Name: determination of the gas-phase acidities of oligopeptides
Uploaded: 2013-06-24T12:00:00
Description: Watch this Scientific Journal Video about Determination of the Gas-phase Acidities of Oligopeptides at JoVE.com

<p class="jove_content"&gt; 작품은 국립 과학 재단 (CHE-0749737)에 의해 지원되었다. 기기의 사용은 태평양의 대학에서 질량 분석 시설에 의해 제공되었다.</p

1. 샘플 솔루션의 준비 <ol><li> 먼저 펩타이드의 주식 솔루션과 1:1 부피비로 메탄올과 물의 혼합 용매를 사용하여 여섯 참조 지방산을 준비합니다. 주식 솔루션은 약 10 -3 M.의 농도가 있어야합니다 </li><li> 1.5 ML 에펜 도르프 튜브에 고체 펩타이드 샘플 1 MG, 3 CH를 밖으로 달아 메탄올과 물의 혼합 용매의 1.0 ML를 추가하고, 소용돌이를 사용하여 섞는다. </li><li> difluoroacetic 산 (DFAH) 1 ?...

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펩타이드의 기체 산도의 성공적인 측정은 주로 적절한 참조 지방산의 선택에 의존한다. 이상적인 레퍼런스 산 노포 기체 산도 값이 구조적으로 유사한 유기 화합물이다. 참고 지방산은 서로 유사한 구조를 가지고 있어야합니다. 이 세트의 참조 지방산 각각의 탈 양성자의 유사한 엔트로피를 보장합니다. 참고 아미노산은 펩티드의 그들에 가까운 산도 값이 있어야합니다. amidated C-말단에 짧은 시?...

