저자 아무 승인 있다.

1. 샘플 접시 늘어진
<ol><li>
샘플 접시의 청소
	<ol>
<li>청소 하는 동안 샘플 플레이트의 오염을 피하기 위하여 니트 릴 장갑을 착용 한다.</li>
		<li>손 세척 세제 솔루션 (1.0 mg/mL)의 100.0 mL과 샘플 접시.</li>
		<li>손-세척-이온 증 류 물 (DDW) 샘플 접시.</li>
		<li>씻어 MeOH의 30.0 mL과 샘플 접시 표면.</li>
		<li>샘플 접시는 완전히 물에 잠기 다 때까지 600 mL 비이 커에 채우기 DDW와 샘플 접시를 넣어.</li>
		<li>비 커는 초음파에 넣어 ( 테이블의 자료를 참조) 목욕 하 고 15 분 (200 W, 40 kHz)의 샘플 접시를 sonicate.</li>
		<li>샘플 접시를 꺼내와 물 방울 가압된 질소를 사용 하 여 날 려 버릴.</li>
		<li>0.2 µ L MeOH의 MeOH 다른 장소에 확산 여부를 확인 하려면 샘플 접시에 입금. 참고: 만약 다른 명소 MeOH 병합, 반복 단계 1.1.3-1.1.5; 그렇지 않은 경우에 다음 단계를 수행 하십시오.</li>
	</ol>
</li>
	<li>
챔버 온도 건조의 규제
	<ol>
<li>안정적인 조건에서 건조 챔버를 사용 하 여 앞에서 설명한11,,1213방울, 건조. 특히, Ou, Y. M의 2.1-2.5 단계에서 설명 하는 자세한 절차를 사용 하 여. 외. 201612. 간단히:<ol>
<li>낮은 상대 습도 환경을 유지 하기 위하여 일정 한 유량에서 실내 온도 질소에 의해 건조 챔버를 제거.</li>
			<li>샘플 접시 온도 상수 정기적으로 (25 ° C) 또는 빠른 건조 조건 (50 ° C), 건조 챔버에서 온도 제어 구리 블록에 의해 규제를 유지.</li>
		</ol>
</li>
	</ol>
</li>
	<li>
매트릭스 및 분석 솔루션의 준비
	<ol>
<li>
매트릭스 솔루션의 준비
		<ol>
<li>DHB 50% 이기 (ACN)에서 해산: 50% DDW 0.1 M 솔루션을 준비 하.</li>
		</ol>
</li>
		<li>
탄수화물 analytes의 준비
		<ol>
<li>SLeA 10-4 M 솔루션을 준비 하는 DDW에서 디졸브.</li>
			<li>DDW 10-4 M 솔루션을 준비 하에서 수소를 분해.</li>
		</ol>
</li>
	</ol>
</li>
</ol>2. 샘플 증 착 및 Recrystallization
참고: 작고 일반 양의 샘플을 분석 하기 위한 최적화 된 절차는 여기에 설명 되어 있습니다. 샘플 판 온도 솔루션을 입금 하기 전에 원하는 온도에서 안정 된 확인 하십시오. 샘플 recrystallization 동안 다른 샘플 명소를 충당 하기 위해 큰 영역 확산, 새로운 샘플 준비 또는 반복 단계 1.1.
<ol><li>
샘플의 소량 (0.1 µ L)의 분석에 대 한 참고: 다음 단계 샘플 및 시간 소비 최소화에 대 한 개발 되었습니다. 그것은 제한 된 금액 또는 정량화 분석에 대 한 빠른 IMS 실제 샘플의 정량 분석에 적합 합니다.<ol>
<li>0.25 µ L DHB 솔루션 및 0.25 µ L SLeA 의 또는 microcentrifuge 관에서 수소 솔루션의 공기로</li>
		<li>소용돌이 소용돌이 믹서를 사용 하 여 3 혼합된 솔루션 s.</li>
		<li>스핀 2 미니 원심 분리기에서 혼합된 솔루션 다운 s (2000 x g).</li>
		<li>Premixed 솔루션의 0.1 µ L에 밖으로 그리고 즉시 샘플 접시에 그것을 예금 하는 피 펫을 사용 합니다. 참고: 샘플의 작은 금액을 입금 하지 마십시오 유지 premixed 솔루션 10에 대 한 피 펫의 끝 s.</li>
		<li>밖으로 건조에 샘플에 대 한 기다립니다. 전형적인 건조 시간은 표 1에 나열 됩니다.</li>
