이 작품은 캐나다 자연 과학 및 공학 연구 위원회, 퐁드 드 레체슈 뒤 퀘벡 자연 에 테크놀로지, 혁신을위한 캐나다 재단, 맥길 대학 창업 기금에 의해 지원되었다.

<ol>
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우리는 공개 할 것이 없습니다.

이 프로토콜에 제시된 HDX-MS 워크플로우는 단백질의 구조적으로 역동적인 요소의 공간 분포를 매핑하고 이러한 역학이 변투(리간드 결합, 효소 돌연변이 발생 등)에 대한 응답으로 어떻게 변화하는지 조사하기 위한 매우 견고한 플랫폼을 제공합니다. HDX-MS는 일반적으로 형성 역학을 조사하는 데 사용되는 다른 구조 생물학 접근 방식에 비해 몇 가지 뚜렷한 이점을 보유하고 있습니다. 특히 소량의...

단백질은 펨토초 규모의 결합 진동에서부터 여러 초 동안 발생할 수 있는 전체 단백질 도메인의 재배열에 이르기까지 시간 척도에 따라 다른 적합성을 샘플링하는 구조적으로 역동적인분자입니다. 이러한 형태 적 변동은 종종 효소 / 단백질 기능의 중요한 측면입니다. 예를 들어, 리간드 결합에 의해 유도된 형태 변화는 촉매에 필요한 활성 부위 잔류물을 구성하거나, 순차적 운동 메커니즘에서 기판 결합 부위를 정의하거나, 환경에서 반응성 중간체를 차폐하거나, 알로스티아 네트워크를 통해 효소 기능을 조절함으로써 효소 기능을 조절하는 데 매우 중요합니다. 최근 연구에 따르면 형태 역학은 진화 전반에 걸쳐 보존될 수 있으며 분자 운동을 보존하는 혼란은 기판 특이성의 변화와 새로운 효소 기능2,3의출현과 상관관계가 있을 수 있음을 보여주었다.
최근 몇 년 동안 수소-중수소 교환 질량 분광법(HDX-MS)은 단백질 형성 경관이 리간드 결합 또는 돌연변이 발생4,5,6,7과같은 동요에 어떻게 반응하는지 조사하는 강력한 기술로 급속히부상하고 있다. 전형적인 HDX-MS실험(도 1)에서관심 있는 단백질은 D2O로 제조된 완충제에 배치되어, 이는 신테리아를 가진 용매 교환 성수기의 교체를 유발한다. 펩티드 결합의 아미드 모이티의 교환 속도는 pH, 국소 아미노산 서열 및 아미드8의국소 구조 환경에 크게 의존한다. 수소 결합 상호 작용에 종사하는 아미드 (예 : α-헬릭 및 β 시트에 존재하는 것과 같은)은 대량 용매에 노출되는 단백질의 구조화되지 않은 영역에서 아미드보다 더 느리게 교환됩니다. 따라서, 중수소 섭취의 정도는 효소의 구조를 반영한다. 형태역학적 이거나 리간드 결합 시 구조적 전이를 겪는 효소는 측정 가능한 HDX 응답을 얻을 것으로 예상됩니다.
구조화된 아마이드의 느린 환율에 대한 기계론적 기초는 도 25,8,9로도시된다. HDX를 받기 위해서는, 구조화된 영역은 먼저 전개된 형성을 일시적으로 샘플링해야 하며, 이러한 용매 분자는 특정 산/염기 화학 메커니즘을 통해 HDX 교환을 촉매하는, 교환 가능한 아미드에 접근할 수 있다. 궁극적으로,화학환율(kchem)과접이식 및 재접이식 비율(kopen 및 kclose)의상대적 크기는 실험5,8에서측정된 HDX 속도를 결정한다. 이 간단한 운동 모델에서, 중수소 섭취량의 정도는 기본 형태 역학을 반영할 것이 분명하다 (k열기 및 k닫기에의해 정의). 대부분의 HDX-MS 실험은 상향식 워크플로우에서 수행되며, 교환 반응에 따라 관심 있는 단백질이 펩타이드로 소화되고 각 펩티드에 의한 중수소 흡수량은 질량7의증가로 측정된다. 이러한 방식으로, HDX-MS는 효소 형성 역학에 대한 교란을 펩티드의 로컬 공간 척도에 매핑할 수 있게 하여, 연구자가 동요가 관심 있는 효소의 다른 영역에서 역학을 어떻게 변화시키는지 평가할 수 있게 합니다.