아미노산 잔기의 산성도는 구조에 영향을 미치는 가장 중요한 열 화학적 성질, 반응성 단백질 9,19의 폴딩 전개 과정 중입니다. 각각의 아미노산 잔기들은 단백질에 자신의 위치에 따라 다른 효과 산성도를 보여줍니다. 특히, 활성 부위에있는 잔류 물은 종종 전시는 크게 산성도를 교란. 하나의 예는 효소 20,21의 티 오레 독신 슈퍼 가족의 활성 부위에 존재하는 시스테인 잔류 물이다. 활성 사이트 시스테인 단백질에게 3-5 펼쳐있는 사람에 비해 비정상적으로 산성이다. 그것은 나선형 형태가 특이한 산성에 상당한 기여가있을 수 있습니다 것을 제안했습니다. 특히 수용액 2,6-8에 솔루션을 수행 펩티드의 산 - 염기 특성에 대한 광범위한 실험 연구가있다. 결과는 종종 용매 효과에 의해 복잡하게되었다7. 사실, 단백질의 활성 사이트의 대부분은 용매 효과는 9,10를 최소화하는 내부 영역 근처에 위치하고 있습니다. 펩티드와 단백질의 고유 산 - 염기 특성을 이해하기 위해서는 용제가없는 환경에서 연구를 수행하는 것이 중요합니다. 여기서 우리는 기체 상 산도 측정을위한 질량 분석 기반 방법을 소개합니다. 접근 방식은 확장 요리사 운동 방법이라고합니다. 이 방법은 성공적으로 같은 기체 상 산도, 양성자 친화, 금속 이온 친 화성, 전자 친화, 그리고 이온화 에너지 11-15 등 다양한 열 화학적 특성의 측정을위한 화학 시스템의 넓은 범위에 적용되었습니다 22-26. 우리는 올리고 시스테인 polyalanine과 시스테인 polyglycine 펩티드 17,18,27 시리즈의 기체 상 산성도를 결정하기 위해이 방법을 적용했습니다. 이러한 연구는 N-말단 시스테인 펩타이드 보여ES는 해당 C-말단 것보다 훨씬 더 산성이다. 이전의 높은 산성도 가능성이 thiolate의 음이온이 강하게 나선 매크로 쌍극자와의 상호 작용에 의해 안정화되는 나선형 구조적인 효과 때문이다. 때문에 펩티드의 비 휘발성과 열적으로 불안정한 성격, 운동 방법은 펩티드 28의 비교적 정확한 산 - 염기 열화학 수량을 생산하기 위해 현재 사용할 수있는 가장 실용적인 방법입니다. 일반 체계 및 운동 방법과 관련된 방정식은 그림 1에 나타내었다. 펩타이드의 기체 상 산성의 결정은 (AH) 양성자 바인딩 클러스터 음이온의 일련의 형성으로 시작 [A • H • I] ¯ (또는 [A ¯ • H + • I ¯] ¯), 질량 분석기의 이온 소스 지역에서 어디 ¯와 나 ¯ 펩타이드의 탈수 소화 형태이며,각각 참조 지방산. 참고 지방산 알려진 기체의 산성도를 가진 유기 화합물이다. 참고 지방산 (그러나 펩타이드의 그것과 반드시​​ 유사하지) 서로 유사한 구조를 가지고 있어야합니다. 참고 지방산 사이의 구조의 유사성은 그 (것)들의 사이에서 탈 양성자의 엔트로피의 유사성을 보장합니다. 양성자 바인딩 클러스터 음이온은 해당 단량체 음이온을 산출하기 위해 충돌에 의한 해리 (CID) 실험을 사용하여 대량 선정 collisionally 활성화 이후 해리되어 ¯ 그리고 난 ¯, K와 K의 속도 상수 나, 각각 그림 1a와 같이와. 차 fragmentations가 무시할 경우, CID 조각 이온의 풍부한 비율 [A ¯] / [I ¯], 속도 상수, K / K I의 비율의 대략적인 측정을 나타냅니다. 어떤 역 activat가 없다는 가정하에모두 분리 채널 비율을 분기 CID 제품 이온 이온 장벽, LN [A ¯] / [I ¯], 선형 기체 상 펩타이드의 산도 (Δ 산 H)와 그 상관 관계가 될 것입니다 참조 지방산 (Δ 산 H I), 같은 그림 1b와 같이. 