이 작품은 NCI 임상 Proteomic 기술 평가 센터 (CPTAC) 부여 (# U24 CA126476)뿐만 아니라 엔터테인먼트 산업 재단 (EIF)과 EIF 여성 암 연구 기금에서 유방암 Biomarker 디스커버리 컨소시엄에 부여로 투자하고,되었다 켁 재단 카나리 재단, 그리고 폴 G. 알렌 가족 재단에서 후한 선물.

실험 절차 : 분석은 합성 펩티드 및 안티 펩티드 항체가 필요합니다. 선택된 펩티드 관심의 단백질 고유, 8시 사이에 22 아미노산을 포함하고, 더 알려진 사후 translational 수정을해서는 안됩니다. 메티오닌 잔류물은 일반적으로 피할 및 이염 기성 아미노산 (예 : 주식, KR, RR)이있는 펩티드는 바람직하지입니다. 이 기법에 대한, 그것은 펩타이드의 C - 말단 (예 : K 또는 R은 표시)에서 아미노산을 레이블 (13 C, 15 N) 무거운 통합, 내부 표준으로 안정 동위 원소 분류 펩티드를 사용하는 것이 일반적입니다. 다음 프로토콜은 Epitomics 주식 회사 (버링게임, CA)과 뉴 잉글랜드 펩타이드 (가드너, MA)에서 합성 펩티드에서 얻은 방지 펩티드​​ 항체를 사용하여 마우스 단백질 Osteopontin의 펩타이드 GDSLAYGLR를 측정하기 위해 개발된 분석을 설명합니다. 1) 펩티드 3 농축) 질량 분광법으로 분석 복잡한 단백질 혼합물, 2 트립신의 소화를) 프로토콜은 세 가지 주요 단계 (그림 1)으로 구성되어 있습니다. 이것은 마우스 Osteopontin 단백질과 아군 인간의 플라즈마 샘플을 시연한다. 1. 트립신 효소 소화 및 정리 <ol><li> 서부 유럽 표준시 얼음 10 μL 깔끔한 플라즈마 나누어지는를 녹여. </li><li> BCA 분석하여 총 단백질 농도를 결정하고 정지 고체를 제거하기 위해 시료를 원심 분리기. </li><li> 자사의 스토리지 관에서 1000 μL 딥웰 플레이트와 피어스 가능한 필름으로 덮어 Pipet 10 μL 나누어지는. </li><li> 각 샘플에 우레아 / 30mM dithiothreitol (DTT) (최종 농도 6M의 요소 / 20mM DTT) 신선한 9M 20 μL를 추가합니다. </li><li> 37 30 분 품어 ° C. </li><li> 각 샘플에 신선한 500 MM의 iodoacetamide (최종 IAM 50mM) 3 μL를 추가합니다. </li><li> 상온에서 어두운 곳에 30 분 알을 품다. </li><li> 100 MM 트리스 (산도 8) (~ 0.6M로 dilutes의 요소)의 257 μL를 추가합니다. </li><li> 트립신 주식 솔루션 (; 기판 비율 1시 50분 효소 1 μg / μL) 10 μL를 추가합니다. </li><li> 부화 37 ° C 야간 (12-16시간). </li><li> 깔끔한 개미의 산성 (1 %의 최종 농도) 3 μL를 추가합니다. </li><li> 안정 동위 원소 표준 (다중 분석을 수행, 일반적으로이 표준 isotopically - 분류 펩타이드 10 μL가 포함된 50-100 fmol에 관한 것이라면 여러 기준 추가) 추가합니다. </li><li> 80 % acetonitrile, 폐기 흐름 통해 0.1 % 개미 산성 500 μL 잘 오아시스 카트리지 플레이트 씻으십시오. 이 3 번 반복합니다. </li><li> 물에 0.1 % 개미 산성 500 μL를 추가하여 카트리지 판 평형과 흐름을 통해 폐기하십시오. 이 4 번 반복합니다. </li><li> 카트리지 플레이트에 다이제스트 샘플을로드하고 흐름이 매우 느리게 때문에 진공을 조정합니다. </li><li> 물에 0.1 % 개미 산성 500 μL로 씻어하고, 흐름을 통해 폐기하십시오. 이 3 번 반복합니다. </li><li> 1000 μl 깊이 잘 플레이트에 80 % acetonitrile의 0.1 % 개미 산성 2 X 500 μL를 추가하여 Elute 펩티드는 (흐름을 통해 폐기하지 않음). </li><li> 건조 (또는 speedvac) eluate를 Lyophilize. (Lyophilization이 선호하는 방법입니다) </li><li> Reconstitute 50 μL PBS + 0.03 % 챕스을 추가하여 펩티드를 건조. </li></ol> 2. 펩타이드의 면역 친 화성의 농축 <ol><li> 표준 킹피셔 96 잘 접시에 샘플을 전송합니다. </li><li> 1 μg 항체와 표적 단백질 당 1.5 μL - G 코팅 자기 구슬을 (이것은 이전뿐만 아니라 비즈에 항체를 crosslink에게 옵션으로 제공됩니다) 추가합니다. 확인 구슬이 잘 흔들어 또는 vortexing으로 중지됩니다. </li><li> 호일로 접시를 커버. </li><li> 부드러운는 구슬이 정지되도록하는 텀블링와 (과 12~16시간) 밤새 품어. </li><li> 포일 표면에서 어떤 액체를 제거하기 위해 5 초 동안 32 XG에 접시를 원심 분리기. </li><li> 호일 커버를 제거합니다. </li><li> 구슬의 세탁과 용출은 수동 또는 자동 방식으로 수행할 수 있습니다. 