는 프랑스어 프레스, 구슬 박동, 또는 초음파 등의 물리적 방법에 의해, 용해하는 세포에 의해 세포 단백질의 1 밀리그램을 준비합니다. 효소 질량 분석 측정을 혼동하기 때문에 라이소자임 - 매개 세포 용해를 방지. 단백질의 2. TCA 강수량 단백질을 침전 10 분 동안 얼음에 4 권의 단백질과 진정으로 1 볼륨 트리클로로 아세트산 (TCA)를 추가합니다. 원심 분리기 4 ° C에서 5 분 12,000 XG에 상층 액을 제거합니다. 4 ℃에서 5 분 동안 12,000 XG에 얼음처럼 차가운 아세톤 및 원심 분리기 1 ㎖에 펠렛을 재현 탁 상층 액을 제거하고 15 분 동안 펠렛을 건조 벤치에 튜브를 반전. -80 ° C에서 저장 단백질 펠렛 3. 변성 단백질과 이황화 채권을 감소 다시단백질을 중단 500 ㎕의 변성 및 감소 버퍼에 1 ~ 2 ㎎ / ㎖ (50 mM의 HEPES 산도 8.5, 5 mM의 DTT 4 M 요소 또는 3 % SDS 중 하나)에. 선택적으로, 버퍼에있는 단백질 분해 효소 억제제 (가) 있습니다. 실온에서 10 분 뒤에 56 ° C에서 30 분, 위해 단백질을 품어. 4. Aklylate 무료 설 프히 드릴 그룹은 돌이킬 수 이황화 결합 형성을 방해하는 물에 신선한 0.3 M의 요오도 아세트 아미드를 준비합니다. 주의! 요오도 아세트 아미드는 매우 독성이다. 500 ㎕의 단백질을 25 ㎕의 0.3 M 요오도 아세트 아미드 (15 mM의 최종 농도)를 추가하고 실온에서 어둠 속에서 20 분 동안 품어. 300 mM의 DTT (5 mM의 DTT 최종 농도)의 10 μl를 추가하여 요오도 아세트 아미드 끄다. C. -80 ° 알킬화 단백질을 저장 5. 단백질 소화 TCA는 단백질을 침전 (2 단계에 설명 된대로), 50 mM의 HEPES (산도 8.2) 1 ㎖, 1 M 요소의에 resuspend. 리실의 endoprotein의 재고 솔루션을 준비합니다2 ㎍ / μL의 농도와 단백질 용액에 5 μl를 추가 물에 ASE (리스-C). 실온에서 16 시간 동안 혼합물을 배양한다. 최종리스-C 농도가 10 NG / μL 및 단백질 - 투 -의 lysC 비율 (W / W) 1 / 200 ~ 1 / 50입니다 확인합니다. 50 mM의 아세트산의 40 μL에 20 μg 시퀀싱 등급 트립신을 재현 탁리스 - C 다이제스트에 5 μL (10 μg 트립신)를 추가하고, 37 ℃에서 6 시간 동안 배양 트립신에 사용되는리스-C에 대해 같은 단백질 분해 효소의 농도와 단백질 분해 효소 - 투 - 단백질 비율을 사용합니다. 6. 역상 펩타이드 추출 <ol><li> 0.5 %의 최종 농도 (산도 ≈ 2)에 트리 플루오로 아세트산 (TFA)를 첨가하여 펩티드를 산성화. 추출 매니 폴드에 C18 컬럼을 연결합니다. 펩티드의로드 및 용출를 제외한 모든 단계에 대한 가장 높은 유량을 사용합니다. </li><li> 6 ㎖의 아세토 니트릴 (ACN) 젖은 열입니다. 6 ㎖의 80 % ACN, 0.1 % TFA로 열을 세척 한 다음 10 ㎖의 0.1 % TFA와 평형. 허용하지 않음단계 사이에 건조 실행하는 열입니다. </li><li> 진공 압력을 정지하고 약 1 ㎖ / 분의 유속으로 칼럼에 펩타이드 500 μg를로드. 펩티드는 열, 다시 시작 진공에 바인딩 한 다음 (0.09 M 구연산, 0.23 M 나 2 HPO 4, pH를 5.5) 구연산 버퍼의 3 ㎖로, 10 ㎖의 0.1 % TFA로 세척 한 후. 참고 : 높은 펩티드 량이 표시 될 수 있지만 결합능 C18 컬럼이 적어도 샘플 손실을 피하기 위하여 펩티드 양보다 두 배이어야한다. </li></ol> 7.에 열 환원성 디메틸 화에 의해 펩타이드 라벨링 (REDI 라벨링) 시안화 수소는 레테르를 붙이는 과정에서 낮은 농도로 방출 등의 화학 후드에서이 단계를 수행합니다. <ol><li> 펩타이드 무료 아민을 메틸화하는 &quot;빛&quot;과 &quot;무거운&quot;REDI 버퍼 12 ML을 준비합니다. 밝은 REDI 버퍼는 0.8 %의 포름 알데히드 및​​ 번째에 수소를 운반하는 0.12 M 소듐 사이 아 노보로 하이드 라이드로 구성구연산 버퍼에 EIR 천연 동위 원소의 분포. 무거운 REDI 버퍼는 0.8 % 중수 소화 포름 알데히드와 구연산 완충액에 0.12 M 중수 소화 나트륨 시아 노보로 구성된다. </li><li> 1 ㎖ / 분의 유속으로 펩티드 함유 컬럼에 10 ㎖의 광 또는 농후 한 Redi 버퍼 하나를 첨가하여 펩티드를 함유하는 컬럼을 부화하고 완전한 라벨링을 위해 반복한다. 워시 6 ㎖의 0.1 % TFA와 열 한 후 1 ㎖의 0.5 % 아세트산. </li><li> 진공을 중단하고 약 0.5 ㎖ / 분의 유속을 사용하여 다음 1 ㎖의 80 % ACN으로, 0.5 % 초산 1 ml의 40 % ACN, 0.5 % 초산과 제 표지 펩티드​​를 용리 하였다. 원하는 경우, 무겁고 가벼운 샘플을 혼합하기 전에 질량 분석법에 의한 개별 샘플 라벨링 효율을 측정 ( &quot;대표 결과&quot;참조). 질량 분석에 의해 정량화 될 수 1:1 무겁고 가벼운 표지 펩타이드 샘플을 섞는다. </li></ol> 기본 pH를 역상 (BPRP) 크로마토 그래피 &lt;8. 별도의 펩타이드 혼합물/ P&gt; 기본 pH는 독립적으로 프로테옴 범위를 확대하기 위해 LC-MS/MS로 분석되는 여러 개의 분수로 펩타이드 혼합물을 분리하는 단계 (BPRP) 크로마토 그래피를 반전. <ol><li> 10mM의 중탄산 암모늄 (pH가 13)에서 증가 ACN 농도 구배를 적용하여 C18-HPLC 컬럼에서 펩타이드 혼합물을 분별. 5 분 5 % (V / V) ACN 시작, 60 분에 35 % ACN으로 증가하고, 1 분에 다음에 90 % ACN. 9 분 다시 평형 컬럼에 5 % ACN을 감소하기 전에 4 분 동안 90 % ACN을 유지합니다. 