이 방정식에서, Δ 산 H 평균이 기준 지방산의 평균 기체 산도이며, Δ (Δ S)는 엔트로피 용어 (참조 지방산은 구조적으로 서로 유사하다 일정하게 가정 할 수있다)이며, R은 유니버설 가스 상수, 그리고 T EFF는 시스템의 유효 온도입니다. 유효 온도는 충돌 에너지를 포함한 여러 실험 변수에 따라 경험적 매개 변수입니다. 기체 산도의 값은 열 운동 플롯의 두 세트를 구성에 의해 결정됩니다. 첫 번째 세트는 OB입니다LN 음모를 꾸미고에 의한 것 ([A ¯] / [I ¯]) Δ 산 H 나에 대하여 - Δ 산 H 평균, 그림 4A에 표시. 선형 회귀는 Y의 X = 1 / RT EFF와 차단의 경사면과 직선의 집합을 얻을 것입니다 = - [Δ 산 H - Δ 산 H 평균] / RT EFF - Δ (Δ S) / R. 플롯의 두 번째 세트는 그림 4B에 표시된 해당 슬로프에 첫 번째 집합 (X)에서 발생 차단 (Y)를 플로팅하여 얻을 수 있습니다. Δ 산 H 평균과 Δ (Δ S) / R의 절편 - 선형 회귀 분석 Δ 산 H의 경사를 가진 새로운 라인을 생산하고 있습니다. Δ 산 H의 값은 다음의 기울기로부터 구할 때, 엔트로피 용어, Δ (Δ S는)에서 얻을 수있다차단. 실험은 전기 분무 이온화 (ESI) 이온 소스 인터페이스 삼중 사중 극자 질량 분석기를 사용하여 수행됩니다. 질량 분석기의 개략도는 그림 2에 표시됩니다. CID 실험은 질량이 첫 번째 사중 극자 기기와 양성자 바인딩 클러스터 음이온을 선택하고 그 주위에 0.5 mTorr의의 압력에서 개최되는 충돌 챔버로 유출 아르곤 원자와 충돌을받을 수 있도록하여 수행됩니다. 해리 제품 이온은 질량 번째 사중 극자 단위로 분석됩니다. CID 스펙트럼은 모든 가능한 보조 조각을 커버 할 수있을만큼 넓은 M / Z 범위를 여러 충돌 에너지에 기록됩니다. CID 제품 이온 농도는 검사가 선택한 제품 이온에 집중되는 선택된 반응 모니터링 (SRM) 모드로 기기를 설정하여 측정됩니다. CID 실험에 해당하는 네 가지 충돌 에너지에서 수행됩니다각각 1.0, 1.5, 2.0 및 2.5 eV로의 중심의 질량 에너지 (E cm). 중심의 질량 에너지 방정식을 사용하여 계산됩니다 : E cm = E 실험실 [M / (M + m)은] E 실험실 실험실 프레임의 충돌 에너지이고, M은 아르곤의 질량이고, M은 양성자 바인딩 클러스터 이온의 질량. 이 문서에서는, 우리는 모델 화합물로 올리고 펩티드 알라모 3 CysNH 2 (3 CH)를 사용합니다. C-말단은 amidated과 시스테인 잔기의 티올 그룹 (SH)은 산성 사이트가 될 것입니다. 적절한 참조 지방산의 선택은 기체 상 산성의 성공적인 측정을 위해 매우 중요합니다. 이상적인 기준 지방산은 구조적으로 유사하다 (서로에게) 잘 확립 된 기체 산도 값을 가진 유기 화합물. 참고 아미노산은 펩티드의 가까운 산도 값이 있어야합니다. 펩타이드 3 CH 들어, 여섯 carboxyli 할로겐화C 지방산은 참조 지방산으로 선택됩니다. 여섯 참고 지방산 클로로 산 (MCAH), bromoacetic 산 (MBAH), difluoroacetic 산 (DFAH), 디클로로 산 (DCAH), dibromoacetic 산 (DBAH) 및 트리 플루오 (TFAH)입니다. 그 중 두 DFAH 및 MBAH이 프로토콜을 설명하는 데 사용됩니다.