단백질 구조 역학을 해명하기 위한 HDX-MS 접근법의 장점은 다양합니다. 먼저, 이 방법은 사분위구조(10)를가진 시스템에서 소량의 토종 단백질 또는 단백질 복합체로 수행될 수 있다. 분석에서 사용되는 효소 제제는11,12,상향식 HDX-MS 워크플로우가 단백질 서열을 커버하는 충분한 수의 자신있게 확인된 펩티드를 제공하는 한, 고도로 정제될 필요가 없다. 더욱이, HDX-MS는 단일 분자 형광연구에서사용되는 바와 같이 현장별 단백질 라벨링필요 없이 근사 조건하에서 형성 역학에 대한 정보를 제공할 수 있으며, 조사할 수 있는 단백질 또는 단백질 복합체에 대한 크기 제한은 없다(핵자기 공명[NMR] 분광법 도전과 같은 접근법을 만드는)7,14. 마지막으로, 시간 해결된 HDX-MS 방법은 X선결정학15,16,17,18로공부하기 어려운 본질적으로 무질서한 단백질을 연구하기 위하여 이용될 수 있다. HDX-MS의 주요 제한사항은 데이터가 낮은 구조적 해상도라는 것입니다. HDX-MS 데이터는 형태 역학이 변화하는 곳을 가리키고 결합된 형태 변화를 드러내는 데 유용하지만 관찰된 변화를 유도하는 정확한 분자 메커니즘에 대한 많은 통찰력을 제공하지는 않습니다. 단백질 HDX-MS 데이터와 전자 포획 해리 방법의 조합에 있는 최근 발전은 단 하나 아미노산 잔류물19에교환 사이트를 매핑하는 약속을 보여주었습니다, 그러나 후속 생화확적인 및 구조 적인 연구는 아직도 HDX-MS 데이터에 의해 전달된 구조 모형에 명확성을 제공하기 위하여 수시로 필요합니다.
아래, HDX-MS 분석기의 개발을 위한 자세한 프로토콜이20을제시한다. 아래에 제시된 견본 준비 프로토콜은 수성 완충제에 있는 좋은 용해도를 전시하는 어떤 단백질든지 일반적으로 적용되어야 합니다. 세제 또는 인지질(21,22,23,24)의존재에서 분석될 필요가 없는 것보다 더 전문적인 시료 제조 방법및HDX-MS 워크플로우를 단백질에 사용할 수있다. HDX-MS 데이터 수집을 위한 기악 설정은 액체 크로마토그래피 시스템에 결합된 고해상도 쿼드러폴 비행 시간 질량 분광계에 대해 설명됩니다. 유사한 복잡성 과 해상도의 데이터는 상업적으로 이용 가능한 다수의 액체 크로마토그래피 질량 분석법(LC-MS) 시스템에서 수집될 수 있습니다. 시판되는 소프트웨어 패키지를 사용하여 데이터 처리의 주요 측면도 제공됩니다. 또한 광범위한 HDX-MS 커뮤니티12의권장 사항과 일치하는 데이터 수집 및 분석에 대한 지침을 제시합니다. 기재된 프로토콜은 항균 펩티드천연물(20)의다단계 성숙을 촉매하는 란티펩타이드 합성물인 HalM2의 동적 구조적 특성을 연구하는 데 사용된다. 우리는 HDX-MS가 기판 바인딩 사이트와 이전 특성을 벗어난 알로스터 속성을 공개하는 데 어떻게 사용할 수 있는지 보여줍니다. 단백질 HDX-MS에 대한 몇몇 그밖 프로토콜은 최근25,26에서간행되었습니다. 현재 작업과 함께 이러한 초기 기여는 독자에게 실험 설계에 약간의 유연성을 제공해야 합니다.

