이 작품은 건강 부여 GM - 26738 국립 연구소 (잣하는)에서 일부 지원됩니다.

1. Pak2 활성화 <ol><li> 버퍼에 12 밀리그램 / ML Pak2 20 μl에 caspase 3의 적절한 사전 테스트 금액을 추가 (산도 7.5 50 MM 트리스 - HCL, 150 MM NaCl, 2 MM의 DTT) 34시 품어 ° C를 30 분 완전히 다니엘과 활성화 Pak2 수 있습니다. </li><li> SDS - PAGE (10 %)과 Coommassie 푸른 얼룩에 의해 절단 제품을 분석합니다. </li><li> 20 μl 10 밀리그램 / ML Pak2의 최종 농도, pH를 7.5에서 (50 MM 트리스 - HCL, 150 MM NaCl, 15 MM MgCl 2, 10 MM ATP) 활성화 버퍼 B에 죽습 Pak2를 추가, 34에서 부화 ° C 30 분에 대해 전적으로 8 사이트에서 autophosphorylation로 Pak2를 활성화합니다. </li><li> 로 SDS - 10 페이지에 의해 결정 p34과 p27 조각의 마이 그 레이션에 의해 Pak2의 전체 절단을 확인합니다. autophosphorylation 조건 적절한 검증 phosphopeptides를 감지할 수 (32 P) ATP 또는 질량 분석계를 사용하여 별도의 실험에서 autoradiography를 통해 이루어집니다. </li></ol> 2. 신분증의 펩신 - 소화 Pak2 파편 <ol><li> 사용하기 전에 두 번 아가로 오스 비즈를 씻어 산도 2.5에서 0.1 % Trifluroacetic 산성 (TFA) 1 ML을 추가합니다. 그런 다음 비즈를 제거하는 2,000 XG에 15 초를위한 샘플을 원심. </li><li> 0.1 % TFA 100 μl에 Pak2 (150 MM NaCl 2 μl 20 μg, 산도 7.5 50 MM 트리스 - HCL 2 MM의 DTT)을 추가하고 펩신 - 복합 아가로 오스 비즈 30 μl로 5 분 샘플을 품어 . </li><li> 산도 2.5에서 0.1 %의 TFA의 기본 용매 시스템에 반대 위상 HPLC C18 컬럼을 평형. 0.2 ML / 분의 유량에서 0.1 % TFA를 포함하는 80 % acetonitrile - 펩신 소화 (50 μl)이 0 선형 100 분 그라데이션을 사용하여 eluted 있습니다. 분수는 매 2 분을 수집하고 있습니다. </li><li> (3 H) 속도 VAC와 HPLC의 분수를 건조하고 acetonitrile (5 μl)와 산도 2.5에서 0.1 % TFA (5 μl)의 솔루션에 샘플을 resuspend. </li><li> 처음에는 각 분획이 든 - TOF PerSeptive Biosystems 보이저 드 STR (PE 동성를 사용하여 펩티드를위한 검사입니다osystems, 포스터 시티, CA, 미국). </li><li> 펩티드를 포함하는 16 분수는 조각을 식별할 수있는 Q - TOF 질량 분석기와 QSTAR XL oMALDI MS / MS를 사용하여 탠덤 질량 분광법를 받게됩니다. </li><li> 각 펩타이드의 시퀀스 단백질 광부 웹사이트 (사용 Pak2의 기본 순서에 따라 이론적 M / Z 비율로 탠덤 질량 분광법에 의해 생성된 제품의 이온에 의해 확인 <a href="http://prospector.ucsf.edu/">http://prospector.ucsf.edu/</a> ) 11. </li></ol> 3. 아미드 H / D 교환의 측정 <ol><li> 물 목욕 25에서 H / D 교환에 필요한 모든 솔루션 및 시약 ° C를 잡아. ATP는 물속에 녹아이며, 산도는 사용하기 전에 7.0으로 조정됩니다. D 2 O 버퍼에 추가 4mm짜리 ATP는 희석 후 Pak2에서 dissociated ATP를 방지하는 것입니다. </li><li> (20 μg 2 μl로 버퍼 D 2 O (50 MM의 맙스의 산도 6.98 125 MM NaCl) 18 μl를 추가하여 H / D 교환을 개시) 150 MM를 포함하는 버퍼 Pak2의 NaCl, 0 50 MM 트리스 - HCL (산도 7.5) 및 2 MM의 10 MG / ML의 Pak2 수준 결과 DTT, 및 7.0의 산도 ° C. </li><li> 0, 0.5, 1, 3, 5, 10 분, 또는 24 H.