<table border="1">
		<tbody>
		 <tr>
			<td>Name of the reagent</td>
			<td>Company</td>
			<td>Catalogue number</td>
			<td>Comments (optional)</td>
		 </tr>
		 <tr>
			<td>Mass Spectrometer</td>
			<td>Varian</td>
			<td>1200 L and 320 L</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Chloroacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>402923</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Bromoacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>B56307</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Difluoroacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>142859</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Dichloroacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>D54702</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Dibromoacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>242357</td>
			<td></td>
		 </tr>
		 <tr>
			<td>Trifluoroacetic acid</td>
			<td>Sigma-Aldrich</td>
			<td>T6508</td>
			<td></td>
		 </tr>
		</tbody>
 </table>

determination of the gas-phase acidities of oligopeptides

접힌 단백질의 다른 위치에있는 아미노산 잔기들은 산성도의 다른 정도를 나타냅니다. 예를 들어, 나선의 N-말단에 또는 근처에있는 시스테인 잔류 물은 종종 C-말단 1-6로 인근보다 더 산성이다. 펩티드의 산 - 염기 특성에 대한 광범위한 실험 연구는 수용액 6-8 특히 응축 단계에서 실시되어 있지만, 결과는 종종 용매 효과 7에 의해 복잡하게된다. 사실, 단백질의 활성 사이트의 대부분은 용매 효과 9,10 최소화 된 내부 영역 근처에 위치하고 있습니다. 펩티드와 단백질의 고유 산 - 염기 특성을 이해하기 위해서는 용제가없는 환경에서 연구를 수행하는 것이 중요합니다. 우리는 가스 단계에 올리고 펩타이드의 산성도를 측정하는 방법을 제시한다. 우리는 시스테인 함유 올리고 펩티드, 알라모 3 CysNH를 사용 (3 CH). 측정은 잘 확립 확장 요리사 운동 방법 (그림 1) 11-16를 기준으로합니다. 실험은 전기 분무 이온화 (ESI) 이온 소스 (그림 2)와 인터페이스 트리플 사중 극자 질량 분석기를 사용하여 수행됩니다. 각 펩타이드 샘플은 여러 가지 기준 지방산이 선택됩니다. 참고 지방산 알려진 기체 상 산성도와 구조적으로 유사한 유기 화합물이다. 펩타이드 및 레퍼런스 산의 혼합 용액을 질량 분석기에 도입되고, 펩타이드 참조 산의 기체 상 수소 이온 바인딩 음이온 클러스터가 형성된다. 양성자 바인딩 클러스터 대량 고립 이후 충돌에 의한 해리 (CID) 실험을 통해 조각화되어 있습니다. 결과 조각 이온 농도는 산성도와 클러스터 이온 해리 반응 속도 사이의 관계를 사용하여 분석됩니다. 펩타이드의 기체 산도는 obtai입니다열 운동 플롯 17,18 선형 회귀 네드. 이 방법은 유기 화합물, 아미노산과 그들의 유도체, 올리고 뉴클레오티드 및 올리고 펩타이드 등의 분자 시스템의 다양한 적용 할 수 있습니다. 다른 conformers 계산하는 값을 실험적으로 측정 된 가스 단계 산성도를 비교하여, 산성도에 구조적 효과를 평가할 수 있습니다.

<ol><li> CID 브라케팅 실험은 선택된 참조 지방산에 비해 펩타이드의 상대적인 산성도에 대한 정보를 제공합니다. 두 개의 레퍼런스 산, DFAH 및 MBAH와 펩티드 (3 CH)의 두 가지 대표적인 CID 스펙트럼은 그림 3에 나와 있습니다. 그림 3A에서 펩타이드 이온의 이온 풍부 (최대 높이) DFA의 ¯, 그리고 그림 3b에서, 펩타이드 이온의 이온 풍부 MBA ¯보다 강한 것보다 약한 것입니다. ?...

Watch this Scientific Journal Video about Determination of the Gas-phase Acidities of Oligopeptides at JoVE.com

Determination of the Gas-phase Acidities of Oligopeptides

시스테인 함유의 올리고 펩타이드의 가스 단계 산성도의 결정은 설명합니다. 실험은 트리플 사중 극자 질량 분석기를 사용하여 수행됩니다. 펩티드의 상대적인 산성도는 충돌에 의한 분리 실험을 사용하여 측정하고 있으며, 양적 산성도가 확장 요리사에게 운동 방법을 사용하여 결정됩니다.

올리고 펩타이드의 가스 단계 산성도의 결정

Amino acid residues located at different positions in folded proteins often exhibit different degrees of acidities. For example, a cysteine residue ...

determination-of-the-gas-phase-acidities-of-oligopeptides

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Chemistry

Rapid High Throughput Amylose Determination in Freeze Dried Potato Tuber Samples

This protocol describes a high through put colorimetric method that relies on the formation of a complex between iodine and chains of glucose molecules in starch. Iodine forms complexes with both amylose and long chains within amylopectin. After the addition of iodine to a starch sample, the maximum absorption of amylose and amylopectin occurs at 620 and 550 nm, respectively. The amylose/amylopectin ratio can be estimated from the ratio of the 620 and 550 nm absorbance values and comparing them to a standard curve in which specific known concentrations are plotted against absorption values. This high throughput, inexpensive method is reliable and reproducible, allowing the evaluation of large populations of potato clones.&#160;

냉동 건조 된 감자 괴경 샘플의 신속한 높은 처리량 아밀로스 결정

This protocol describes a high through put colorimetric method that relies on the formation of a complex between iodine and chains of glucose molecules in ...