96 - 웰 플레이트 (H12에 A1)에 같은 부피의 96 분수를 수집합니다. 펩티드는 열 (여기에 설명 된 조건에 대한 10 ~ 70 분) 떨어져 용출하는 동안 220 nm에서 UV 검출기를 사용하여 분별을 모니터링합니다. </li><li> 우물 A2, C2, E2, 및 G2 (분수 A2)에서하고 그에 따라 들어, 우물 B1, D1, F1 및 H1 (분수 B1)에서, 우물 A1, C1, E1, 및 G1 (분수 A1)에서 분수를 결합 나머지 분수. solven를 제거진공 원심 분리기를 이용하여 t. 분수에서 재현 탁 펩티드는 A1, B2, A3, B4, A5, B6, A7, B8, A9, B10, A11 및 B12 1 M urea/0.5 %의 TFA의 130 μL와 9 단계에 설명 된대로 StageTips을 사용하여 정제. 저장 분수 B1, A2, B3, A4, B5, A6, B7, A8, B9, A10, B11, -20에서 A12 º C. </li></ol> 9. 정지 및 이동 추출로 정제 펩티드 (StageTips) 1.07 mm의 내부 직경 (ID)와 두 개의 C18 디스크 200 μL 피펫 팁을 포장하여 C18-StageTip 7 마이크로 컬럼을 준비합니다. 에펜 도르프 튜브에 무대 팁을 넣어. 메탄올 130 μL, 다음 130 UL 80 % ACN, 0.5 % 아세트산과 팁을 씻어 마이크로 원심을 사용합니다. (130) 0.1 μL % TFA로 StageTips을 평형. StageTips에 펩티드 혼합물을 전송하고 40 ㎕의 0.5 % 초산 후, 40 ㎕의 0.1 % TFA 후, 130 ㎕의 0.1 % TFA로 세척. 20 ㎕의 40 % ACN, 0.5 % 아세트산, 다음 20 ㎕의 80 % ACN, 0.5 % 아세트산으로 제 펩타이드 용출. 용출액 결합차 진공 여과하여 건조. 10. 마이크로 캐 필러 LC-MS/MS <ol><li> 약 1 ㎍ / ㎕의 농도에 1 ~ 5 ㎕의 5 % 포름산, 5 % ACN의 펩티드를 녹인다. 에서 ~ 1 μg 펩티드를 해결 100 μM × C18 반전 0.125 % 포름산에서 6~22% ACN의 구배로 위상 HPLC 컬럼 ~ 300 NL / 분의 유속으로 75 또는 100 분에 걸쳐 적용될 20cm. </li><li> LTQ Orbitrap VELOS (12) 또는 질량 분석계는 고해상도 및 높은 질량 정확도를 제공하는 것과 유사한 액체 크로마토 그래피 - 질량 분석 플랫폼을 사용하여 펩티드를 식별한다. Orbitrap 분석기에서 획득 한 전체 MS 스캔 (60,000의 해상도)과 데이터 종속 모드에서 질량 분석기를 사용하십시오. 전체 MS 스펙트럼에서 검출 된 20 가장 풍부한 이온 선형 이온 트랩 MS / MS 스펙트럼을 생성합니다. MS / MS에 1 × 10의 전체 MS 6 2,000에 자동 이득 제어 (AGC) 목표를 설정합니다. 1000 밀리 초에 최대 이온 축적 시간을 설정할MS 및 MS / MS 150 밀리 초에 대한. 20 ~ 60 초 동안 MS / MS에서 추가 선택에서 조각난 펩타이드 전구체 이온을 제외합니다. </li></ol> 11. MS / MS 데이터 수집 이러한 매개 변수 (표 1)를 사용하여 같은 SEQUEST 8과 같은 알고리즘을 이론적으로 데이터베이스에 MS / MS 스펙트럼 RAW 파일을 비교하여 펩티드를 확인합니다. 표 1. 펩타이드 데이터베이스 검색 매개 변수를. <table border="0" cellpadding="0" cellspacing="0" width="600"><tbody><tr height="19"><td height="19" rowspan="3" style="height:19px;" width="180"> 일반 매개 변수 </td><td> 최대 2와 완벽하게 트립신 소화 분열을 놓친 </td></tr><tr height="19"><td> 25 ppm의 전구체 이온 허용 </td></tr><tr height="19"><td> 1.0 다 조각 이온 허용 </td></tr><tr height="35"><td height="35" rowspan="2" style="height:36px;"> 정적 수정 </strong&gt; </td><td> 시스테인, carboxyamidomethylation에 57.02146 다 </td></tr><tr height="19"><td> 라이신과 펩타이드 N-말단에 28.03130 다, 빛 디메틸 화 라벨 </td></tr><tr height="19"><td height="19" rowspan="2" style="height:19px;"> 동적 수정 </td><td> 메티오닌, 산화에 15.99491 다 </td></tr><tr height="19"><td> 라이신과 펩타이드 N-말단, 무거운 디메틸 화 라벨 6.03766 다 </td></tr></tbody></table><ol><li> 실제와 반대 방향에서 오픈 리딩 프레임의 데이터베이스를 사용하여 이러한 목표 미끼 9 전략 등의 방법으로 1 %의 거짓 발견 속도에 필터 펩티드. </li></ol> 12. 펩티드 정량 계산 MS1의 중사 슬 및 경 쌍의 영역은 이온 크로마토 그램 (MS1의 피크 면적)을 추출하고, 펩티드 신호 대 잡음 (S / N) 비 10. 펩타이드 쌍을 포함하는 경우에만 자신의 평균 신호 - 대 - 노이SE 비율은 다섯 이상입니다. 동일한 펩티드 (MS1 피크 면적비)의 중사 슬 및 경 버전의 MS1의 피크 면적의 비율로서 두 개의 샘플에서 펩티드의 상대적인 풍부함을 계량화한다. 단백질의 모든 펩티드에 대한 평균 MS1의 피크 면적 비율로 상대적으로 단백질의 개체수를 계산합니다.