위한 H / D 교환 세중의에서 샘플을 품어 </li><li> 200 μl의 최종 볼륨에서 2.5 산도을 제공 산도 2.2에서 얼음처럼 차가운 0.1 % TFA 180 μl을 추가하여 H / D 교환 과정을 끄다. ATP, 산도 2.2에서 얼음처럼 차가운 0.11 %의 TFA 180 μl를 포함하는 샘플 침묵 상태에서 2.5에서 산도를 유지하는 데 사용됩니다. </li><li> 활성화된 펩신 - 복합 아가로 오스 비즈 30 μl 각 침묵 샘플의 나누어지는 (100 μl)를 추가합니다. 펩신의 소화 다섯 분 얼음 진행하는 허용되고 샘플은 매 30 초 vortexed 있습니다. </li><li> 4 5000 XG에 15 초를위한 시료의 원심 분리하여 소화를 종료할 ° C는 펩신을 제거합니다. </li><li> 신속하게 더 이상 둘 이상을 -80 ° C에서 액체 N 2와 저장소에 샘플을 동결든 - TOF 분석하기 전에 일. </li><li> 든에 대한 매트릭스는 5 밀리그램 / ML α - cyano - 4 - hydroxycinnamic acetonitrile, 에탄올과 pH를 2.5에서 0.1 %의 TFA의 솔루션 (1시 1분 1초)에 용해와 0 ° C.에서 개최되는 산 (CHCA)입니다 </li><li> 부분적으로 신속하게 각각의 샘플을 해동하고 4 ° C 든 대상 접시에 매트릭스와 현장 1 μl 각 샘플 1 μl를 섞는다. </li><li> 이전 든 - TOF 분석 30 초에있는 명소를 건조 판에 중간 진공을 적용합니다. 샘플 및 스펙트럼 검색의 해동 사이의 시간은 3 분 정도입니다. </li><li> PerSeptive Biosystems 보이저 드 STR과 함께 든 - TOF 질량 스펙트럼을 취득. 20000 - V 가속 전압 및 180 NS 펄스 지연과 지연 추출을 사용하여 65 % 그리드 전압, 100,000 데이터 채널에서 2 GHz의 샘플링 속도에서 데이터를 수집. 백 검사는 2 분 안에 평균입니다. 든 - TOF 장비는 모든 브랜드 또는 특정 소프트웨어에 한정되지 않습니다. </li><li> calcul에 데이터 Explorer 4.0을 컴퓨터 프로그램을 사용하여각 조각의 deuteration을 먹었어요. </li><li> N - 터미널 아미드 (그림 1)를 제외하고 백본 아미드있는 모든 가능한 교환 사이트 나눈 H / D 교환의 24 시간에서 실제 통합 deuteron 번호의 비율에 따라 다시 교환을 계산합니다. </li></ol> 4. 대표 결과 : caspase 절단 및 autophosphorylation은 SDS - PAGE Commassie의 얼룩에 의해 확인됩니다. 비활성 Pak2 58 kDa의 단일 밴드입니다. Pak2의 Caspase 절단이 완료되며, 별도로 마이 그 레이션이 조각, p27과 p34을 생산하고 있습니다. p27과 p34 autophosphorylated 보여주는 완전히 phosphorylated p27 조각 (그림 2)의 지진아 마이 그 레이션에 의한 겔에 중복의 마이 그 레이션. H / D 교환 실험은 0과 10 분, (그림 3) 사이를 여러 번 수행하고 있습니다. 예를 들어, M / zp에 중수소 설립의 시간 코스비활성 Pak2의 eak 1697.85는 같은 시간이 지남에 따라 대량 봉투의 변화에​​ 의해 나타난 여러 isotopic 봉우리로 구성되어 있습니다. <img alt="그림 1" src="/files/ftp_upload/3602/3602fig1.jpg" /> 그림 1. deuteration의 계산을위한 수식. <img alt="그림 2" src="/files/ftp_upload/3602/3602fig2.jpg" /> 그림 2. Autophosphorylation과 Pak2의 caspase 절단. Caspase - 죽습 Pak2 (왼쪽 차선), 손상 비활성 Pak2 (중앙 차선)와 caspase - 죽습 autophosphorylated Pak2 (오른쪽 차선)는 SDS - PAGE와 Coommassie 푸른 얼룩에 의해 분석되었다. 수의, YH 외 2 적응. <img alt="그림 3" src="/files/ftp_upload/3602/3602fig3.jpg" /> 그림 3. 비활성 Pak2에 대한 중수소 설립의 시간 코스의 질량 스펙트럼. 비활성 Pak2는 H / D excha를 받게되었다00-10 분 nge 및 든 - TOF로 분석했다. H / D 교환의 시간 코스 중에 최고 M / Z 1697.85의 든 - TOF의 확장 isotopic 배포판의 예제는 표시됩니다. 빨간색 점선은 대량 봉투의 평균을 나타냅니다하고 대량 봉투의 변화는 펩티드의 deuteration를 보여줍니다.