연구

화학

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox reactions. The Cu (I) complex of the peptide model of a Cu (I) binding metallochaperone protein, which includes the sequence MTCSGCSRPG (underlined is conserved), was determined in solution under inert conditions by NMR spectroscopy.
NMR is a widely accepted technique for the determination of solution structures of proteins and peptides. Due to difficulty in crystallization to provide single crystals suitable for X-ray crystallography, the NMR technique is extremely valuable, especially as it provides information on the solution state rather than the solid state. Herein we describe all steps that are required for full three-dimensional structure determinations by NMR. The protocol includes sample preparation in an NMR tube, 1D and 2D data collection and processing, peak assignment and integration, molecular mechanics calculations, and structure analysis. Importantly, the analysis was first conducted without any preset metal-ligand bonds, to assure a reliable structure determination in an unbiased manner.

Structure and Coordination Determination of Peptide-metal Complexes Using 1D and 2D 1H NMR

The NMR-solution structure of a metallochaperone model peptide with Cu (I) was determined, and a detailed protocol from sample preparation and 1D and 2D data collection to a three-dimensional structure is described.

Copper (I) binding by metallochaperone transport proteins prevents copper oxidation and release of the toxic ions that may participate in harmful redox ...

From Constructs to Crystals &#8211; Towards Structure Determination of &#946;-barrel Outer Membrane Proteins

Membrane proteins serve important functions in cells such as nutrient transport, motility, signaling, survival and virulence, yet constitute only ~1% percent of known structures. There are two types of membrane proteins, &#945;-helical and &#946;-barrel. While &#945;-helical membrane proteins can be found in nearly all cellular membranes, &#946;-barrel membrane proteins can only be found in the outer membranes of mitochondria, chloroplasts, and Gram-negative bacteria. One common bottleneck in structural studies of membrane proteins in general is getting enough pure sample for analysis. In hopes of assisting those interested in solving the structure of their favorite &#946;-barrel outer membrane protein (OMP), general protocols are presented for the production of target &#946;-barrel OMPs at levels useful for structure determination by either X-ray crystallography and/or NMR spectroscopy. Here, we outline construct design for both native expression and for expression into inclusion bodies, purification using an affinity tag, and crystallization using detergent screening, bicelle, and lipidic cubic phase techniques. These protocols have been tested and found to work for most OMPs from Gram-negative bacteria; however, there are some targets, particularly for mitochondria and chloroplasts that may require other methods for expression and purification. As such, the methods here should be applicable for most projects that involve OMPs from Gram-negative bacteria, yet the expression levels and amount of purified sample will vary depending on the target OMP.

From Constructs to Crystals &amp;#8211; Towards Structure Determination of &amp;#946;-barrel Outer Membrane Proteins

&#946;-barrel outer membrane proteins (OMPs) serve many functions within the outer membranes of Gram-negative bacteria, mitochondria, and chloroplasts. Here, we hope to alleviate a known bottleneck in structural studies by presenting protocols for the production of &#946;-barrel OMPs in sufficient quantities for structure determination by X-ray crystallography or NMR spectroscopy.

β-배럴 외부 막 단백질의 구조 결정을 향해 - 구축해에서 결정에

Membrane proteins serve important functions in cells such as nutrient transport, motility, signaling, survival and virulence, yet constitute only ~1% ...

Determination of Carbonyl Functional Groups in Bio-oils by Potentiometric Titration: The Faix Method

Carbonyl compounds present in bio-oils are known to be responsible for bio-oil property changes upon storage and during upgrading. Specifically, carbonyls cause an increase in viscosity (often referred to as &#39;aging&#39;) during storage of bio-oils. As such, carbonyl content has previously been used as a method of tracking bio-oil aging and condensation reactions with less variability than viscosity measurements. Additionally, carbonyls are also responsible for coke formation in bio-oil upgrading processes. Given the importance of carbonyls in bio-oils, accurate analytical methods for their quantification are very important for the bio-oil community. Potentiometric titration methods based on carbonyl oximation have long been used for the determination of carbonyl content in pyrolysis bio-oils. Here, we present a modification of the traditional carbonyl oximation procedures that results in less reaction time, smaller sample size, higher precision, and more accurate carbonyl determinations. While traditional carbonyl oximation methods occur at room temperature, the Faix method presented here occurs at an elevated temperature of 80 &#176;C.