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저자는 더 경쟁 재정적 이익 또는 그 밖의 충돌 선언하지 않습니다.

높은 저렴한 라벨 시약 (시약은 샘플 당 1 달러 미만의 비용), 빠른 반응 속도 (~ 10 분), 부산물의 부재 : 몇 가지 점은 양적 proteomics의 매력적인 방법 환원 디메틸 화 (REDI 라벨)를 사용하여 펩티드의 안정 동위 원소 라벨링을 재현성 (도 3,도 4), 안정적인 반응 생성물, 임의의 단백질 분해 효소를 사용하는 능력 및 표지 된 펩티드의 높은 이온화 효율. 이 합성 중간에 특정 아미노산 auxotrop...

복잡한 시료 사이에 많은 단백질의 농도 차이를 측정하는 것은 단백질 체학의 중심 과제이다. 점점이 서로 다른 동위 원소의 태그를 각 샘플에서 단백질을 라벨 샘플을 결합하고, 농도의 차이를 정량화하기 위해 질량 분석기를 사용하여 수행되고있다. 몇 가지 방법은 단백질과 펩타이드의 안정 동위 원소 표지 존재한다. ICAT 3, iTRAQ 4, 환원 디메틸 화 5 단백질 추출 및 소화 후 안정 동위 원소의 태그를 추가하는 반면 15 N 표시 1 SILAC 2, 생체 내에서 대사 동위 원소 라벨을 소개합니다. 이러한 방법 중에서, 환원 디메틸 화 (REDI 라벨링)는 샘플의 거의 모든 종류의 단백질 농도 차이를 정량화 할 수있는 저렴하고 재현성있는 방법으로 인기를 얻고있다. REDI 라벨은 감소 쉬프 기반을 형성하는 포름 알데히드와 반응 펩티드를 포함노보로 하이드 라이드로. 이 반응은 N-말단 및 리신 측쇄 monomethylates 및 N-말단에 프롤린 자유 아미노기를 dimethylates. 여기에 설명 된 프로토콜은 중수 소화 포름 알데히드와 노보로 하이드 라이드 (그림 1)를 사용하여 &quot;무거운&quot;레이블이 자연 동위 원소 분포와 샘플 2의 수소 원자와 시약을 사용하여 &quot;빛&quot;라벨 샘플 1 펩티드를 methylates. 빛과 질량 분석기를 이용하여 두 형태를 구별하기 위해 사용되는 무거운 형태 사이 6.0377 다의 질량 차이로 펩티드 결과의 각 dimethylated 아미노기. 구체적으로는, 상대 펩티드 존재비는 광의 MS1 추출 이온 크로마토 영역 비율 (MS1 피크 면적비) 및 각 펩티드 이온 쌍 무거운 버전으로 정량화된다. 단백질의 상대적인 풍부함이 단백질에있는 펩티드들 중앙값 MS1 피크 면적의 비율로서 계산된다. 이 보고서에서, 우리는 행위에 대한 강력한 프로토콜을 설명역상 펩타이드 고체상 추출에 열 REDI 라벨링, 펩티드 분획 염기성 pH 기준 위상 (BPRP) 크로마토 그래피를 반전하고 StageTips를 이용 펩타이드 혼합물의 정제 (도 2)를 포함 LC-MS/MS에 의해 REDI 라벨링 실험을 보내고 . 우리는 정량 프로테오믹스에 대한 REDI 라벨 사용의 장점과 한계에 대해 설명합니다.