<table>
	<tbody>
		<tr>
			<td>Reagents</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>[glu-1]-fibrinopeptide B (Glu-Fib)</td>
			<td>BioBasic</td>
			<td>NA</td>
			<td></td>
		</tr>
		<tr>
			<td>0.5 mL Amicon Ultracel 10k centrifugal filtration device (Millipore)</td>
			<td>Milipore Sigma</td>
			<td>UFC501096</td>
			<td></td>
		</tr>
		<tr>
			<td>acetonitrile</td>
			<td>Fisher</td>
			<td>A955-1</td>
			<td></td>
		</tr>
		<tr>
			<td>AMP-PNP</td>
			<td>SIGMA</td>
			<td>A2647-25MG</td>
			<td></td>
		</tr>
		<tr>
			<td>ATP</td>
			<td>SIGMA</td>
			<td>a2383-5G</td>
			<td></td>
		</tr>
		<tr>
			<td>D2O</td>
			<td>ALDRICH</td>
			<td>435767-100G</td>
			<td></td>
		</tr>
		<tr>
			<td>formic acid</td>
			<td>Thermo Fisher</td>
			<td>28905</td>
			<td></td>
		</tr>
		<tr>
			<td>guanidine-HCl</td>
			<td>VWR</td>
			<td>97063-764</td>
			<td></td>
		</tr>
		<tr>
			<td>HEPES</td>
			<td>Fisher</td>
			<td>BP310-1</td>
			<td></td>
		</tr>
		<tr>
			<td>Magnesium chloride</td>
			<td>SiGMA-Aldrich</td>
			<td>63068-250G</td>
			<td></td>
		</tr>
		<tr>
			<td>Potassium chloride</td>
			<td>BioBasic</td>
			<td>PB0440</td>
			<td></td>
		</tr>
		<tr>
			<td>potassium phosphate</td>
			<td>BioBasic</td>
			<td>PB0445</td>
			<td></td>
		</tr>
		<tr>
			<td>TCEP Hydrochloride</td>
			<td>TRC Canada</td>
			<td>T012500</td>
			<td>peptide was synthesized upon request</td>
		</tr>
		<tr>
			<td>Name of Material/ Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>software</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>Deuteros</td>
			<td>Andy M C Lau, et al</td>
			<td>version 1.08</td>
			<td></td>
		</tr>
		<tr>
			<td>DynamX</td>
			<td>Waters</td>
			<td>version 3.0</td>
			<td></td>
		</tr>
		<tr>
			<td>MassLynx</td>
			<td>Waters</td>
			<td>version 4.1</td>
			<td></td>
		</tr>
		<tr>
			<td>Protein Lynx 
			Global Server (PLGS)</td>
			<td>Waters</td>
			<td>version 3.0.3</td>
			<td></td>
		</tr>
		<tr>
			<td>PyMOL</td>
			<td>Schr&#246;dinger</td>
			<td>version 2.2.2</td>
			<td></td>
		</tr>
		<tr>
			<td>Name of Material/ Equipment</td>
			<td>Company</td>
			<td>Catalog Number</td>
			<td>Comments/Description</td>
		</tr>
		<tr>
			<td>Instrument and equipment</td>
			<td></td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>ACQUITY UPLC BEH C18 analytical Column</td>
			<td>Waters</td>
			<td>186002346</td>
			<td></td>
		</tr>
		<tr>
			<td>ACQUITY UPLC BEH C8 VanGuard Pre-column</td>
			<td>Waters</td>
			<td>186003978</td>
			<td></td>
		</tr>
		<tr>
			<td>ACQUITY UPLC M-Class HDX System</td>
			<td>Waters</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>HDX Manager</td>
			<td>Waters</td>
			<td></td>
			<td></td>
		</tr>
		<tr>
			<td>microtip pH electrode</td>
			<td>Thermo Fisher</td>
			<td>13-620-291</td>
			<td></td>
		</tr>
		<tr>
			<td>Waters Enzymate BEH column or Pepsin solumn</td>
			<td>Waters</td>
			<td>186007233</td>
			<td></td>
		</tr>
		<tr>
			<td>Waters Synapt G2-Si</td>
			<td>Waters</td>
			<td></td>
			<td></td>
		</tr>
	</tbody>
</table>

a hydrogen-deuterium exchange mass spectrometry (hdx-ms) platform for investigating peptide biosynthetic enzymes