이 절차는 킹피셔 플랫폼에서 자동으로 단계를 설명합니다. </li><li> 200 μL PBS + 0.03 % 챕스 (세척 당 일분)와 비즈에게 2 번 씻으십시오. </li><li> PBS 200 μL 1시 10분 희석 + 0.03 % 챕스 (1 분)와 비즈 1 시간을 씻으십시오. </li><li> 펩티드 5 % 초산 + 0.03 % 챕스 25 UL에 eluted 있습니다. </li><li> 자석에 용출 플레이트를 삽입하고 남은 비즈를 전송하지를 돌보는 96 잘 접시에 eluate을 전송하기만하면됩니다. </li><li> 실링 매트와 접시를 커버. </li><li> eluate가 포함된 플레이트는 분석을 위해 세 quadrapole 질량 분석기로 전달됩니다. </li></ol> 3. 다중 반응 모니터링에 의해 분석 - 질량 분광법 <ol><li> SRM / MRM 분석을위한 전환은 0.5 μL / 분 질량 분석기에 30% acetonitrile/0.1 % 개미 산성에서 펩타이드 표준의 microliter 솔루션 당 1 picomole을 일으키는 선정 및 최적화할 수 있습니다. 일단 안정 스프레이를 얻을 수 있습니다, MS / MS 스펙트럼을 수집합니다. </li><li> 전환은 thre을 식별하여 MS / MS 스펙트럼에서 선택됩니다전자 약간의 노이즈가 스펙트럼의 영역에 풍부한 조각 이온. 번식 - 충전 펩타이드 이온 들어, Y 이온 조각 M /에서 감지 Z&gt; 전구체 M / z는 일반적으로 SRM / MRM 분석 개발을위한 최선입니다. 그림 2 보여줍니다 476.3 MS / MS 스펙트럼 선택한 전환 이온&gt; 508.3, 579.3 , 펩타이드 GDSLAYGLR에 대한 692.4 (무거운 안정 동위 원소 표준 481.3에 대한 전환이&gt; 518.3, 589.3, 702.4 표시되지 않습니다.) </li><li> 사용되는 질량 분석계의 제조 업체 및 모델에 따라 각 전환에 대해 최적화할 수 있습니다 여러 개의 매개 변수가 없습니다. 여기, 우리는 충돌 에너지를 올렸 및 신호의 수준을 모니터링하여 선택한 각 전환의 충돌 에너지를 최적화. 25 충돌 에너지는 각 전환에 대해 사용되었습니다. </li><li> 다음과 같이 간단히, SRM / MRM 분석을위한 일반적인 구성은 다음과 같습니다 : 모바일 단계 (A) 0.1 % 개미 산성, (B) 90 % Acetonitrile / 0.1 % 개미 산성, 0.3 X 5mm C18 트랩 열, 75 μm의 ID를 X 15cm C18 분석 열 (Reprosil - PUR C18 AQ, 120 A · 기공). 주사 량은 10 μL이며, 샘플은 총 유량에서 3 %의 B에서 5 분 장전 3 μL / 분 및 3 선형 그라데이션으로 eluted - 300에서 45 % B - 10 분 400 NL / 분. 4000 QTRAP (ABSCIEX, 포스터 시티, CA)에 조건이 스프레이 전압 2.3kV, 이온 소스 온도 150 ° C했다, 12의 GS1, 그리고 커튼 가스 15. </li><li> 샘플은 샘플의 10 μL를 주입하여 SRM / MRM 의해 분석됩니다. 피크 영역은 프로그램 스카이 라인을 사용하여 빛과 무거운 펩티드를 위해 통합되어 있습니다. 14이 경우, 배경 잡음 무료입니다 가장 풍부한 전환을 사용하여 각 샘플에 레이블이 지정되지 않은 / 레이블 펩타이드의 최고 지역 비율 (PAR), Y5 전환을 계산 (가벼운 펩티드 476.3&gt; 579.3, 무거운 표준 펩티드 481.3&gt; 589.3). </li></ol> 4. 대표 결과 : 측정된 피크 면적 비율 (아군 무거운 isotopically - 표시 펩타이드에 가벼운 내생 펩타이드의 상대)는 대상 펩타이드의 양적 측정을 제공합니다. 그림 3 SISCAPA - 풍부한 샘플의 빛과 무거운 펩티드의 예제 크로마토 그램을 보여줍니다. 동시에 여러 전환에 elute 가볍고 무거운 펩티드가 신원을 확인하여 각 펩타이드에 대해 모니터링할 수 수 있습니다. <img src="/files/ftp_upload/2812/2812fig1.jpg" alt="그림 1"/> SISCAPA 과정을 그림 1. 도식 개요. 복잡한 단백질 혼합물은 펩티드로 소화합니다. 타겟 펩티드 analytes는 (내생 analyte과 아군 안정 동위 원소 - 라벨이 내부 표준) 단백질 G - 코팅 자성 입자에 고정화 방지 펩티드​​ 항체를 사용하여 풍부하게하고 있습니다. 절연 다음, 대상 펩티드는 자성 입자에서 eluted되고 내부 표준에 상대적으로 quantitation에 대한 질량 분광법으로 분석했다. <img src="/files/ftp_upload/2812/2812fig2.jpg" alt="그림 2"/> 그림 2. SRM / MRM 변환 세 조각의 선택을 보여주는 펩타이드 GDSLAYGLR의 MS / MS 스펙트럼. <img src="/files/ftp_upload/2812/2812fig3.jpg" alt="그림 3"/> 그림 3. 가벼운 펩티드 analyte의 최고 프로필 (빨간색)와 무거운 안정 동위 원소 - 라벨 내부 표준 (파란색)을 보여주는 예제 chromatograms. 그들은 크로마 토그래피 시스템에서 elute으로 가벼운 펩티드 (476.3&gt; 579.3)와 라벨 무거운 동위 원소에서 안정 Y5 전환 Chromatograms는 표준 (481.3&gt; 589.3) 시간이 지남에 따라 꾸몄다 있습니다.