<table border="0" cellpadding="0" cellspacing="0" width="973">
	<tbody>
		<tr height="19">
			<td height="19" style="height:19px;width:377px;">trichloroacetic acid</td>
			<td style="width:141px;">Sigma-Aldrich</td>
			<td style="width:136px;">T9159</td>
			<td style="width:319px;">protein precipitation</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">acetone</td>
			<td>Sigma-Aldrich</td>
			<td style="width:136px;">650501</td>
			<td>protein precipitation</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">Sodium dodecyl sulfate</td>
			<td>Sigma-Aldrich</td>
			<td style="width:136px;">71736</td>
			<td>denature, reduce protein</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">sodium hydroxide</td>
			<td>Sigma-Aldrich</td>
			<td style="width:136px;">S8045</td>
			<td>denature, reduce protein</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">DL-Dithiothreitol</td>
			<td>Sigma-Aldrich</td>
			<td style="width:136px;">43816</td>
			<td>denature, reduce, alkylate protein</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">protease Inhibitor Complete Mini Cocktail</td>
			<td>Roche</td>
			<td>4693124001</td>
			<td>denature, reduce protein</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">iodoacetamide</td>
			<td>Sigma-Aldrich</td>
			<td>I6125</td>
			<td>alkylate protein</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">HEPES</td>
			<td>Sigma-Aldrich</td>
			<td style="width:136px;">H7523</td>
			<td>resuspend, extract, label protein</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">calcium chloride</td>
			<td>Sigma-Aldrich</td>
			<td style="width:136px;">C5670</td>
			<td>resuspend protein</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;width:377px;">Lysyl Endoprotease</td>
			<td>Wako Chemicals</td>
			<td style="width:136px;">129-02541</td>
			<td>protein digestion</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">sequencing grade trypsin</td>
			<td>Promega</td>
			<td style="width:136px;">V5111</td>
			<td>protein digestion</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">acetic acid</td>
			<td>Sigma-Aldrich</td>
			<td style="width:136px;">320099</td>
			<td>protein digestion</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">trifluoroacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>299537</td>
			<td>Reversed-phase peptide extraction</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">tC18 Sep-Pak C18 cartridges</td>
			<td>Waters</td>
			<td>WAT054960</td>
			<td>Reversed-phase peptide extraction</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">extraction manifold</td>
			<td>Waters</td>
			<td>WAT200609</td>
			<td>Reversed-phase peptide extraction</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">acetonitrile</td>
			<td>Sigma-Aldrich</td>
			<td style="width:136px;">14261</td>
			<td>various</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">formaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>252549</td>
			<td>&#8220;light&#8221; peptide labeling</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">cyanoborohydride</td>
			<td>Sigma-Aldrich</td>
			<td style="width:136px;">71435</td>
			<td>&#8220;light&#8221; peptide labeling</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">deuterated formaldehyde</td>
			<td>Sigma-Aldrich</td>
			<td>492620</td>
			<td>&#8220;heavy&#8221; peptide labeling</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">sodium cyanoborodeuteride</td>
			<td>CDN isotopes</td>
			<td style="width:136px;">D-1797</td>
			<td>&#8220;heavy&#8221; peptide labeling</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">MES</td>
			<td>Sigma-Aldrich</td>
			<td>M3671</td>
			<td>peptide labeling</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">C18-HPLC column (4.6 x 250 mm, 5 &#181;m particle size)</td>
			<td>Agilent</td>
			<td style="width:136px;">770450-902</td>
			<td>basic pH reversed-phase chromatography</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">formic acid</td>
			<td>Sigma-Aldrich</td>
			<td style="width:136px;">399388</td>
			<td>various</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">C18 Empore Disks</td>
			<td>3M</td>
			<td style="width:136px;">14-386-3&#160;</td>
			<td>STAGE tips</td>
		</tr>
		<tr height="19">
			<td height="19" style="height:19px;">methanol</td>
			<td>Sigma-Aldrich</td>
			<td style="width:136px;">494437</td>
			<td>STAGE tips</td>
		</tr>
	</tbody>
</table>

quantitative proteomics using reductive dimethylation for stable isotope labeling

환원 디메틸 화 (REDI 라벨)에 의해 펩티드의 안정 동위 원소 표지 정확하게 질량 분석기를 사용하여 샘​​플 간의 단백질 발현 차이를 정량화하는 방법입니다. REDI 라벨은 각각의 유리 아민에 두 개의 메틸 그룹을 추가하기 위해 정기적 (빛) 또는 포름 알데히드와 나트륨 시아 노보로 하이드 라이드의 중수 (무거운) 양식을 사용하여 수행됩니다. 여기에서 우리는 REDI 표시와 복잡한 단백질 혼합물의 정량 비교를위한 강력한 프로토콜을 보여줍니다. 비교를 위해 단백질 시료는 빛 또는 무거운 메틸 태그, 혼합하고, LC-MS/MS 공동 분석 중 하나를 수행하는 레이블 펩타이드로 분해된다. 상대 단백질의 개체수는 전체 MS 스펙트럼에서 추출 된 성분 펩타이드의 무겁고 가벼운 표시된 버전의 이온 크로마토 그램의 피크 면적을 비교하여 정량화. 여기에서 설명하는 방법은 샘플 역상 고상 추출에 의한 제조, 펩타이드에 열 REDI 라벨, 기본 산도 회전에 의해 펩타이드 분획을 포함ersed 상 (BPRP) 크로마토 그래피 StageTip 펩티드 정제. 우리는 안정 동위 원소의 설립을위한 다른 방법에 대한 REDI 라벨의 장점과 한계에 대해 설명합니다. 우리는 샘플의 거의 모든 종류의 단백질의 개체수를 비교하는 빠르고, 저렴하고 정확한 방법으로 REDI 라벨을 사용하여 새로운 응용 프로그램을 강조 표시합니다.