<ol>
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관심 없음 충돌 선언하지 않습니다.

펩신 - 소화 파편의 확인은 H / D 교환 실험의 중요한 단계입니다. 펩타이드의 잘못된 인식은 잘못된 결론으로​​ 이어질 수 있습니다. 펩신 - 소화 Pak2은 깨끗한 배경을 얻기 위해 반대 위상 HPLC C18 컬럼을 통해 eluted입니다. 우리의 실험에서 40 분수의 총 회수되었고 든 - TOF를 받게. 여러 개의 펩티드는 분수의 각 식별 수 있습니다. 펩티드들은 시퀀스를 식별하는 탠덤 질량 분광법 (Q - TOF MS / MS 및 oMALDI MS / MS)를 받게되었습니다. 펩타이드의 MS / MS 스펙트럼의 펩타이드의 신분을 확인하기 위해 적어도 세 제품 이온이 있어야합니다. 데이터 Explorer 4.0을 컴퓨터 프로그램 (응용 Biosystems)는 초기 기준 보정과 노이즈 여과를 수행합니다. 각 스펙트럼은 두 개의 봉우리 합성을 기반으로 보정해야합니다. 우리는 923.45과 1697.84있는 undeuterated M / Z의 이론적인 대량하여 스펙트럼을 보정. 티그는 교정 후 대량의 정확성은 10 PPM에 도달하실 수 있습니다. deuteration시 모노 동위 원소는 높은 미사로 이동됩니다. 이러한 M / Z 비율로 하나의 단계 증가 deuteration와 관련된 향상된 대중이 나왔고. 대량 봉투의 정상 농도는 deuteration 수준에 영향을 미치지 않습니다. 그러나, 특정 집단 봉투에 isotopic 봉우리의 정상 농도는 평균 질량 계산 중요하다. 통합 deuteron의 계산의 첫 번째 단계는 진정제를 맞았을 샘플에서 비 진정제를 맞았 샘플의 펩티드 질량을 빼서입니다. 세 가지 다른 요인, D 2 O 희석, 사이드 체인과 다시 교환 잔류 deuterons은 더 (그림 1) 계산에 고려해야합니다. D 2 O의 희석은 샘플을 믹싱 및 D 2 O 버퍼 H / D 교환을 시작 후 D 2 O의 희석 요소입니다. 펩신 - 다이제스트의 측면 체인의 잔류 deuteron (4.5 %)에드 펩티드는 빼서해야합니다. 다시 교환은 질량 측정 과정에있는 모든 진정제를 맞았 및 펩신 - 소화 펩티드의 deuteration의 피할 수없는 손실이다. H / D 교환 24 시간 후 가장 용매 접근 펩타이드 (M / Z = 1105.60)은 전체 deuteration 지역을 나타냅니다. 펩티드의 각 통합 deuteron 번호는 진정제를 맞았와 비 진정제를 맞았 Pak2 소화제의 펩티드의 중심 사이의 차이입니다. 가장 높은 진정제를 맞았 펩타이드 M / Z 1105은 Pak2은 H / D 교환의 24 시간에 있었을 때 전체 deuteration을 대표로 선정되었다. 뒤로 교환 비율은 펩타이드 M / Z 1105에서 가능한 모든 교환 사이트로 나눈 H / D 교환의 24 시간에서 실제 통합 deuteron 번호로 계산되었다.