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Proteome Res. 9, 5422-5437 (2010).</li><li>Berna, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Online+immunoaffinity+liquid+chromatography/tandem+mass+spectrometry+determination+of+a+type+II+collagen+peptide+biomarker+in+rat+urine:+Investigation+of+the+impact+of+collision-induced+dissociation+fluctuation+on+peptide+quantitation.">Online immunoaffinity liquid chromatography/tandem mass spectrometry determination of a type II collagen peptide biomarker in rat urine: Investigation of the impact of collision-induced dissociation fluctuation on peptide quantitation.</a> Anal. Biochem. 356, 235-243 (2006).</li></ol>

관심 없음 충돌 선언하지 않습니다.

로 설명한 프로토콜에서 가장 중요한 단계는 구슬이 부화 기간 동안 잘 혼합 남아 보장합니다. 구슬이 잘 / 유리병의 바닥에 정착 수 있도록하는 것은 다양성을 증가 발생합니다. 또한 중요하기 잠복기 다음 잘 / 유리병의 상단에 남아있을 수있는 액체 다운 스핀. 재현성 트립신의 소화도 중요합니다. 그러나, 그것은 소화의 가능성이 다른 방법이 대상 단백질의 주어진 집합에 대한 최적화할 수 있습니다 무료로 항체의 15 용리가의 검색을 방해하지 않는다;. 소화에 대한 절차는 SISCAPA와 함께 우리의 연구실에 광범위하게 활용되어 여기에서 설명한 항체가 나중에 펩티드보다 열 또는 elutes에서 제외되어 있지만 항체가 이전에 대규모 실험 또는 무료 항체 경우 친화 농축에 단백질 G 비즈에 가교 될 수 있기 때문에 가능성이 펩티드는 문제가된다. 우리는 또한 잔여 구슬이나 입자를 제거하는 예제 autosampler에 접시뿐만 아니라 흐름의 반대 방향으로 트랩 열을 씻는 아래 자석을 (로딩에 비해) 장소에 유용하게 나타났습니다. 설명 마그네틱 비드 접근뿐만 아니라, 기술도 칼럼 형식 (즉, 친화 크로마 토그래피)에 적용할 수 있습니다. 12, 16 사용자가 전체 프로토콜과 함께 편안한되면, 기술은 또한 전체적인 분석을 강화 몇 가지 수정 의무가 있습니다. 우선 11, 그것이 강화 단계 (즉 멀티플렉싱)에 항체를 결합하여 하나의 분석에서 여러 analytes를 분석 할 수 있습니다. 질량 분석계는 동시에 analytes 다수의 분석이 가능합니다. 둘째, 원래의 샘플의 볼륨을 증가하는 것은 분석의 감도를 향상시킵니다.

<table border="1"><tbody><tr><th> 자료 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th></tr><tr><td> 1000ul 딥 잘 96 - 웰 플레이트 </td><td> Eppendorf </td><td> 951032786 </td></tr><tr><td> 오아시스 HLB 카트리지 CC 1 판, 9 월 - 박, 40 MG 96 웰 플레이트 </td><td> 워터스 </td><td> 186003966 </td></tr><tr><td> 킹피셔 96 웰 플레이트 </td><td> 써모 피셔 사이 언티픽 </td><td> 97002540 </td></tr><tr><td> 96 음, 흰 벽에 접시를 지우고 </td><td> 바이오 래드 </td><td> HSP9601 </td></tr><tr><td> 96 잘 접시 커버 호일 </td><td> Excelscientific </td><td> 12-169 </td></tr><tr><td> 96 잘 접시 Axymat 씰링 매트 </td><td> Axygen </td><td> 521-01-151 </td></tr><tr><td> X 피어스 필름 </td><td> 시그마 - 알드리치 </td><td> Z722502 </td></tr><tr><td> PCR 자석의 머리와 킹피셔 자석 구슬 프로세서 </td><td> 써모 피셔 사이 언티픽 </td><td> HSP 9601 </td></tr></tbody></table> 표 1 : 자료 <table border="1"><tbody><tr><th> 자료 이름 </th><th> 회사 </th><th> 카탈로그 번호 </th><th> 댓글 (옵션) </th></tr><tr><td> 요소 </td><td> 시그마 알드리치 </td><td> U6031 </td><td></td></tr><tr><td> Trizma베이스 </td><td> 시그마 알드리치 </td><td> T1503 </td><td></td></tr><tr><td> Dithiothreitol (DTT) </td><td> 찌르다 </td><td> 20,291 </td><td> &quot;dithiothreitol 노 달다&quot; </td></tr><tr><td> Iodoacetamide (IAM) </td><td> 시그마 알드리치 </td><td> I1149 </td><td></td></tr><tr><td> 트립신 골드 </td><td> Promega </td><td> V5280 </td><td></td></tr><tr><td> 단백질 G 자석 구슬, 2.8um </td><td> Invitrogen </td><td> 10004D </td><td></td></tr><tr><td> 인산 버퍼 호수 (PBS) </td><td> 써모 피셔 사이 언티픽 </td><td> L5401 </td><td></td></tr><tr><td> 챕스 </td><td> 써모 피셔 사이 언티픽 </td><td> 28,300 </td><td></td></tr><tr><td> 개미 산성 </td><td> EMD </td><td> 11,670 </td><td></td></tr><tr><td> Acetonitrile </td><td> 써모 피셔 사이 언티픽 </td><td> A998 - 1 </td><td></td></tr><tr><td> 아세트산 </td><td> 시그마 알드리치 </td><td> 242853 </td><td></td></tr></tbody></table> 표 2 : 시약 <table border="1"><tbody><tr><th> 용액 </th><th> 댓글 (옵션) </th></tr><tr><td> 100 MM 트리스의 산도 8 </td><td></td></tr><tr><td> 9 M 100 MM 트리스 (산도 8)의 요소 / 30 MM의 DTT </td><td> 매번 신선한 준비를해야합니다 </td></tr><tr><td> 100 MM 500 MM 트리스의 IAM (산도 8) </td><td> 매번 신선한 준비를해야합니다 </td></tr><tr><td> 100 MM 트리스의 산도 8 트립신 1mg/mL </td><td></td></tr><tr><td> 100 MM 트리스의 산도 8 트립신 억제제 1mg/mL </td><td></td></tr><tr><td> 안정 동위 원소 표준 mastermix </td><td></td></tr><tr><td> 안티 펩티드 항체 mastermix </td><td></td></tr></tbody></table> 표 3 : 솔루션 준비에