우리는 사카로 마이 세스 세레 비시 애를 사용하여 정확도, 정밀도 및 REDI 라벨의 재현성을 평가하고 클로 스트 리듐 속의 세균은 전체 세포 용 해물을 phytofermentans. 우리는 먼저 C의 혼합의 REDI 라벨의 효율성을 정량화 셀룰로오스 (무거운 표시, H)과 포도당 (빛 라벨, L) 문화에서 단백질 해물을 phytofermentans. 1 %의 펩타이드 거짓 발견의 속도로 여과 할 때,이 샘플은 ...

Watch this Scientific Journal Video about Quantitative Proteomics Using Reductive Dimethylation for Stable Isotope Labeling at JoVE.com

Quantitative Proteomics Using Reductive Dimethylation for Stable Isotope Labeling

환원 디메틸 화 (REDI 라벨)에 의해 펩티드의 안정 동위 원소 표지 정확한 질량 분석 기반의 정량 프로테오믹스에 대한 신속하고 저렴한 전략이다. 여기에서 우리는 거의 모든 샘플 유형에 적용 할 수있는 한 Redi 방식을 사용하여 단백질의 혼합물의 제조 및 분석을위한 강력한 방법을 보여준다.

안정 동위 원소 라벨링 환원성 디메틸 화를 사용하여 정량 프로테오믹스

Stable isotope labeling of peptides by reductive dimethylation (ReDi labeling) is a method to accurately quantify protein expression differences between ...

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JoVE Journal

Chemistry

16.0K 조회수.  Genoscope.  환원 디메틸 화 (REDI 라벨)에 의해 펩티드의 안정 동위 원소 표지 정확한 질량 분석 기반의 정량 프로테오믹스에 대한 신속하고 저렴한 전략이다. 여기에서 우리는 거의 모든 샘플 유형에 적용 할 수있는 한 Redi 방식을 사용하여 단백질의 혼합물의 제조 및 분석을위한 강력한 방법을 보여준다. 

비디오: Quantitative Proteomics Using Reductive Dimethylation for Stable Isotope Labeling

Large Scale Non-targeted Metabolomic Profiling of Serum by Ultra Performance Liquid Chromatography-Mass Spectrometry (UPLC-MS)

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to investigate metabolism. The approach offers an unbiased and in-depth analysis that can enable the development of diagnostic tests, novel therapies, and further our understanding of disease processes. The inherent chemical diversity of the metabolome creates significant analytical challenges and there is no single experimental approach that can detect all metabolites. Additionally, the biological variation in individual metabolism and the dependence of metabolism on environmental factors necessitates large sample numbers to achieve the appropriate statistical power required for meaningful biological interpretation. To address these challenges, this tutorial outlines an analytical workflow for large scale non-targeted metabolite profiling of serum by UPLC-MS. The procedure includes guidelines for sample organization and preparation, data acquisition, quality control, and metabolite identification and will enable reliable acquisition of data for large experiments and provide a starting point for laboratories new to non-targeted metabolite profiling by UPLC-MS.

Large Scale Non-targeted Metabolomic Profiling of Serum by Ultra Performance Liquid Chromatography-Mass Spectrometry UPLC-MS

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to investigate metabolism. This article outlines a typical workflow utilized for non-targeted metabolite profiling of serum including sample organization and preparation, data acquisition, data analysis, quality control, and metabolite identification.

울트라 성능 액체 크로마토 그래피 - 질량 분석법 (UPLC-MS)에 의해 혈청 대규모 비 타겟 Metabolomic 프로파일

Non-targeted metabolite profiling by ultra performance liquid chromatography coupled with mass spectrometry (UPLC-MS) is a powerful technique to ...

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Improved In-gel Reductive &#946;-Elimination for Comprehensive O-linked and Sulfo-glycomics by Mass Spectrometry

Separation of proteins by SDS-PAGE followed by in-gel proteolytic digestion of resolved protein bands has produced high-resolution proteomic analysis of biological samples. Similar approaches, that would allow in-depth analysis of the glycans carried by glycoproteins resolved by SDS-PAGE, require special considerations in order to maximize recovery and sensitivity when using mass spectrometry (MS) as the detection method. A major hurdle to be overcome in achieving high-quality data is the removal of gel-derived contaminants that interfere with MS analysis. The sample workflow presented here is robust, efficient, and eliminates the need for in-line HPLC clean-up prior to MS. Gel pieces containing target proteins are washed in acetonitrile, water, and ethyl acetate to remove contaminants, including polymeric acrylamide fragments. O-linked glycans are released from target proteins by in-gel reductive &#946;-elimination and recovered through robust, simple clean-up procedures. An advantage of this workflow is that it improves sensitivity for detecting and characterizing sulfated glycans. These procedures produce an efficient separation of sulfated permethylated glycans from non-sulfated (sialylated and neutral) permethylated glycans by a rapid phase-partition prior to MS analysis, and thereby enhance glycomic and sulfoglycomic analyses of glycoproteins resolved by SDS-PAGE.