<table border="1"></table><table border="1"><tbody><tr><td> 시약의 이름 </td><td> 회사 </td><td> 카탈로그 번호 </td><td> 댓글 (옵션) </td></tr><tr><td> D 2 O </td><td> 올드 리치 </td><td> 7789-20-0 </td><td></td></tr><tr><td> α - cyano - 4 - hydroxycinnamic 산 </td><td> 시그마 </td><td> 28166-41-8 </td><td></td></tr><tr><td> 든 - TOF </td><td> PE Biosystems </td><td> 보이저 드 STR </td><td></td></tr><tr><td> Q - TOF 질량 분석계 </td><td> Q - TOF 울티마 - 글로벌 </td><td></td><td></td></tr><tr><td> QSTAR XL oMALDI MS / MS </td><td> 응용 Biosystems </td><td></td><td></td></tr><tr><td> 데이터 익스플로러 4.0 컴퓨터 프로그램 </td><td> 응용 Biosystems </td><td></td><td></td></tR&gt; </tbody></table>

amide hydrogen/deuterium exchange &amp; maldi-tof mass spectrometry analysis of pak2 activation

질량 분석계와 결합 아미드 수소 / 중수소 교환 (H / D 교환)은 광범위하게 단백질 - 단백질 상호 작용, 단백질 conformational 변화, 단백질 역학 및 단백질 - 리간드 상호 작용의 인터페이스를 분석하는 데 사용되었습니다. 백본 아미드 위치에 H / D 교환 대량 분석법 1,2,3하여 단백질의 마이크로 영역의 deuteration 속도를 측정하는 이용되었습니다. 이 방법의 해상도는 일반적으로 30-20 잔류물에서 다양한 펩티드에 대한 관심의 진정제를 맞았 단백질의 펩신의 소화에 따라 달라집니다. 질량 분석법에 의해 측정 H / D 교환의 해상도 NMR, H / D 교환에서 질량 분석계 측정의 헤테로핵 단일 양자 결맞음 (HSQC) 방법으로 측정 단일 잔여물 해상도보다 낮은이지만,의 크기에 의해 제한되지 않습니다 단백질 4. H / D 교환은 단백질 형태를 유지 수용액에서 수행됩니다. 우리는 방법을 제공 UTIL대신 HPLC / ESI (전기 분무 이온화) - MS 시스템 5,6의 검출 2 든 - TOF를 izes. 든 - TOF이 경우 단백질 키나제 Pak2 (또한 γ - 박라고도 함)에서, 소화 단백질의 펩티드에 대한 정확한 대량 강도 데이터를 제공합니다. 박 2 Proteolysis는 오프라인 펩신의 소화에서 수행됩니다. 사용자가 질량 분광법에 연결된 HPLC 및 펩신 컬럼에 액세스할 때 또는 HPLC에있는 펩신 칼럼은 크게 이황화 - 보세 분비 포스 포 라이 페이스 2, 예를 들어, 최적의 소화지도 발생하지 않는가없는이 대체 방법, (sPLA 2). 이 방법을 활용, 우리는 성공적으로 caspase 3 절단 및 autophosphorylation 7, 8, 9에 의해 Pak2의 활성화 동안 deuteration 수준의 변화를 감시.