quantification of proteins using peptide immunoaffinity enrichment coupled with mass spectrometry

단백질을 측정 양적 assays에 대한 큰 필요가있다. 크게 quantitation의 황금 표준으로 간주 전통 샌드위치 immunoassays은, 높은 비용, 긴 리드 타임과 관련된, 그리고 단점 (예 : heterophilic 항체, autoantibody 간섭 &#39;후크 효과&#39;)이 따른 다네입니다. 한 대체 기술 친화 농축집니다 양적 질량 분광법과 결합 펩티드의 일반적 SISCAPA (안티 펩타이드 항체에 의해 안정 동위 원소 표준 및 캡처)이라고도합니다.이 기법 2 다중 / 선택된 반응 모니터링 질량 분석법에 의해 안정 동위 원소 희석 및 검출과 펩티드의 선호도 강화가 ( SRM / MRM - MS)는 각각의 단백질에 대한 surrogates으로 펩티드의 양적 측정을 제공합니다. SRM / MRM - MS 잘 작은 분자 3, 4의 정확한 quantitation을 위해 설립되고 최근에는 플라즈마와 세포 lysates의 단백질의 농도를 측정하는 맞게되었습니다. 단백질의 quantitation를 달성하기 위해 5-7이 큰 분자로 소화 같은 트립신과 같은 효소를 사용하여 구성 요소 펩티드. 누구의 순서대로 해당 종의 대상 단백질 (예 : &quot;proteotypic&quot;펩티드)에 독특한 하나 이상 선택 펩티드는 다음 방지 펩티드​​ 항체를 사용하여 샘​​플에서 농축하여 시료의 단백질 농도에 대한 정량 stoichiometric의 surrogates로 측정됩니다. 따라서, 안정 동위 원소 희석 (SID) 방법 (즉, 아군 인 펩타이드 표준 표시 안정 동위 원소), SRM / MRM 복잡한 생물 학적 매트릭스의 단백질 양을 정함 대한 surrogates로 proteotypic 펩티드의 농도를 측정하는 데 사용할 수 있습니다.을 결합 assays은 전통적인 immunoassays에 비해 몇 가지 장점이 있습니다. 시약은 생성하기 위해 상대적으로 저렴, analyte에 대한 특이성이 우수합니다, assays가 높은 멀티 플렉스 수, 농축은 깔끔한 플라즈마 (더 고갈 필요 없음)에서 수행하고, 기술은 단백질이나 수정의 다양한 의무가있다 할 수있다 자석 구슬 플랫폼에 맞게 본 동영상에 대한 관심. 8-13의 우리는 기본적인 프로토콜을 보여줍니다.

Watch this Scientific Journal Video about Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry at JoVE.com

Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

안정 동위 원소 표준 및 안티 펩타이드 항체 (SISCAPA) 각각의 단백질에 대한 surrogates으로 펩티드의 양적 측정을 제공하기 위해 안정 동위 원소 희석 질량 분석법 (MRM - MS)과 펩티드의 커플 선호도 농축하여 캡처합니다. 여기서 우리는 부분적으로 자동화된 형식으로 자기 입자를 사용하여 프로토콜을 설명합니다.

펩타이드 면역 친 화성 농축을 사용하여 단백질의 양을 정함은 질량 분광법과 함께

There is a great need for quantitative assays in measuring proteins. Traditional sandwich immunoassays, largely considered the gold standard in ...

quantification-proteins-using-peptide-immunoaffinity-enrichment

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Biology

22.7K 조회수.  Fred Hutchinson Cancer Research Center - FHCRC. 안정 동위 원소 표준 및 안티 펩타이드 항체 (SISCAPA) 각각의 단백질에 대한 surrogates으로 펩티드의 양적 측정을 제공하기 위해 안정 동위 원소 희석 질량 분석법 (MRM - MS)과 펩티드의 커플 선호도 농축하여 캡처합니다. 여기서 우리는 부분적으로 자동화된 형식으로 자기 입자를 사용하여 프로토콜을 설명합니다.