수소-황량제 교환 질량 분석법(HDX-MS)은 효소 형태 변화와 효소 기질 상호작용의 생체물리학적 특성화를 위한 강력한 방법입니다. HDX-MS는 소량의 물질만 소비하며, 효소/기질 라벨링 없이 근동 조건하에서 수행할 수 있으며, 큰 효소 및 다단백 복합체에서도 효소 형성 역학에 대한 공간적으로 해결된 정보를 제공할 수 있습니다. 이 방법은D2O에서 제조된 완충제로 관심 있는 효소의 희석에 의해 개시된다. 이것은 중수소 (N-D)를 가진 펩티드 본드 아마이네스 (N-H)에서 프로티움의 교환을 트리거합니다. 원하는 교환 시점에서, 반응 알리쿼트가 담금질되고, 효소는 펩티드로 프로테오리제화되고, 펩타이드는 초고성능 액체 크로마토그래피(UPLC)에 의해 분리되고, 각 펩타이드의 질량의 변화(중수소용 수소의 교환로 인해)는 MS에 의해 기록된다. 각 펩티드에 의한 중수소 섭취량의 양은 해당 펩티드의 국소 수소 결합 환경에 크게 의존한다. 펩타이드는 효소 교환 중음류의 매우 역동적인 부위에 매우 빠르게 존재하는 반면, 잘 정렬된 지역에서 유래된 펩타이드는 훨씬 더 느리게 교환을 받는다. 이러한 방식으로, HDX 속도 로컬 효소 형성 역학에 대 한 보고서. 다른 리간드가 있는 중수소 섭취량 수준에 대한 혼란은 리간드 결합 부위를 매핑하고, 알로스터 네트워크를 식별하고, 효소 기능에서 형성 역학의 역할을 이해하는 데 사용될 수 있다. 여기에서, 우리는 우리가 더 나은 lanthipeptides에게 불린 펩티드 천연 제품의 모형의 생합성을 이해하기 위하여 HDX-MS를 이용한 방법을 보여줍니다. Lanthipeptides는 전통적인 구조 생물학 접근으로 공부하기 어려운 크고 다기능, 형태역학적 효소에 의해 번역 후 변형되는 유전자 로코딩된 펩티드입니다. HDX-MS는 이러한 유형의 효소의 기계적인 특성을 조사하기 위한 이상적이고 적응력이 뛰어난 플랫폼을 제공합니다.

각 샘플 주사 세트에 대한 보철 소화의 품질과 워크플로우의 재현성을 평가할 필요가 있습니다. 따라서, HDX-MS 애사를 수행하기 전에, 관심 있는 단백질의 프로테오리시스에 대한 효과적인 조건을 확립하고, 역상 액체 크로마토그래피 및 가스상 이온 이동성을 이용한 펩티드의 분리를 위해, MS를 이용한 펩타이드검출을 위한 것이 필수적이다. 이를 위해 관심 있는 단백질에 대한 기준 샘플(중수소?...

Watch this Scientific Journal Video about A Hydrogen-Deuterium Exchange Mass Spectrometry HDX-MS Platform for Investigating Peptide Biosynthetic Enzymes at JoVE.com

A Hydrogen-Deuterium Exchange Mass Spectrometry HDX-MS Platform for Investigating Peptide Biosynthetic Enzymes

Lanthipeptide 는 펩타이드 천연 제품의 생합성 중에 다단계 반응을 촉매합니다. 여기서, 우리는 펩타이드 천연 제품 생합성에 관여하는 다른 유사한 효소뿐만 아니라 란티페타이드 신디사이저의 형성 역학을 연구하기 위해 사용될 수 있는 연속, 상향식, 수소 중수소 교환 질량 분석법(HDX-MS) 워크플로우를 설명합니다.