Improved In-gel Reductive &amp;#946;-Elimination for Comprehensive O-linked and Sulfo-glycomics by Mass Spectrometry

In order to comprehensively explore the diversity of O-linked glycans, a new procedure for in-gel reductive &#946;-elimination, combined with permethylation and a rapid phase-partition method, is applied to the analysis of O-linked glycans directly released from glycoproteins resolved by SDS-PAGE and amenable to subsequent glycomic analysis by mass spectrometry.

질량 분석에 의해 - 겔 개선 환원 적 β-제거 포괄적 인 O가 연결된과에 대한 설포 글라 이코믹스

Separation of proteins by SDS-PAGE followed by in-gel proteolytic digestion of resolved protein bands has produced high-resolution proteomic analysis of ...

Quantitative and Qualitative Examination of Particle-particle Interactions Using Colloidal Probe Nanoscopy

Colloidal Probe Nanoscopy (CPN), the study of the nano-scale interactive forces between a specifically prepared colloidal probe and any chosen substrate using the Atomic Force Microscope (AFM), can provide key insights into physical interactions present within colloidal systems. Colloidal systems are widely existent in several applications including, pharmaceuticals, foods, paints, paper, soil and minerals, detergents, printing and much more.1-3 Furthermore, colloids can exist in many states such as emulsions, foams and suspensions. Using colloidal probe nanoscopy one can obtain key information on the adhesive properties, binding energies and even gain insight into the physical stability and coagulation kinetics of the colloids present within. Additionally, colloidal probe nanoscopy can be used with biological cells to aid in drug discovery and formulation development. In this paper we describe a method for conducting colloidal probe nanoscopy, discuss key factors that are important to consider during the measurement, and show that both quantitative and qualitative data that can be obtained from such measurements.

Colloidal probe nanoscopy can be used within a variety of fields to gain insight into the physical stability and coagulation kinetics of colloidal systems and aid in drug discovery and formulation sciences using biological systems. The method described within provides a quantitative and qualitative means to study such systems.

콜로이드 프로브 Nanoscopy을 사용하여 입자 간 상호 작용의 양적 및 질적 시험

Colloidal Probe Nanoscopy (CPN), the study of the nano-scale interactive forces between a specifically prepared colloidal probe and any chosen substrate ...

A Quantitative Glycomics and Proteomics Combined Purification Strategy

There is a growing desire in the biological and clinical sciences to integrate and correlate multiple classes of biomolecules to unravel biology, define pathways, improve treatment, understand disease, and aid biomarker discovery. N-linked glycosylation is one of the most important and robust post-translational modifications on proteins and regulates critical cell functions such as signaling, adhesion, and enzymatic function. Analytical techniques to purify and analyze N-glycans have remained relatively static over the last decade. While accurate and effective, they commonly require significant expertise and resources. Though some high-throughput purification schemes have been developed, they have yet to find widespread adoption and often rely on the enrichment of glycopeptides. One promising method, developed by Thomas-Oates et al., filter aided N-glycan separation (FANGS), was qualitatively demonstrated on tissues. Herein, we adapted FANGS to plasma and coupled it to the individuality normalization when labeling with glycan hydrazide tags strategy in order to achieve accurate relative quantification by liquid chromatography mass spectrometry and enhanced electrospray ionization. Furthermore, we designed new functionality to the protocol by achieving tandem, shotgun proteomics and glycosylation site analysis on hen plasma. We showed that N-glycans purified on filter and derivatized by hydrophobic hydrazide tags were comparable in terms of abundance and class to those by solid phase extraction (SPE); the latter is considered a gold standard in the field. Importantly, the variability in the two protocols was not statistically different. Proteomic data that was collected in-line with glycomic data had the same depth compared to a standard trypsin digest. Peptide deamidation is minimized in the protocol, limiting non-specific deamidation detected at glycosylation motifs. This allowed for direct glycosylation site analysis, though the protocol can accommodate 18O site labeling as well. Overall, we demonstrated a new in-line high-throughput, unbiased, filter based protocol for quantitative glycomics and proteomics analysis.

A high-throughput protocol was developed for combined proteomics and glycomics purification and LC-MS/MS quantification in plasma. Deamidation analysis of N-linked glycosylation motifs was specific to deglycosylated sites. Accurate quantitation of N-glycans was achieved by coupling filter aided N-glycan separation to the individuality normalization when labeling with glycan hydrazide tags strategy.

양적 글리코 믹스와 단백질 체학 복합 정제 전략

There is a growing desire in the biological and clinical sciences to integrate and correlate multiple classes of biomolecules to unravel biology, define ...

Standards for Quantitative Metalloproteomic Analysis Using Size Exclusion ICP-MS

Metals are essential for protein function as cofactors to catalyze chemical reactions. Disruption of metal homeostasis is implicated in a number of diseases including Alzheimer's and Parkinson's disease, but the exact role these metals play is yet to be fully elucidated. Identification of metalloproteins encounters many challenges and difficulties. Here we report an approach that allows metalloproteins in complex samples to be quantified. This is achieved using size exclusion chromatography coupled with inductively coupled plasma - mass spectrometry (SEC-ICP-MS). Using six known metalloproteins, the size exclusion column can be calibrated and the respective trace elements (iron, copper, zinc, cobalt, iodine) can be used for quantification. SEC-ICP-MS traces of human brain and plasma are presented. The use of these metalloprotein standards provides the means to quantitatively compare metalloprotein abundances between biological samples. This technique is poised to help shed light on the role of metalloproteins in neurodegenerative disease as well as other diseases where imbalances in trace elements are implicated.