비디오: Quantification of Proteins Using Peptide Immunoaffinity Enrichment Coupled with Mass Spectrometry

MALDI Sample Preparation: the Ultra Thin Layer Method

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS) 1,2. The ultra-thin layer method involves the production of a substrate layer of matrix crystals (alpha-cyano-4-hydroxycinnamic acid) on the sample plate, which serves as a seeding ground for subsequent crystallization of a matrix/analyte mixture. Advantages of the ultra-thin layer method over other sample deposition approaches (e.g. dried droplet) are that it provides (i) greater tolerance to impurities such as salts and detergents, (ii) better resolution, and (iii) higher spatial uniformity. This method is especially useful for the accurate mass determination of proteins. The protocol was initially developed and optimized for the analysis of membrane proteins and used to successfully analyze ion channels, metabolite transporters, and receptors, containing between 2 and 12 transmembrane domains 2. Since the original publication, it has also shown to be equally useful for the analysis of soluble proteins. Indeed, we have used it for a large number of proteins having a wide range of properties, including those with molecular masses as high as 380 kDa 3. It is currently our method of choice for the molecular mass analysis of all proteins. The described procedure consistently produces high-quality spectra, and it is sensitive, robust, and easy to implement.

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption Ionization Mass Spectrometry (MALDI-MS).

든 샘플 준비 : 울트라 씬 레이어 방법

This video demonstrates the preparation of an ultra-thin matrix/analyte layer for analyzing peptides and proteins by Matrix-Assisted Laser Desorption ...

연구

생물학

A Quantitative Assessment of The Yeast Lipidome using Electrospray Ionization Mass Spectrometry

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast Saccharomyces cerevisiae, a genetically and biochemically manipulable unicellular eukaryote with annotated genome and very simple lipidome, is a valuable model for studying biological functions of various lipid species in multicellular eukaryotes2,3,5. S. cerevisiae has 10 major classes of lipids with chain lengths mainly of 16 or 18 carbon atoms and either zero or one degree of unsaturation6,7. Existing methods for lipid identification and quantification - such as high performance liquid chromatography, thin-layer chromatography, fluorescence microscopy, and gas chromatography followed by MS - are well established but have low sensitivity, insufficiently separate various molecular forms of lipids, require lipid derivitization prior to analysis, or can be quite time consuming. Here we present a detailed description of our experimental approach to solve these inherent limitations by using survey-scan ESI/MS for the identification and quantification of the entire complement of lipids in yeast cells. The described method does not require chromatographic separation of complex lipid mixtures recovered from yeast cells, thereby greatly accelerating the process of data acquisition. This method enables lipid identification and quantification at the concentrations as low as g/ml and has been successfully applied to assessing lipidomes of whole yeast cells and their purified organelles. Lipids extraction from whole yeast cells for using this method of lipid analysis takes two to three hours. It takes only five to ten minutes to run each sample of extracted and dried lipids on a Q-TOF mass spectrometer equipped with a nano-electrospray source.

We describe a new quantitative lipidomics method for identifying numerous lipid species in yeast using survey-scan electrospray ionization mass spectrometry (ESI/MS). This method exceeds currently available methods for lipid identification and quantification in the ability to resolve various molecular forms of lipids, sensitivity, and speed.

전기 분무 이온화 질량 분광법을 사용하여 효모 Lipidome의 양적 평가

Lipids are one of the major classes of biomolecules and play important roles membrane dynamics, energy storage, and signalling1-4. The budding yeast ...

Analyzing Large Protein Complexes by Structural Mass Spectrometry

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an array of dynamic, multi-protein complexes1. To gain a mechanistic understanding of the various cellular processes, it is crucial to determine the structure of such protein complexes, and reveal how their structural organization dictates their function. Many aspects of multi-protein complexes are, however, difficult to characterize, due to their heterogeneous nature, asymmetric structure, and dynamics. Therefore, new approaches are required for the study of the tertiary levels of protein organization.
One of the emerging structural biology tools for analyzing macromolecular complexes is mass spectrometry (MS)2-5. This method yields information on the complex protein composition, subunit stoichiometry, and structural topology. The power of MS derives from its high sensitivity and, as a consequence, low sample requirement, which enables examination of protein complexes expressed at endogenous levels. Another advantage is the speed of analysis, which allows monitoring of reactions in real time. Moreover, the technique can simultaneously measure the characteristics of separate populations co-existing in a mixture. 
Here, we describe a detailed protocol for the application of structural MS to the analysis of large protein assemblies. The procedure begins with the preparation of gold-coated capillaries for nanoflow electrospray ionization (nESI). It then continues with sample preparation, emphasizing the buffer conditions which should be compatible with nESI on the one hand, and enable to maintain complexes intact on the other. We then explain, step-by-step, how to optimize the experimental conditions for high mass measurements and acquire MS and tandem MS spectra. Finally, we chart the data processing and analyses that follow. Rather than attempting to characterize every aspect of protein assemblies, this protocol introduces basic MS procedures, enabling the performance of MS and MS/MS experiments on non-covalent complexes. Overall, our goal is to provide researchers unacquainted with the field of structural MS, with knowledge of the principal experimental tools.