Watch this Scientific Journal Video about Amide Hydrogen/Deuterium Exchange & MALDI-TOF Mass Spectrometry Analysis of Pak2 Activation at JoVE.com

Amide Hydrogen/Deuterium Exchange &amp; MALDI-TOF Mass Spectrometry Analysis of Pak2 Activation

든 - TOF 질량 분석법을 성공적으로 단백질 키나제 Pak2 활성화에 아미드 수소 / 중수소 교환을 모니터 활용했다.

Pak2 활성화의 아미드 수소 / 중수소 교환 및 든 - TOF 질량 분광법 분석

Amide hydrogen/deuterium exchange (H/D exchange) coupled with mass spectrometry has been widely used to analyze the interface of protein-protein ...

amide-hydrogendeuterium-exchange-maldi-tof-mass-spectrometry-analysis

Research

JoVE Journal

Biology

17.7K 조회수.  Tunghai University. 든 - TOF 질량 분석법을 성공적으로 단백질 키나제 Pak2 활성화에 아미드 수소 / 중수소 교환을 모니터 활용했다.

비디오: Amide Hydrogen/Deuterium Exchange & MALDI-TOF Mass Spectrometry Analysis of Pak2 Activation

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

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 ...

T-wave Ion Mobility-mass Spectrometry: Basic Experimental Procedures for Protein Complex Analysis

Ion mobility (IM) is a method that measures the time taken for an ion to travel through a pressurized cell under the influence of a weak electric field. The speed by which the ions traverse the drift region depends on their size: large ions will experience a greater number of collisions with the background inert gas (usually N2) and thus travel more slowly through the IM device than those ions that comprise a smaller cross-section. In general, the time it takes for the ions to migrate though the dense gas phase separates them, according to their collision cross-section (&Omega;). 
Recently, IM spectrometry was coupled with mass spectrometry and a traveling-wave (T-wave) Synapt ion mobility mass spectrometer (IM-MS) was released. Integrating mass spectrometry with ion mobility enables an extra dimension of sample separation and definition, yielding a three-dimensional spectrum (mass to charge, intensity, and drift time). This separation technique allows the spectral overlap to decrease, and enables resolution of heterogeneous complexes with very similar mass, or mass-to-charge ratios, but different drift times. Moreover, the drift time measurements provide an important layer of structural information, as &Omega; is related to the overall shape and topology of the ion. The correlation between the measured drift time values and &Omega; is calculated using a calibration curve generated from calibrant proteins with defined cross-sections1.
The power of the IM-MS approach lies in its ability to define the subunit packing and overall shape of protein assemblies at micromolar concentrations, and near-physiological conditions1. Several recent IM studies of both individual proteins2,3 and non-covalent protein complexes4-9, successfully demonstrated that protein quaternary structure is maintained in the gas phase, and highlighted the potential of this approach in the study of protein assemblies of unknown geometry. Here, we provide a detailed description of IMS-MS analysis of protein complexes using the Synapt (Quadrupole-Ion Mobility-Time-of-Flight) HDMS instrument (Waters Ltd; the only commercial IM-MS instrument currently available)10. We describe the basic optimization steps, the calibration of collision cross-sections, and methods for data processing and interpretation. The final step of the protocol discusses methods for calculating theoretical &Omega; values. Overall, the protocol does not attempt to cover every aspect of IM-MS characterization of protein assemblies; rather, its goal is to introduce the practical aspects of the method to new researchers in the field.