펩타이드 생합성 효소를 조사하기 위한 수소-신투륨 교환 질량 분광법(HDX-MS) 플랫폼

Hydrogen-deuterium exchange mass spectrometry (HDX-MS) is a powerful method for the biophysical characterization of enzyme conformational changes and ...

a-hydrogen-deuterium-exchange-mass-spectrometry-hdx-ms-platform-for

Research

JoVE Journal

Biochemistry

A Hydrogen-Deuterium Exchange Mass Spectrometry (HDX-MS) Platform for Investigating Peptide Biosynthetic Enzymes

7.8K 조회수.  McGill University. Lanthipeptide 는 펩타이드 천연 제품의 생합성 중에 다단계 반응을 촉매합니다. 여기서, 우리는 펩타이드 천연 제품 생합성에 관여하는 다른 유사한 효소뿐만 아니라 란티페타이드 신디사이저의 형성 역학을 연구하기 위해 사용될 수 있는 연속, 상향식, 수소 중수소 교환 질량 분석법(HDX-MS) 워크플로우를 설명합니다.

비디오: A Hydrogen-Deuterium Exchange Mass Spectrometry HDX-MS Platform for Investigating Peptide Biosynthetic Enzymes

Time-resolved ElectroSpray Ionization Hydrogen-deuterium Exchange Mass Spectrometry for Studying Protein Structure and Dynamics

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. Hydrogen-Deuterium Exchange (HDX) measured at rapid time scales is uniquely suited to detect structures and hydrogen bonding networks that are briefly populated, allowing for the characterization of transient conformers in native ensembles. Coupling of HDX to mass spectrometry offers several key advantages, including high sensitivity, low sample consumption and no restriction on protein size. This technique has advanced greatly in the last several decades, including the ability to monitor HDX labeling times on the millisecond time scale. In addition, by incorporating the HDX workflow onto a microfluidic platform housing an acidic protease microreactor, we are able to localize dynamic properties at the peptide level. In this study, Time-Resolved ElectroSpray Ionization Mass Spectrometry (TRESI-MS) coupled to HDX was used to provide a detailed picture of residual structure in the tau protein, as well as the conformational shifts induced upon hyperphosphorylation.

Conformational flexibility plays a critical role in protein function. Herein, we describe the use of time-resolved electrospray ionization mass spectrometry coupled to hydrogen-deuterium exchange for probing the rapid structural changes that drive function in ordered and disordered proteins.

시간 해결 전기 분무 이온화 수소 - 중수소 교환 질량 분석 단백질 구조와 역학 연구를위한

Intrinsically disordered proteins (IDPs) have long been a challenge to structural biologists due to their lack of stable secondary structure elements. ...

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생화학

Resolving Affinity Purified Protein Complexes by Blue Native PAGE and Protein Correlation Profiling

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological processes. Affinity purification coupled to mass spectrometry (AP-MS) has become the method of choice for identifying protein-protein interactions. However, conventional AP-MS studies provide information on protein interactions, but the organizational information is lost. To address this issue, we developed a strategy to unravel the distinct functional assemblies a protein might be involved in, by resolving affinity-purified protein complexes prior to their characterization by mass spectrometry. Protein complexes isolated through affinity purification of a bait protein using an epitope tag and competitive elution are separated through blue native electrophoresis. Comparison of protein migration profiles through correlation profiling using quantitative mass spectrometry allows assignment of interacting proteins to distinct molecular entities. This method is able to resolve protein complexes of close molecular weights that might not be resolved by traditional chromatographic techniques such as gel filtration. With little more work than conventional AP-geLC-MS/MS, we demonstrate this strategy may in many cases be adequate for obtaining protein complex topological information concomitantly to identifying protein interactions.

Here we present protocols for affinity purification of protein complexes and their separation by blue native PAGE, followed by protein correlation profiling using label free quantitative mass spectrometry. This method is useful to resolve interactomes into distinct protein complexes.

블루 기본 페이지와 단백질 상관 관계 프로파일 링에 의한 선호도 정제 단백질 복합체 해결

Most proteins act in association with others; hence, it is crucial to characterize these functional units in order to fully understand biological ...