Mass spectrometry has proven to be a valuable tool for analyzing large protein complexes. This method enables insights into the composition, stoichiometry and overall architecture of multi-subunit assemblies. Here, we describe, step-by-step, how to perform a structural mass spectrometry analysis, and characterize macromolecular structures.

구조 질량 분광법에 의해 큰 단백질 단지 분석

Living cells control and regulate their biological processes through the coordinated action of a large number of proteins that assemble themselves into an ...

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial applications as well as increases basic fundamental biological knowledge. F&ouml;rster (Fluorescence) Resonance Energy Transfer (FRET) between a donor molecule in an electronically excited state and a nearby acceptor molecule has been frequently utilized for studies of protein-protein interactions in living cells. The proteins of interest are tagged with two different types of fluorescent probes and expressed in biological cells. The fluorescent probes are then excited, typically using laser light, and the spectral properties of the fluorescence emission emanating from the fluorescent probes is collected and analyzed. Information regarding the degree of the protein interactions is embedded in the spectral emission data. Typically, the cell must be scanned a number of times in order to accumulate enough spectral information to accurately quantify the extent of the protein interactions for each region of interest within the cell. However, the molecular composition of these regions may change during the course of the acquisition process, limiting the spatial determination of the quantitative values of the apparent FRET efficiencies to an average over entire cells. By means of a spectrally resolved two-photon microscope, we are able to obtain a full set of spectrally resolved images after only one complete excitation scan of the sample of interest. From this pixel-level spectral data, a map of FRET efficiencies throughout the cell is calculated. By applying a simple theory of FRET in oligomeric complexes to the experimentally obtained distribution of FRET efficiencies throughout the cell, a single spectrally resolved scan reveals stoichiometric and structural information about the oligomer complex under study. Here we describe the procedure of preparing biological cells (the yeast Saccharomyces cerevisiae) expressing membrane receptors (sterile 2 &alpha;-factor receptors) tagged with two different types of fluorescent probes. Furthermore, we illustrate critical factors involved in collecting fluorescence data using the spectrally resolved two-photon microscopy imaging system. The use of this protocol may be extended to study any type of protein which can be expressed in a living cell with a fluorescent marker attached to it.

In vivo Quantification of G Protein Coupled Receptor Interactions using Spectrally Resolved Two-photon Microscopy

By employing a spectrally resolved two-photon microscopy imaging system, pixel-level maps of F&ouml;rster Resonance Energy Transfer (FRET) efficiencies are obtained for cells expressing membrane receptors hypothesized to form homo-oligomeric complexes. From the FRET efficiency maps, we are able to estimate stoichiometric information about the oligomer complex under study.

Spectrally 해결된 두 광자 현미경을 사용하여 G 단백질 결합 수용체 상호 작용의 생체내 부량에서

The study of protein interactions in living cells is an important area of research because the information accumulated both benefits industrial ...

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry (CCMS)

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity chromatography 1 or Activity Based Protein Profiling 2. Trifunctional Capture Compounds (CCs, Figure 1A) 3 are the basis for a generic approach, in which the initial equilibrium-driven interaction between a small molecule probe (the selectivity function, here S-adenosyl-L-homocysteine, SAH, Figure 1A) and target proteins is irreversibly fixed upon photo-crosslinking between an independent photo-activable reactivity function (here a phenylazide) of the CC and the surface of the target proteins. The sorting function (here biotin) serves to isolate the CC - protein conjugates from complex biological mixtures with the help of a solid phase (here streptavidin magnetic beads). Two configurations of the experiments are possible: "off-bead" 4 or the presently described "on-bead" configuration (Figure 1B). The selectivity function may be virtually any small molecule of interest (substrates, inhibitors, drug molecules).
S-Adenosyl-L-methionine (SAM, Figure 1A) is probably, second to ATP, the most widely used cofactor in nature 5, 6. It is used as the major methyl group donor in all living organisms with the chemical reaction being catalyzed by SAM-dependent methyltransferases (MTases), which methylate DNA 7, RNA 8, proteins 9, or small molecules 10. Given the crucial role of methylation reactions in diverse physiological scenarios (gene regulation, epigenetics, metabolism), the profiling of MTases can be expected to become of similar importance in functional proteomics as the profiling of kinases. Analytical tools for their profiling, however, have not been available. We recently introduced a CC with SAH as selectivity group to fill this technological gap (Figure 1A).
SAH, the product of SAM after methyl transfer, is a known general MTase product inhibitor 11. For this reason and because the natural cofactor SAM is used by further enzymes transferring other parts of the cofactor or initiating radical reactions as well as because of its chemical instability 12, SAH is an ideal selectivity function for a CC to target MTases. Here, we report the utility of the SAH-CC and CCMS by profiling MTases and other SAH-binding proteins from the strain DH5&alpha; of Escherichia coli (E. coli), one of the best-characterized prokaryotes, which has served as the preferred model organism in countless biochemical, biological, and biotechnological studies. Photo-activated crosslinking enhances yield and sensitivity of the experiment, and the specificity can be readily tested for in competition experiments using an excess of free SAH.