		<li>한 피 펫을 사용 하 여 0.2 µ L MeOH의 말린된 샘플 자리에 바로 입금. 샘플 얻을 것 이다 즉시 밖으로 건조 하 고 젖은. 참고: 증 착 절차는 3에 완료 확인 MeOH의 중요 한 증발 손실을 방지 하기 위해 s.</li>
		<li>현미경을 사용 하 여 샘플을 검사 합니다. 크리스탈 형태학으로 예상된 (원하는 결과의 예를 그림 1 참조) 없는 경우, 새로운 샘플을 준비 하는 단계 2.1.1-2.1.6를 반복 합니다.</li>
		<li>니트 릴 장갑 착용 하 고 신중 하 게 건조 실에서 샘플 접시를 꺼내.</li>
	</ol>
</li>
	<li>
샘플의 일정 금액 (1 µ L)의 분석에 대 한 참고: 다음 단계는 일반적으로 이용한 MALDI 샘플 금액 탄수화물 샘플의 동질성을 극대화 하기 위한 개발 된다. 과정은 루틴 및 정량 분석에 적합 합니다. Recrystallization 프로세스 샘플 및 행렬 큰 지역에 균등 하 게 재분배.<ol>
<li>DHB 솔루션 및 2.5 µ SLeA L 또는 microcentrifuge 관에서 수소 솔루션의 2.5 µ L 공기로</li>
		<li>소용돌이 소용돌이 믹서 5 premixed 솔루션 s.</li>
		<li>스핀 2 미니 원심 분리기에서 혼합된 솔루션 다운 s (2000 x g).</li>
		<li>Premixed 솔루션의 1.0 µ L를 끌어낼 즉시 샘플 접시에 그것을 예금 하는 피 펫을 사용 합니다. 참고: 하지 않습니다 나머지 사용 샘플 입금 후 다시 솔루션을 혼합.</li>
		<li>밖으로 건조에 샘플에 대 한 기다립니다. 전형적인 건조 시간은 표 1에 나열 됩니다.</li>
		<li>1.5 µ L MeOH의 말린된 샘플 자리에 바로 입금 하는 피 펫을 사용 합니다. 샘플 얻을 것 이다 즉시 밖으로 건조 하 고 젖은. 참고: 높은 샘플 접시 온도 (50 ° C)의 경우, 5에서 recrystallization 단계를 수행 피 펫 팁에서 MeOH의 증발을 최소화 하기 위해 s.</li>
		<li>현미경을 사용 하 여 샘플을 검사 합니다. 크리스탈 형태학으로 예상된 (원하는 결과의 예를 그림 1 참조) 없는 경우, 새로운 샘플을 준비 하는 단계 2.2.1-2.2.6를 반복 합니다.</li>
		<li>니트 릴 장갑 착용 하 고 신중 하 게 건조 실에서 샘플 접시를 꺼내.</li>
	</ol>
</li>
</ol>3. 질량 분석 데이터 수집 및 분석
참고: 분석은 상업적인 시간의 비행 질량 분 서 계 (자료 테이블) 갖춘 MALDI 이온 소스를 사용 하 여 수행 됩니다. 악기는 특정 제어 소프트웨어 (자료 테이블) 미리 최적화 된 추출 지연 및 레이저 에너지에 운영 됩니다. 스펙트럼은 m/z의 대량 범위 선형 모드에서 기록 됩니다 = 0-1500. 잠재적인 샘플 격판덮개는 ± 25 케빈과 모든 스펙트럼 평균 10 레이저 샷. 사용자는 악기 최적화와 호환 하는 소프트웨어를 사용 하 여 샘플 분석을 실시 하 고 악기 제조 업체의 지침에 따라 해야 합니다.
<ol><li>인스트루먼트 컨트롤 소프트웨어를 열고 ( 재료의 표참조).</li>
	<li>질량 분석기에 샘플 접시를 삽입 합니다.</li>
	<li>소프트웨어에서 사전 최적화 된 데이터 수집 방법을 선택 합니다.</li>
	<li>이미징 소프트웨어를 사용 하 여 IMS에 대 한 전체 샘플 지역 등록 ( 재료의 표참조). 참고: IMS를 하 고 그렇지 않다면이 단계를 건너뜁니다.</li>
	<li>제어 소프트웨어의 일괄 처리 모드에서 데이터 수집을 시작 합니다.</li>
	<li>데이터 수집 완료 후 이미징 소프트웨어를 사용 하 여 이온 이미지 플롯.</li>
	<li>질량 스펙트럼 분석 소프트웨어를 사용 하 여 분석 ( 재료의 표참조)는 데이터는 이온 이미지 없이 기록 하는 경우.</li>
</ol>