Here we present a potentiometric titration technique for accurately quantifying carbonyl compounds in pyrolysis bio-oils.

Carbonyl compounds present in bio-oils are known to be responsible for bio-oil property changes upon storage and during upgrading. Specifically, carbonyls ...

Determination of Thermodynamic Properties of Alkaline Earth-liquid Metal Alloys Using the Electromotive Force Technique

A novel electrochemical cell based on a CaF2 solid-state electrolyte has been developed to measure the electromotive force (emf) of binary alkaline earth-liquid metal alloys as functions of both composition and temperature in order to acquire thermodynamic data. The cell consists of a chemically stable solid-state CaF2-AF2 electrolyte (where A is the alkaline-earth element such as Ca, Sr, or Ba), with binary A-B alloy (where B is the liquid metal such as Bi or Sb) working electrodes, and a pure A metal reference electrode. Emf data are collected over a temperature range of 723 K to 1,123 K in 25 K increments for multiple alloy compositions per experiment and the results are analyzed to yield activity values, phase transition temperatures, and partial molar entropies/enthalpies for each composition.

This protocol describes the measurement of the electromotive force of alkaline-earth elements in liquid metal alloys at high temperatures (723-1,123 K) to determine their thermodynamic properties, including activity, partial molar entropy, partial molar enthalpy, and phase transition temperatures, over a wide composition range.

A novel electrochemical cell based on a CaF2 solid-state electrolyte has been developed to measure the electromotive force (emf) of binary alkaline ...

Disentangling High Strength Copolymer Aramid Fibers to Enable the Determination of Their Mechanical Properties

Traditionally, soft body armor has been made from poly(p-phenylene terephthalamide) (PPTA) and ultra-high molecular weight polyethylene. However, to diversify the fiber choices in the United States body armor market, copolymer fibers based on the combination of 5-amino-2-(p-aminophenyl) benzimidazole (PBIA) and the more conventional PPTA were introduced. Little is known regarding the long-term stability of these fibers, but as condensation polymers, they are expected to have potential sensitivity to moisture and humidity. Therefore, characterizing the strength of the materials and understanding their vulnerability to environmental conditions is important for evaluating their use lifetime in safety applications. Ballistic resistance and other critical structural properties of these fibers are predicated on their strength. To accurately determine the strength of the individual fibers, it is necessary to disentangle them from the yarn without introducing any damage. Three aramid-based copolymer fibers were selected for the study. The fibers were washed with acetone followed by methanol to remove an organic coating that held the individual fibers in each yarn bundle together. This coating makes it difficult to separate single fibers from the yarn bundle for mechanical testing without damaging the fibers and affecting their strength. After washing, fourier transform infrared (FTIR) spectroscopy was performed on both washed and unwashed samples and the results were compared. This experiment has shown that there are no significant variations in the spectra of poly(p-phenylene-benzimidazole-terephthalamide-co-p-phenylene terephthalamide) (PBIA-co-PPTA1) and PBIA-co-PPTA3 after washing, and only a small variation in intensity for PBIA. This indicates that the acetone and methanol rinses are not adversely affecting the fibers and causing chemical degradation. Additionally, single fiber tensile testing was performed on the washed fibers to characterize their initial tensile strength and strain to failure, and compare those to other reported values. Iterative procedural development was necessary to find a successful method for performing tensile testing on these fibers.

The primary goal of the study is to develop a protocol to prepare consistent specimens for accurate mechanical testing of high strength copolymer aramid fibers, by removing a coating and disentangling the individual fiber strands without introducing significant chemical or physical degradation.

그들의 기계적 특성의 결정 수 있도록 고 강도 공중 합체 Aramid 섬유 disentangling

Traditionally, soft body armor has been made from poly(p-phenylene terephthalamide) (PPTA) and ultra-high molecular weight polyethylene. However, to ...