Profiling of Methyltransferases and Other S-adenosyl-L-homocysteine-binding Proteins by Capture Compound Mass Spectrometry CCMS

Capture Compounds are trifunctional small molecules to reduce the complexity of the proteome by functional reversible small molecule-protein interaction followed by photo-crosslinking and purification. Here we use a Capture Compound with S-adenosyl-L-homocysteine-binding as selectivity function to isolate methyltransferases from an Escherichia coli whole cell lysate and identify them by MS.

Methyltransferases의 프로 파일링 및 기타 S - adenosyl - L</sub캡처 컴파 운드 질량 분광법 (CCMS)에서> - 호모 시스테인 결합 단백질

There is a variety of approaches to reduce the complexity of the proteome on the basis of functional small molecule-protein interactions such as affinity ...

Profiling Thiol Redox Proteome Using Isotope Tagging Mass Spectrometry

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on Arabidopsis thaliana, a genetically tractable host plant1,2. The accumulation of reactive oxygen species (ROS) in cotyledons inoculated with DC3000 indicates a role of ROS in modulating necrotic cell death during bacterial speck disease of tomato3. Hydrogen peroxide, a component of ROS, is produced after inoculation of tomato plants with Pseudomonas3. Hydrogen peroxide can be detected using a histochemical stain 3'-3' diaminobenzidine (DAB)4. DAB staining reacts with hydrogen peroxide to produce a brown stain on the leaf tissue4. ROS has a regulatory role of the cellular redox environment, which can change the redox status of certain proteins5. Cysteine is an important amino acid sensitive to redox changes. Under mild oxidation, reversible oxidation of cysteine sulfhydryl groups serves as redox sensors and signal transducers that regulate a variety of physiological processes6,7. Tandem mass tag (TMT) reagents enable concurrent identification and multiplexed quantitation of proteins in different samples using tandem mass spectrometry8,9. The cysteine-reactive TMT (cysTMT) reagents enable selective labeling and relative quantitation of cysteine-containing peptides from up to six biological samples. Each isobaric cysTMT tag has the same nominal parent mass and is composed of a sulfhydryl-reactive group, a MS-neutral spacer arm and an MS/MS reporter10. After labeling, the samples were subject to protease digestion. The cysteine-labeled peptides were enriched using a resin containing anti-TMT antibody. During MS/MS analysis, a series of reporter ions (i.e., 126-131 Da) emerge in the low mass region, providing information on relative quantitation. The workflow is effective for reducing sample complexity, improving dynamic range and studying cysteine modifications. Here we present redox proteomic analysis of the Pst DC3000 treated tomato (Rio Grande) leaves using cysTMT technology. This high-throughput method has the potential to be applied to studying other redox-regulated physiological processes.

Reactive oxygen species level is elevated when cells encounter stress conditions. Here we show the example of 3'-3' diaminobenzidine staining as well as cysTMT labeling and mass spectrometry to profile the redox proteome in Pseudomonas syringae treated tomato leaves.

동위 원소 태그 질량 분광법을 사용하여 Thiol 산화 환원 프로테옴 프로 파일링

Pseudomonas syringae pv. tomato strain DC3000 not only causes bacterial speck disease in Solanum lycopersicum but also on Brassica species, as well as on ...

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the identification of the subunit composition of multi-protein complexes can provide insight into gene function and enhance understanding of biological systems1, 2. Physical interactions can be mapped with high confidence vialarge-scale isolation and characterization of endogenous protein complexes under near-physiological conditions based on affinity purification of chromosomally-tagged proteins in combination with mass spectrometry (APMS). This approach has been successfully applied in evolutionarily diverse organisms, including yeast, flies, worms, mammalian cells, and bacteria1-6. In particular, we have generated a carboxy-terminal Sequential Peptide Affinity (SPA) dual tagging system for affinity-purifying native protein complexes from cultured gram-negative Escherichia coli, using genetically-tractable host laboratory strains that are well-suited for genome-wide investigations of the fundamental biology and conserved processes of prokaryotes1, 2, 7. Our SPA-tagging system is analogous to the tandem affinity purification method developed originally for yeast8, 9, and consists of a calmodulin binding peptide (CBP) followed by the cleavage site for the highly specific tobacco etch virus (TEV) protease and three copies of the FLAG epitope (3X FLAG), allowing for two consecutive rounds of affinity enrichment. After cassette amplification, sequence-specific linear PCR products encoding the SPA-tag and a selectable marker are integrated and expressed in frame as carboxy-terminal fusions in a DY330 background that is induced to transiently express a highly efficient heterologous bacteriophage lambda recombination system10. Subsequent dual-step purification using calmodulin and anti-FLAG affinity beads enables the highly selective and efficient recovery of even low abundance protein complexes from large-scale cultures. Tandem mass spectrometry is then used to identify the stably co-purifying proteins with high sensitivity (low nanogram detection limits).
Here, we describe detailed step-by-step procedures we commonly use for systematic protein tagging, purification and mass spectrometry-based analysis of soluble protein complexes from E. coli, which can be scaled up and potentially tailored to other bacterial species, including certain opportunistic pathogens that are amenable to recombineering. The resulting physical interactions can often reveal interesting unexpected components and connections suggesting novel mechanistic links. Integration of the PPI data with alternate molecular association data such as genetic (gene-gene) interactions and genomic-context (GC) predictions can facilitate elucidation of the global molecular organization of multi-protein complexes within biological pathways. The networks generated for E. coli can be used to gain insight into the functional architecture of orthologous gene products in other microbes for which functional annotations are currently lacking.