Lipid Droplet Isolation for Quantitative Mass Spectrometry Analysis

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of virion morphogenesis. Quantitative lipid droplet proteome analysis can be used to identify proteins that localize to or are displaced from lipid droplets under conditions such as virus infections. Here, we describe a protocol that has been successfully used to characterize the changes in the lipid droplet proteome following infection with HCV. We use Stable Isotope Labeling with Amino Acids in Cell Culture (SILAC) and thus label the complete proteome of one population of cells with &#34;heavy&#34; amino acids to quantitate the proteins by mass spectrometry. For lipid droplet isolation, the two cell populations (i.e.&#160;HCV-infected/&#34;light&#34; amino acids and uninfected control/&#34;heavy&#34; amino acids) are mixed 1:1 and lysed mechanically in hypotonic buffer. After removing the nuclei and cell debris by low speed centrifugation, lipid droplet-associated proteins are enriched by two subsequent ultracentrifugation steps followed by three washing steps in isotonic buffer. The purity of the lipid droplet fractions is analyzed by western blotting with antibodies recognizing different subcellular compartments. Lipid droplet-associated proteins are then separated by SDS-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie staining. After tryptic digest, the peptides are quantified by liquid chromatography-electrospray ionization-tandem mass spectrometry (LC-ESI-MS/MS). Using this method, we identified proteins recruited to lipid droplets upon HCV infection that might represent pro- or antiviral host factors. Our method can be applied to a variety of different cells and culture conditions, such as infection with pathogens, environmental stress, or drug treatment.

Lipid droplets are important organelles for the replication of several pathogens, including the Hepatitis C Virus (HCV). We describe a method to isolate lipid droplets for quantitative mass spectrometry of associated proteins; it can be used under a variety of conditions, such as virus infection, environmental stress, or drug treatment.

양적 질량 분광법 분석을위한 지질 물방울 절연

Lipid droplets are vital to the replication of a variety of different pathogens, most prominently the Hepatitis C Virus (HCV), as the putative site of ...

Sample Preparation for Mass Spectrometry-based Identification of RNA-binding Regions

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most cases, the action of noncoding RNAs is mediated by proteins whose functions are in turn regulated by these interactions with noncoding RNAs. Consistent with this, a growing number of proteins involved in nuclear functions have been reported to bind RNA and in a few cases the RNA-binding regions of these proteins have been mapped, often through laborious, candidate-based methods.
Here, we report a detailed protocol to perform a high-throughput, proteome-wide unbiased identification of RNA-binding proteins and their RNA-binding regions. The methodology relies on the incorporation of a photoreactive uridine analog in the cellular RNA, followed by UV-mediated protein-RNA crosslinking, and mass spectrometry analyses to reveal RNA-crosslinked peptides within the proteome. Although we describe the procedure for mouse embryonic stem cells, the protocol should be easily adapted to a variety of cultured cells.

We describe a protocol to identify RNA-binding proteins and map their RNA-binding regions in live cells using UV-mediated photocrosslinking and mass spectrometry.

질량 분석 기반 식별 RNA 바인딩 영역에 대 한 샘플 준비

Noncoding RNAs play important roles in several nuclear processes, including regulating gene expression, chromatin structure, and DNA repair. In most ...

Optimal Preparation of Formalin Fixed Samples for Peptide Based Matrix Assisted Laser Desorption/Ionization Mass Spectrometry Imaging Workflows

The use of matrix-assisted laser desorption/ionization, mass spectrometry imaging (MALDI MSI) has rapidly expanded, since this technique analyzes a host of biomolecules from drugs and lipids to N-glycans. Although various sample preparation techniques exist, detecting peptides from formaldehyde preserved tissues remains one of the most difficult challenges for this type of mass spectrometric analysis. For this reason, we have created and optimized a robust methodology that preserves the spatial information contained within the sample, while eliciting the greatest number of ionizable peptides. We have also aimed to achieve this in a cost effective and simple way, thereby eliminating potential bias or preparation error, which can occur when using automated instrumentation. The end result is a reproducible and inexpensive protocol.

This protocol describes a reproducible and reliable method for the sublimation-based preparation of formalin fixed tissue destined for imaging mass spectrometry.