Synthesis and Structure Determination of &#181;-Conotoxin PIIIA Isomers with Different Disulfide Connectivities

Peptides with a high number of cysteines are usually influenced regarding the three-dimensional structure by their disulfide connectivity. It is thus highly important to avoid undesired disulfide bond formation during peptide synthesis, because it may result in a completely different peptide structure, and consequently altered bioactivity. However, the correct formation of multiple disulfide bonds in a peptide is difficult to obtain by using standard self-folding methods such as conventional buffer oxidation protocols, because several disulfide connectivities can be formed. This protocol represents an advanced strategy required for the targeted synthesis of multiple disulfide-bridged peptides which cannot be synthesized via buffer oxidation in high quality and quantity. The study demonstrates the application of a distinct protecting group strategy for the synthesis of all possible 3-disulfide-bonded peptide isomers of &#181;-conotoxin PIIIA in a targeted way. The peptides are prepared by Fmoc-based solid phase peptide synthesis using a protecting group strategy for defined disulfide bond formation. The respective pairs of cysteines are protected with trityl (Trt), acetamidomethyl (Acm), and tert-butyl (tBu) protecting groups to make sure that during every oxidation step only the required cysteines are deprotected and linked. In addition to the targeted synthesis, a combination of several analytical methods is used to clarify the correct folding and generation of the desired peptide structures. The comparison of the different 3-disulfide-bonded isomers indicates the importance of accurate determination and knowledge of the disulfide connectivity for the calculation of the three-dimensional structure and for interpretation of the biological activity of the peptide isomers. The analytical characterization includes the exact disulfide bond elucidation via tandem mass spectrometry (MS/MS) analysis which is performed with partially reduced and alkylated derivatives of the intact peptide isomer produced by an adapted protocol. Furthermore, the peptide structures are determined using 2D nuclear magnetic resonance (NMR) experiments and the knowledge obtained from MS/MS analysis.

Synthesis and Structure Determination of &amp;#181;-Conotoxin PIIIA Isomers with Different Disulfide Connectivities

Cysteine-rich peptides fold into distinct three-dimensional structures depending on their disulfide connectivity. Targeted synthesis of individual disulfide isomers is required when buffer oxidation does not lead to the desired disulfide connectivity. The protocol deals with the selective synthesis of 3-disulfide-bonded peptides and their structural analysis using NMR and MS/MS studies.

합성 및 다른 아 황산 연결 된 μ-Conotoxin PIIIA이 성체의 구조 결정

Peptides with a high number of cysteines are usually influenced regarding the three-dimensional structure by their disulfide connectivity. It is thus ...

Ion Mobility-Mass Spectrometry Techniques for Determining the Structure and Mechanisms of Metal Ion Recognition and Redox Activity of Metal Binding Oligopeptides

Electrospray ionization (ESI) can transfer an aqueous-phase peptide or peptide complex to the gas-phase while conserving its mass, overall charge, metal-binding interactions, and conformational shape. Coupling ESI with ion mobility-mass spectrometry (IM-MS) provides an instrumental technique that allows for simultaneous measurement of a peptide&#8217;s mass-to-charge (m/z) and collision cross section (CCS) that relate to its stoichiometry, protonation state, and conformational shape. The overall charge of a peptide complex is controlled by the protonation of 1) the peptide&#8217;s acidic and basic sites and 2) the oxidation state of the metal ion(s). Therefore, the overall charge state of a complex is a function of the pH of the solution that affects the peptides metal ion binding affinity. For ESI-IM-MS analyses, peptide and metal ions solutions are prepared from aqueous-only solutions, with the pH adjusted with dilute aqueous acetic acid or ammonium hydroxide. This allows for pH dependence and metal ion selectivity to be determined for a specific peptide. Furthermore, the m/z and CCS of a peptide complex can be used with B3LYP/LanL2DZ molecular modeling to discern binding sites of the metal ion coordination and tertiary structure of the complex. The results show how ESI-IM-MS can characterize the selective chelating performance of a set of alternative methanobactin peptides and compare them to the copper-binding peptide methanobactin.

Ion mobility-mass spectrometry and molecular modeling techniques can characterize the selective metal chelating performance of designed metal-binding peptides and the copper-binding peptide methanobactin. Developing new classes of metal chelating peptides will help lead to therapeutics for diseases associated with metal ion misbalance.