Ion mobility-mass spectrometry is an emerging gas-phase technology that separates ions, based on their collision cross-section and mass. The method provides three-dimensional information on the overall topology and shape of protein complexes. Here, we outline a basic procedure for instrument setting and optimization, calibration of drift times, and data interpretation.

T - 파 이온 모빌리티 분석법 - 질량 : 단백질 복잡한 분석을위한 기본 실험 절차

Ion mobility (IM) is a method that measures the time taken for an ion to travel through a pressurized cell under the influence of a weak electric field. ...

Direct Analysis of Single Cells by Mass Spectrometry at Atmospheric Pressure

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell types exhibit heterogeneous biochemical makeup depending on their physiological conditions and interactions with the environment. Conventional methods of mass spectrometry (MS) used for the analysis of biomolecules in single cells rely on extensive sample preparation. Removing the cells from their natural environment and extensive sample processing could lead to changes in the cellular composition. Ambient ionization methods enable the analysis of samples in their native environment and without extensive sample preparation.1 The techniques based on the mid infrared (mid-IR) laser ablation of biological materials at 2.94 &mu;m wavelength utilize the sudden excitation of water that results in phase explosion.2 Ambient ionization techniques based on mid-IR laser radiation, such as laser ablation electrospray ionization (LAESI) and atmospheric pressure infrared matrix-assisted laser desorption ionization (AP IR-MALDI), have successfully demonstrated the ability to directly analyze water-rich tissues and biofluids at atmospheric pressure.3-11 In LAESI the mid-IR laser ablation plume that mostly consists of neutral particulate matter from the sample coalesces with highly charged electrospray droplets to produce ions. Recently, mid-IR ablation of single cells was performed by delivering the mid-IR radiation through an etched fiber. The plume generated from this ablation was postionized by an electrospray enabling the analysis of diverse metabolites in single cells by LAESI-MS.12 This article describes the detailed protocol for single cell analysis using LAESI-MS. The presented video demonstrates the analysis of a single epidermal cell from the skin of an Allium cepa bulb. The schematic of the system is shown in Figure 1. A representative example of single cell ablation and a LAESI mass spectrum from the cell are provided in Figure 2.

Single cell analysis is performed by mass spectrometry on plant and animal cells at atmospheric pressure by using a sharpened optical fiber to sample the cells for laser ablation electrospray ionization (LAESI) mass spectrometry.

대기압에서 질량 분석법에 의해 단일 ​​세포의 직접 분석

Analysis of biochemicals in single cells is important for understanding cell metabolism, cell cycle, adaptation, disease states, etc. Even the same cell ...

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 ...

Use of MALDI-TOF Mass Spectrometry and a Custom Database to Characterize Bacteria Indigenous to a Unique Cave Environment (Kartchner Caverns, AZ, USA)

MALDI-TOF mass spectrometry has been shown to be a rapid and reliable tool for identification of bacteria at the genus and species, and in some cases, strain levels. Commercially available and open source software tools have been developed to facilitate identification; however, no universal/standardized data analysis pipeline has been described in the literature. Here, we provide a comprehensive and detailed demonstration of bacterial identification procedures using a MALDI-TOF mass spectrometer. Mass spectra were collected from 15 diverse bacteria isolated from Kartchner Caverns, AZ, USA, and identified by 16S rDNA sequencing. Databases were constructed in BioNumerics 7.1. Follow-up analyses of mass spectra were performed, including cluster analyses, peak matching, and statistical analyses. Identification was performed using blind-coded samples randomly selected from these 15 bacteria. Two identification methods are presented: similarity coefficient-based and biomarker-based methods. Results show that both identification methods can identify the bacteria to the species level.

Use of MALDI-TOF Mass Spectrometry and a Custom Database to Characterize Bacteria Indigenous to a Unique Cave Environment Kartchner Caverns, AZ, USA

This work details procedures for rapid identification of bacteria using MALDI-TOF MS. The identification procedures include spectrum acquisition, database construction, and follow up analyses. Two identification methods, similarity coefficient-based and biomarker-based methods, are presented.