펩타이드 기반 매트릭스 샘플을 고정 하는 포 르 말린의 최적의 준비 지원 레이저 탈 착/이온화 질량 분석 이미징 워크플로

The use of matrix-assisted laser desorption/ionization, mass spectrometry imaging (MALDI MSI) has rapidly expanded, since this technique analyzes a host ...

Defining Hsp33's Redox-regulated Chaperone Activity and Mapping Conformational Changes on Hsp33 Using Hydrogen-deuterium Exchange Mass Spectrometry

Living organisms regularly need to cope with fluctuating environments during their life cycle, including changes in temperature, pH, the accumulation of reactive oxygen species, and more. These fluctuations can lead to a widespread protein unfolding, aggregation, and cell death. Therefore, cells have evolved a dynamic and stress-specific network of molecular chaperones, which maintain a "healthy" proteome during stress conditions. ATP-independent chaperones constitute one major class of molecular chaperones, which serve as first-line defense molecules, protecting against protein aggregation in a stress-dependent manner. One feature these chaperones have in common is their ability to utilize structural plasticity for their stress-specific activation, recognition, and release of the misfolded client. 
In this paper, we focus on the functional and structural analysis of one such intrinsically disordered chaperone, the bacterial redox-regulated Hsp33, which protects proteins against aggregation during oxidative stress. Here, we present a toolbox of diverse techniques for studying redox-regulated chaperone activity, as well as for mapping conformational changes of the chaperone, underlying its activity. Specifically, we describe a workflow which includes the preparation of fully reduced and fully oxidized proteins, followed by an analysis of the chaperone anti-aggregation activity in vitro using light-scattering, focusing on the degree of the anti-aggregation activity and its kinetics. To overcome frequent outliers accumulated during aggregation assays, we describe the usage of Kfits, a novel graphical tool which allows easy processing of kinetic measurements. This tool can be easily applied to other types of kinetic measurements for removing outliers and fitting kinetic parameters. To correlate the function with the protein structure, we describe the setup and workflow of a structural mass spectrometry technique, hydrogen-deuterium exchange mass spectrometry, that allows the mapping of conformational changes on the chaperone and substrate during different stages of Hsp33 activity. The same methodology can be applied to other protein-protein and protein-ligand interactions.

Defining Hsp33&#039;s Redox-regulated Chaperone Activity and Mapping Conformational Changes on Hsp33 Using Hydrogen-deuterium Exchange Mass Spectrometry

One of the most challenging stress conditions that organisms encounter during their lifetime involves the accumulation of oxidants. During oxidative stress, cells heavily rely on molecular chaperones. Here, we present methods used to investigate the redox-regulated anti-aggregation activity, as well as to monitor structural changes governing the chaperone function using HDX-MS.

Hsp33의 Redox 규제 보호자 활동을 정의 하 고 수소 중수소 교환 질량 분석을 사용 하 여 Hsp33에 구조적 변화를 매핑

Living organisms regularly need to cope with fluctuating environments during their life cycle, including changes in temperature, pH, the accumulation of ...

Detection of Protein Ubiquitination Sites by Peptide Enrichment and Mass Spectrometry

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ubiquitinated proteins, peptides with a diglycine remnant conjugated to the epsilon amino group of lysine (&#39;K-&#949;-diglycine&#39; or simply &#39;diGly&#39;) can be used to track back the original modification site. Efficient immunopurification of diGly peptides combined with sensitive detection by mass spectrometry has resulted in a huge increase in the number of ubiquitination sites identified up to date. We have made several improvements to this workflow, including offline high pH reverse-phase fractionation of peptides prior to the enrichment procedure, and the inclusion of more advanced peptide fragmentation settings in the ion routing multipole. Also, more efficient cleanup of the sample using a filter-based plug in order to retain the antibody beads results in a greater specificity for diGly peptides. These improvements result in the routine detection of more than 23,000 diGly peptides from human cervical cancer cells (HeLa) cell lysates upon proteasome inhibition in the cell. We show the efficacy of this strategy for in-depth analysis of the ubiquitinome profiles of several different cell types and of in vivo samples, such as brain tissue. This study presents an original addition to the toolbox for protein ubiquitination analysis to uncover the deep cellular ubiquitinome.