금속 결합 올리고펩타이드의 금속 이온 인식 및 Redox 활성의 구조 및 메커니즘을 결정하기 위한 이온 이동성 질량 분광법 기술

Electrospray ionization (ESI) can transfer an aqueous-phase peptide or peptide complex to the gas-phase while conserving its mass, overall charge, ...

Determination of the Photoisomerization Quantum Yield of a Hydrazone Photoswitch

Photoswitching organic molecules that undergo light-driven structural transformations are key components to construct adaptive molecular systems, and they are utilized in a wide variety of applications. In most studies employing photoswitches, several important photophysical properties such as maximum wavelengths of absorption and emission, molar attenuation coefficient, fluorescence lifetime, and photoisomerization quantum yield are carefully determined to investigate their electronic states and transition processes. However, measurement of the photoisomerization quantum yield, the efficiency of photoisomerization with respect to the absorbed photons, in a typical laboratory setting is often complicated and prone to error because it requires the implementation of rigorous spectroscopic measurements and calculations based on an appropriate integration method. This article introduces a set of procedures to measure the photoisomerization quantum yield of a bistable photoswitch using a photochromic hydrazone. We anticipate that this article will be a useful guide for the investigation of bistable photoswitches that are being increasingly developed.

Photoisomerization quantum yield is a fundamental photophysical property that should be accurately determined in the investigation of newly developed photoswitches. Here, we describe a set of procedures to measure the photoisomerization quantum yield of a photochromic hydrazone as a model bistable photoswitch.

히드라존 포토스위치의 광이성체화 양자 수율 결정

Photoswitching organic molecules that undergo light-driven structural transformations are key components to construct adaptive molecular systems, and they ...

QuEChERS 분석법 및 CG-MS/MS를 사용한 아보카도의 살충제 분석

Determination of 45 Pesticides in Avocado Varieties by the QuEChERS Method and Gas Chromatography-Tandem Mass Spectrometry

Gas chromatography (GC) tandem mass spectrometry (MS/MS) stands as a preeminent analytical instrument extensively employed for the surveillance of pesticide residues in food. Nevertheless, these methods are vulnerable to matrix effects (MEs), which can potentially affect accurate quantification depending on the specific combination of analyte and matrix. Among the various strategies to mitigate MEs, matrix-matched calibration represents the prevailing approach in pesticide residue applications due to its cost-effectiveness and straightforward implementation. In this study, a total of 45 representative pesticides were analyzed in three different varieties of avocado (i.e., Criollo, Hass, and Lorena) using the Quick-Easy-Cheap-Effective-Rugged-Safe (QuEChERS) method with ammonium formate and GC-MS/MS. 
For this purpose, 5 g of the avocado sample was extracted with 10 mL of acetonitrile, and then 2.5 g of ammonium formate was added to induce phase separation. Subsequently, the supernatant underwent a cleanup process via dispersive solid-phase extraction employing 150 mg of anhydrous MgSO4, 50 mg of primary-secondary amine, 50 mg of octadecylsilane, 10 mg of graphitized carbon black, and 60 mg of a zirconium oxide-based sorbent (Z-Sep+). The GC-MS/MS analysis was successfully performed in less than 25 min. Rigorous validation experiments were carried out to assess the performance of the method. The examination of a matrix-matched calibration curve for each variety of avocado revealed that the ME remained relatively consistent and less than 20% (considered as a soft ME) for most pesticide/variety combinations. Furthermore, the method&#180;s limits of quantification were lower than 5 &#181;g/kg for all three varieties. Finally, the recovery values for most pesticides fell within the acceptable range of 70-120%, with relative standard deviation values below 20%.

살충제 분석을 위해 포름산암모늄과 함께 QuEChERS 분석법을 사용한 아보카도 시료 전처리

The present protocol describes the analysis of multiclass pesticide residues in avocado varieties using the Quick-Easy-Cheap-Effective-Rugged-Safe (QuEChERS) method with ammonium formate, followed by gas chromatography-tandem mass spectrometry.