Size exclusion chromatography hyphenated with inductively coupled plasma - mass spectrometry (ICP-MS) is a powerful tool to measure changes in the abundance of metalloproteins directly from biological samples. Here we describe a set of metalloprotein standards used to estimate molecular mass and the amount of metal associated with unknown proteins.

Metals are essential for protein function as cofactors to catalyze chemical reactions. Disruption of metal homeostasis is implicated in a number of ...

Highly Stable, Functional Hairy Nanoparticles and Biopolymers from Wood Fibers: Towards Sustainable Nanotechnology

Nanoparticles, as one of the key materials in nanotechnology and nanomedicine, have gained significant importance during the past decade. While metal-based nanoparticles are associated with synthetic and environmental hassles, cellulose introduces a green, sustainable alternative for nanoparticle synthesis. Here, we present the chemical synthesis and separation procedures to produce new classes of hairy nanoparticles (bearing both amorphous and crystalline regions) and biopolymers based on wood fibers. Through periodate oxidation of soft wood pulp, the glucose ring of cellulose is opened at the C2-C3 bond to form 2,3-dialdehyde groups. Further heating of the partially oxidized fibers (e.g., T = 80 &#176;C) results in three products, namely fibrous oxidized cellulose, sterically stabilized nanocrystalline cellulose (SNCC), and dissolved dialdehyde modified cellulose (DAMC), which are well separated by intermittent centrifugation and co-solvent addition. The partially oxidized fibers (without heating) were used as a highly reactive intermediate to react with chlorite for converting almost all aldehyde to carboxyl groups. Co-solvent precipitation and centrifugation resulted in electrosterically stabilized nanocrystalline cellulose (ENCC) and dicarboxylated cellulose (DCC). The aldehyde content of SNCC and consequently surface charge of ENCC (carboxyl content) were precisely controlled by controlling the periodate oxidation reaction time, resulting in highly stable nanoparticles bearing more than 7 mmol functional groups per gram of nanoparticles (e.g., as compared to conventional NCC bearing &#60;&#60; 1 mmol functional group/g). Atomic force microscopy (AFM), transmission electron microscopy (TEM), and scanning electron microscopy (SEM) attested to the rod-like morphology. Conductometric titration, Fourier transform infrared spectroscopy (FTIR), nuclear magnetic resonance (NMR), dynamic light scattering (DLS), electrokinetic-sonic-amplitude (ESA) and acoustic attenuation spectroscopy shed light on the superior properties of these nanomaterials.

Synthesis schemes to prepare highly stable wood fiber-based hairy nanoparticles and functional cellulose-based biopolymers have been detailed.

매우 안정, 기능 털이 나노 입자와 목재 섬유에서 생체 고분자 : 지속 가능한 나노 기술쪽으로

Nanoparticles, as one of the key materials in nanotechnology and nanomedicine, have gained significant importance during the past decade. While ...

Peptide and Protein Quantification Using Automated Immuno-MALDI (iMALDI)

Mass spectrometry (MS) is one of the most commonly used technologies for quantifying proteins in complex samples, with excellent assay specificity as a result of the direct detection of the mass-to-charge ratio of each target molecule. However, MS-based proteomics, like most other analytical techniques, has a bias towards measuring high-abundance analytes, so it is challenging to achieve detection limits of low ng/mL or pg/mL in complex samples, and this is the concentration range for many disease-relevant proteins in biofluids such as human plasma. To assist in the detection of low-abundance analytes, immuno-enrichment has been integrated into the assay to concentrate and purify the analyte before MS measurement, significantly improving assay sensitivity. In this work, the immuno- Matrix-Assisted Laser Desorption/Ionization (iMALDI) technology is presented for the quantification of proteins and peptides in biofluids, based on immuno-enrichment on beads, followed by MALDI-MS measurement without prior elution. The anti-peptide antibodies are functionalized on magnetic beads, and incubated with samples. After washing, the beads are directly transferred onto a MALDI target plate, and the signals are measured by a MALDI-Time of Flight (MALDI-TOF) instrument after the matrix solution has been applied to the beads. The sample preparation procedure is simplified compared to other immuno-MS assays, and the MALDI measurement is fast. The whole sample preparation is automated with a liquid handling system, with improved assay reproducibility and higher throughput. In this article, the iMALDI assay is used for determining the peptide angiotensin I (Ang I) concentration in plasma, which is used clinically as readout of plasma renin activity for the screening of primary aldosteronism (PA).

Peptide and Protein Quantification Using Automated Immuno-MALDI iMALDI

A protocol for the protein quantification in complex biological fluids using automated immuno-MALDI (iMALDI) technology is presented.

펩 티 드 및 단백질 정량화를 사용 하 여 자동 면역-MALDI (iMALDI)

Mass spectrometry (MS) is one of the most commonly used technologies for quantifying proteins in complex samples, with excellent assay specificity as a ...

Large-scale Top-down Proteomics Using Capillary Zone Electrophoresis Tandem Mass Spectrometry

Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) has been recognized as a useful tool for top-down proteomics that aims to characterize proteoforms in complex proteomes. However, the application of CZE-MS/MS for large-scale top-down proteomics has been impeded by the low sample-loading capacity and narrow separation window of CZE. Here, a protocol is described using CZE-MS/MS with a microliter-scale sample-loading volume and a 90-min separation window for large-scale top-down proteomics. The CZE-MS/MS platform is based on a linear polyacrylamide (LPA)-coated separation capillary with extremely low electroosmotic flow, a dynamic pH-junction-based online sample concentration method with a high efficiency for protein stacking, an electro-kinetically pumped sheath flow CE-MS interface with extremely high sensitivity, and an ion trap mass spectrometer with high mass resolution and scan speed. The platform can be used for the high-resolution characterization of simple intact protein samples and the large-scale characterization of proteoforms in various complex proteomes. As an example, a highly efficient separation of a standard protein mixture and a highly sensitive detection of many impurities using the platform is demonstrated. As another example, this platform can produce over 500 proteoform and 190 protein identifications from an Escherichia coli proteome in a single CZE-MS/MS run.