MALDI-TOF 질량 분석 및 사용자 지정 데이터베이스의 사용은 고유 동굴 환경 (Kartchner 동굴, AZ, USA)에 토착 박테리아의 특성을

MALDI-TOF mass spectrometry has been shown to be a rapid and reliable tool for identification of bacteria at the genus and species, and in some cases, ...

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 ...

Applications of the Single-probe: Mass Spectrometry Imaging and Single Cell Analysis under Ambient Conditions

Mass spectrometry imaging (MSI) and in-situ single cell mass spectrometry (SCMS) analysis under ambient conditions are two emerging fields with great potential for the detailed mass spectrometry (MS) analysis of biomolecules from biological samples. The single-probe, a miniaturized device with integrated sampling and ionization capabilities, is capable of performing both ambient MSI and in-situ SCMS analysis. For ambient MSI, the single-probe uses surface micro-extraction to continually conduct MS analysis of the sample, and this technique allows the creation of MS images with high spatial resolution (8.5 &#181;m) from biological samples such as mouse brain and kidney sections. Ambient MSI has the advantage that little to no sample preparation is needed before the analysis, which reduces the amount of potential artifacts present in data acquisition and allows a more representative analysis of the sample to be acquired. For in-situ SCMS, the single-probe tip can be directly inserted into live eukaryotic cells such as HeLa cells, due to the small sampling tip size (&lt; 10 &#181;m), and this technique is capable of detecting a wide range of metabolites inside individual cells at near real-time. SCMS enables a greater sensitivity and accuracy of chemical information to be acquired at the single cell level, which could improve our understanding of biological processes at a more fundamental level than previously possible. The single-probe device can be potentially coupled with a variety of mass spectrometers for broad ranges of MSI and SCMS studies.

Here, we present protocols to perform both ambient mass spectrometry imaging (MSI) of tissues and in-situ live single cell MS (SCMS) analysis using the single-probe, which is a miniaturized multifunctional device for MS analysis.

단일 프로브의 응용 프로그램 : 주변 조건에서 질량 분석 이미징 및 단일 세포 분석

Mass spectrometry imaging (MSI) and in-situ single cell mass spectrometry (SCMS) analysis under ambient conditions are two emerging fields with great ...

Complete Workflow for Analysis of Histone Post-translational Modifications Using Bottom-up Mass Spectrometry: From Histone Extraction to Data Analysis

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone function is mediated by extensive post-translational modification by a myriad of nuclear proteins. These modifications are critical for nuclear integrity as they regulate chromatin structure and recruit enzymes involved in gene regulation, DNA repair and chromosome condensation. Even though a large part of the scientific community adopts antibody-based techniques to characterize histone PTM abundance, these approaches are low throughput and biased against hypermodified proteins, as the epitope might be obstructed by nearby modifications. This protocol describes the use of nano liquid chromatography (nLC) and mass spectrometry (MS) for accurate quantification of histone modifications. This method is designed to characterize a large variety of histone PTMs and the relative abundance of several histone variants within single analyses. In this protocol, histones are derivatized with propionic anhydride followed by digestion with trypsin to generate peptides of 5 - 20 aa in length. After digestion, the newly exposed N-termini of the histone peptides are derivatized to improve chromatographic retention during nLC-MS. This method allows for the relative quantification of histone PTMs spanning four orders of magnitude.

This protocol outlines a fully integrated workflow for characterizing histone post-translational modifications using mass spectrometry (MS). The workflow includes histone purification from cell cultures or tissues, histone derivatization and digestion, MS analysis using nano-flow liquid chromatography and instructions for data analysis. The protocol is designed for completion within 2 - 3 days.

히스톤 추출에서 데이터 분석에 : 상향식 질량 분석 사용 히스톤 번역 후 변형 분석을위한 완벽한 워크 플로우

Nucleosomes are the smallest structural unit of chromatin, composed of 147 base pairs of DNA wrapped around an octamer of histone proteins. Histone ...