Identification of Protein Complexes in Escherichia coli using Sequential Peptide Affinity Purification in Combination with Tandem Mass Spectrometry

Affinity purification of tagged proteins in combination with mass spectrometry (APMS) is a powerful method for the systematic mapping of protein interaction networks and for investigating the mechanistic basis of biological processes. Here, we describe an optimized sequential peptide affinity (SPA) APMS procedure developed for the bacterium Escherichia coli that can be used to isolate and characterize stable multi-protein complexes to near homogeneity even starting from low copy numbers per cell.

의 단백질 단지의 식별<em&gt; 대장균</em&gt; 직렬 질량 분석법과 함께 순차적 펩타이드의 동질 정화를 사용하여

Since most cellular processes are mediated by macromolecular assemblies, the systematic identification of protein-protein interactions (PPI) and the ...

Capture Compound Mass Spectrometry - A Powerful Tool to Identify Novel c-di-GMP Effector Proteins

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis (diguanylate cyclases) and degradation (phosphodiesterases) of the second messenger c-di-GMP. In contrast, little information is available regarding the molecular mechanisms and cellular components through which this signaling molecule regulates a diverse range of cellular processes. Most of the known effector proteins belong to the PilZ family or are degenerated diguanylate cyclases or phosphodiesterases that have given up on catalysis and have adopted effector function. Thus, to better define the cellular c-di-GMP network in a wide range of bacteria experimental methods are required to identify and validate novel effectors for which reliable in silico predictions fail.
We have recently developed a novel Capture Compound Mass Spectrometry (CCMS) based technology as a powerful tool to biochemically identify and characterize c-di-GMP binding proteins. This technique has previously been reported to be applicable to a wide range of organisms1. Here we give a detailed description of the protocol that we utilize to probe such signaling components. As an example, we use Pseudomonas aeruginosa, an opportunistic pathogen in which c-di-GMP plays a critical role in virulence and biofilm control. CCMS identified 74% (38/51) of the known or predicted components of the c-di-GMP network. This study explains the CCMS procedure in detail, and establishes it as a powerful and versatile tool to identify novel components involved in small molecule signaling.

The ubiquitous second messenger c-di-GMP controls growth and behavior of many bacteria. We have developed a novel Capture Compound Mass Spectrometry based technology to biochemically identify and characterize c-di-GMP binding proteins in virtually any bacterial species.

캡처 복합 질량 분석 - 소설의 C 디 GMP 이펙터 단백질을 식별 할 수있는 강력한 도구

Considerable progress has been made during the last decade towards the identification and characterization of enzymes involved in the synthesis ...

Selected Reaction Monitoring Mass Spectrometry for Absolute Protein Quantification

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This protocol provides step-by-step instructions for the development and application of quantitative assays using selected reaction monitoring (SRM) mass spectrometry (MS). First, likely quantotypic target peptides are identified based on numerous criteria. This includes identifying proteotypic peptides, avoiding sites of posttranslational modification, and analyzing the uniqueness of the target peptide to the target protein. Next, crude external peptide standards are synthesized and used to develop SRM assays, and the resulting assays are used to perform qualitative analyses of the biological samples. Finally, purified, quantified, heavy isotope labeled internal peptide standards are prepared and used to perform isotope dilution series SRM assays. Analysis of all of the resulting MS data is presented. This protocol was used to accurately assay the absolute abundance of proteins of the chemotaxis signaling pathway within RAW 264.7 cells (a mouse monocyte/macrophage cell line). The quantification of Gi2 (a heterotrimeric G-protein &#945;-subunit) is described in detail.

This protocol describes how to perform absolute quantification assays of target proteins within complex biological samples using selected reaction monitoring. It was used to accurately quantify proteins of the mouse macrophage chemotaxis signaling pathway. Target peptide selection, assay development, and qualitative and quantitative assays are described in detail.

절대 단백질 정량 질량 분석을 모니터링 선정 반응

Absolute quantification of target proteins within complex biological samples is critical to a wide range of research and clinical applications. This ...

Enrichment of Extracellular Matrix Proteins from Tissues and Digestion into Peptides for Mass Spectrometry Analysis

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of cell proliferation, survival, migration, etc. The ECM plays important roles in development and in diverse pathologies including cardio-vascular and musculo-skeletal diseases, fibrosis, and cancer. Thus, characterizing the composition of ECMs of normal and diseased tissues could lead to the identification of novel prognostic and diagnostic biomarkers and potential novel therapeutic targets. However, the very nature of ECM proteins (large in size, cross-linked and covalently bound, heavily glycosylated) has rendered biochemical analyses of ECMs challenging. To overcome this challenge, we developed a method to enrich ECMs from fresh or frozen tissues and tumors that takes advantage of the insolubility of ECM proteins. We describe here in detail the decellularization procedure that consists of sequential incubations in buffers of different pH and salt and detergent concentrations and that results in 1) the extraction of intracellular (cytosolic, nuclear, membrane and cytoskeletal) proteins and 2) the enrichment of ECM proteins. We then describe how to deglycosylate and digest ECM-enriched protein preparations into peptides for subsequent analysis by mass spectrometry.

This protocol describes a procedure for enriching ECM proteins from tissues or tumors and deglycosylating and digesting the ECM-enriched preparations into peptides to analyze their protein composition by mass spectrometry.

질량 분석기 분석을위한 펩티드로 조직과 소화에서 세포 외 기질 단백질의 농축

The extracellular matrix (ECM) is a complex meshwork of cross-linked proteins that provides biophysical and biochemical cues that are major regulators of ...