A detailed protocol is described for the separation, identification, and characterization of proteoforms in protein samples using capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS). The protocol can be used for the high-resolution characterization of proteoforms in simple protein samples and the large-scale identification of proteoforms in complex proteome samples.

모 세관 지역 전기 이동 법 탠덤 질량 분석을 사용 하 여 대규모 하향식 단백질

Capillary zone electrophoresis-electrospray ionization-tandem mass spectrometry (CZE-ESI-MS/MS) has been recognized as a useful tool for top-down ...

Stable Aqueous Suspensions of Manganese Ferrite Clusters with Tunable Nanoscale Dimension and Composition

Manganese ferrite clusters (MFCs) are spherical assemblies of tens to hundreds of primary nanocrystals whose magnetic properties are valuable in diverse applications. Here we describe how to form these materials in a hydrothermal process that permits the independent control of product cluster size (from 30 to 120 nm) and manganese content of the resulting material. Parameters such as the total amount of water added to the alcoholic reaction media and the ratio of manganese to iron precursor are important factors in achieving multiple types of MFC nanoscale products. A fast purification method uses magnetic separation to recover the materials making production of grams of magnetic nanomaterials quite efficient. We overcome the challenge of magnetic nanomaterial aggregation by applying highly charged sulfonate polymers to the surface of these nanomaterials yielding colloidally stable MFCs that remain non-aggregating even in highly saline environments. These non-aggregating, uniform, and tunable materials are excellent prospective materials for biomedical and environmental applications.

We report a one-pot hydrothermal synthesis of manganese ferrite clusters (MFCs) that offers independent control over material dimension and composition. Magnetic separation allows rapid purification while surface functionalization using sulfonated polymers ensures the materials are non-aggregating in biologically relevant medium. The resulting products are well positioned for biomedical applications.

튜블 가능한 나노 스케일 치수 및 구성망간 페릿 클러스터의 안정적인 수성 현탁액

Manganese ferrite clusters (MFCs) are spherical assemblies of tens to hundreds of primary nanocrystals whose magnetic properties are valuable in diverse ...

13C 6-Glucose Labeling을 사용한 약용 식물의 일차 장기 식별

13C6&#45;Glucose Labeling Associated with LC&#45;MS&#58; Identification of Plant Primary Organs in Secondary Metabolite Synthesis

This paper presents a novel and efficient method for certifying primary organs involved in secondary metabolite synthesis. As the most important secondary metabolite in Parispolyphylla var. yunnanensis (Franch.) Hand. -Mzt. (PPY), Paris saponin (PS) has a variety of pharmacological activities and PPY is in increasing demand. This study established leaf, rhizome, and stem-vascular-bundle 13C6-Glucose feeding and non-feeding four treatments to precisely certify the primary organs involved in Paris saponins VII (PS VII) synthesis. By combining liquid chromatography-mass spectrometry (LC-MS), the 13C/12C ratios of leaf, rhizome, stem, and root in different treatments were quickly and accurately calculated, and four types of PS isotopic ion peak(M&#8722;) ratios were found: (M+1) &#8722;/M&#8722;, (M+2) &#8722;/M&#8722;, (M+3) &#8722;/M&#8722; and (M+4) &#8722;/M&#8722;. The results showed that the ratio of 13C/12C in the rhizomes of the stem-vascular-bundle and rhizome feeding treatments was significantly higher than that in the non-feeding treatment. Compared to the non-feeding treatment, the ratio of PS VII molecules (M+2) &#8722;/M&#8722; in the leaves increased significantly under leaf and stem-vascular-bundle feeding treatments. Simultaneously, compared to the non-feeding treatment, the ratio of PS VII molecules (M+2) &#8722;/M&#8722; in the leaves under rhizome treatment showed no significant difference. Furthermore, the ratio of PS VII molecules (M+2) &#8722;/M&#8722; in the stem, root, and rhizome showed no differences among the four treatments. Compared to the non-feeding treatment, the ratio of the Paris saponin II (PS II) molecule (M+2) &#8722;/M&#8722; in leaves under leaf feeding treatment showed no significant difference, and the (M+3) &#8722;/M&#8722; ratio of PS II molecules in leaves under leaf feeding treatment were lower. The data confirmed that the primary organ for the synthesizing of PS VII is the leaves. It lays the foundation for future identification of the primary organs and pathways involved in the synthesis of secondary metabolites in medicinal plants.

저자 집중 조명: 13C6-Glucose Labeling 및 LC-MS를 사용한 식물 주요 장기 프로파일링

The developed method of 13C6-Glucose labeling combined with liquid chromatography high-resolution mass spectrometry is versatile and lays the foundation for future studies on the primary organs and pathways involved in the synthesis of secondary metabolites in medicinal plants, as well as the comprehensive utilization of these secondary metabolites.

13명LC-MS와 관련된 C6-포도당 라벨링: 2차 대사산물 합성에서 식물 일차 장기 식별

This paper presents a novel and efficient method for certifying primary organs involved in secondary metabolite synthesis. As the most important secondary ...