We present a method for the purification, detection, and identification of diGly peptides that originate from ubiquitinated proteins from complex biological samples. The presented method is reproducible, robust, and outperforms published methods with respect to the level of depth of the ubiquitinome analysis.

펩타이드 농축 및 질량 분광법에 의한 단백질 유비퀴틴화 부위 검출

The posttranslational modification of proteins by the small protein ubiquitin is involved in many cellular events. After tryptic digestion of ...

Nitropeptide Profiling and Identification Illustrated by Angiotensin II

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

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

안지오텐신 II에 의해 설명 니트로 펩티드 프로파일링 및 식별

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

A Plasma Sample Preparation for Mass Spectrometry using an Automated Workstation

Sample preparation for mass spectrometry analysis in proteomics requires enzymatic cleavage of proteins into a peptide mixture. This process involves numerous incubation and liquid transfer steps in order to achieve denaturation, reduction, alkylation, and cleavage. Adapting this workflow onto an automated workstation can increase efficiency and reduce coefficients of variance, thereby providing more reliable data for statistical comparisons between sample types. We previously described an automated proteomic sample preparation workflow1. Here, we report the development of a more efficient and better controlled workflow with the following advantages: 1) The number of liquid transfer steps is reduced from nine to six by combining reagents; 2) Pipetting time is reduced by selective tip pipetting using a 96-position pipetting head with multiple channels; 3) Potential throughput is increased by the availability of up to 45 deck positions; 4) Complete enclosure of the system provides improved temperature and environmental control and reduces the potential for contamination of samples or reagents; and 5) The addition of stable isotope labeled peptides, as well as &#946;-galactosidase protein, to each sample makes monitoring and quality control possible throughout the entire process. These hardware and process improvements provide good reproducibility and improve intra-assay and inter-assay precision (CV of less than 20%) for LC-MS based protein and peptide quantification. The entire workflow for digesting 96 samples in a 96-well plate can be completed in approximately 5 hours.

Here, we present an automated plasma protein digestion method for mass spectrometry-based quantitative proteomic analysis. In this protocol, the liquid transferring and incubation steps for protein denaturation, reduction, alkylation, and trypsin digestion reactions are streamlined and automated. It takes approximately five hours to prepare a 96-well plate with desired precision.

자동화된 워크스테이션을 사용한 질량 분석법을 위한 플라즈마 시료 준비

Sample preparation for mass spectrometry analysis in proteomics requires enzymatic cleavage of proteins into a peptide mixture. This process involves ...

Sample Preparation for Probe Electrospray Ionization Mass Spectrometry

Mass spectrometry (MS) is a powerful tool in analytical chemistry because it provides very accurate information about molecules, such as mass-to-charge ratios (m/z), which are useful to deduce molecular weights and structures. While it is essentially a destructive analytical method, recent advancements in the ambient ionization technique have enabled us to acquire data while leaving tissue in a relatively intact state in terms of integrity. Probe electrospray ionization (PESI) is a so-called direct method because it does not require complex and time-consuming pretreatment of samples. A fine needle serves as a sample picker, as well as an ionization emitter. Based on the very sharp and fine property of the probe tip, destruction of the samples is minimal, allowing us to acquire the real-time molecular information from living things in situ. Herein, we introduce three applications of PESI-MS technique that will be useful for biomedical research and development. One involves the application to solid tissue, which is the basic application of this technique for the medical diagnosis. As this technique requires only 10 mg of the sample, it may be very useful in the routine clinical settings. The second application is for in vitro medical diagnostics where human blood serum is measured. The ability to measure fluid samples is also valuable in various biological experiments where a sufficient volume of sample for conventional analytical techniques cannot be provided. The third application leans toward the direct application of probe needles in living animals, where we can obtain real-time dynamics of metabolites or drugs in specific organs. In each application, we can infer the molecules that have been detected by MS or use artificial intelligence to obtain a medical diagnosis.

This article introduces sample preparation methods for a unique real-time analytical method based on the ambient mass spectrometry. This method lets us perform real-time analysis of the biological molecules in vivo without any special pretreatments.

Mass spectrometry (MS) is a powerful tool in analytical chemistry because it provides very accurate information about molecules, such as mass-to-charge ...