<ol>
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저자는 공개 없다.

샘플이 MALDI 양 DD에서 중요 한 문제는 가장 일반적으로 사용 되는 샘플 준비 방법, 결과 결정은 매우 이질적인 것 이다. 이러한 샘플 가난한 쏜 총에 및 샘플 샘플 신호 재현성을 보여줍니다. 따라서, 데이터 수집 하는 동안 "달콤한 명소" 샘플 영역에 대 한 검색 MALDI 실험에서 일반적인 절차 이다. 이러한 다른 유형의 샘플 루틴 분석에 정량화를 위한 적합 하지 않습니다.
현재 ?...

탄수화물 분석 한 중요 하 고 어려운 주제 이다입니다. 탄수화물과 그들의 유도체 재생 중요 한 역할 살고 있는 생물1,2,3. 이러한 분자 구조를 복잡 하 고 분해 하는 경향이 있다. 그들의 많은 명확 하 게 분리와 검출 때문 특징 될 수 없습니다. 매트릭스 보조 레이저 탈 착 또는 이온화 (MALDI) 질량 분석 (MS)의 다양 한 감도 및 이해할 수 있는 결과4, 생체 분석에 적용 된 있지만 계속 탄수화물 MALDI MS를 사용 하 여 분석 이러한 분자5의 낮은 이온화 효율 인해 주요 도전 하. 화학 derivatization 탄수화물6,7, 이온화 효율을 개선 하는 일반적인 방법 이지만 이러한 절차는 시간 및 샘플 사용. 게다가, derivatized 탄수화물의 이온화 효율의 단백질 보다 여전히 낮습니다. 따라서, 복잡 한 절차 없이 MALDI MS에서 탄수화물 신호를 개선 하는 방법의 개발이 필요 하다.
정량 분석에 MALDI MS의 응용 프로그램은 또 다른 도전 주제. MALDI MS의 주요 문제는 그것의 감도 및 데이터 재현성 실험적인 매개 변수 및 예제 준비 프로토콜에 비판적으로 의존. 대부분의 경우, MALDI MS에 의해 정량 분석 이기종 샘플 형태학 및 분석 배포 안정적입니다. 잘 알려진 예를 들어 2, 5-dihydroxybenzoic 산 (DHB) MALDI 매트릭스와 함께 준비 하는 샘플입니다. DHB는 주위 환경에서 천천히 결정은, 하는 때에 때문에 결과 샘플은 불규칙 한 형태학을 보여 매트릭스 결정으로 분석 법인의 범위 예측 가능한, 하지 않습니다. 큰 바늘 모양 및 결정이 이러한 샘플에 의하여 일반적으로 이루어져 있다. DHB는 휘발성 용 매 또는 온수 샘플 접시를 사용 하 여, 준비 하는 때 빠른 건조 과정 결과 더 균질 정밀한 결정 더 나은 양적 결과8,,910. 이 기술은 MALDI 샘플의 "recrystallization"로 알려져 있다. 개선 빠른 결정 화 과정에서 잘 매트릭스 결정으로 analytes의 더 나은 설립에 기인 된다. 우리 또한 그 탄수화물 신호 및 향상 된 양적 결과11,12의 감소 샘플 준비 환경 조정 시연 했다. 이 작품에서 발견 샘플 형태학 탄수화물 신호 품질을 결정 하는 중요 한 요소는 건의 한다. 매일 분석을 위한 일반적인 전략을 개발, 향상 된 탄수화물 감도 제공 하는 효율적인 샘플 개혁 방법이 필요 합니다.
우리는 체계적으로 최근 보고서13MALDI MS에서 샘플 형태학 및 탄수화물 감도 간의 상관 관계를 시험 했다. 결과 몇 가지 중요 한 탄수화물을 사용 하 여 얻은 고 행렬 표시 recrystallizing에 의해 최고의 신호 향상 실현은 MALDI 샘플 건조. 기존의 말린된 방울 (DD) 메서드를 사용 하 여 준비 하는 샘플의 형태는 메탄올 (MeOH)와 빠른 recrystallization에 의해 개혁 이다. 자세한 샘플 준비 프로토콜은 여기에 설명 했다. 샘플 접시 늘어진, 샘플 증 착 및 recrystallization, 및 질량 분석 분석을 포함 하 여 세 가지 주요 단계는 프로토콜에 의하여 이루어져 있다. 이용한 탄수화물 sialyl-루이스 (SLeA) 및 maltoheptaose (수소)를 포함합니다. DHB는 모델 매트릭스로 사용 됩니다. 결과 탄수화물 신호 강도 및 공간 유통 개선 현저 하 게 recrystallization 후 보여. 이러한 메서드는 샘플을 2,4,6-trihydroxyacetophenone (탑)와 α-cyano-4-hydroxycinnamic 산을 포함 하 여 다른 인기 행렬에 적용할 수 있습니다. 이 방법은 탄수화물 분석을 위한 실험실 루틴에 쉽게 통합 될 수 있는 일반적인 접근 방식으로 제공 합니다.

<table><tbody><tr><td>&lt;strong&gt;Reagent&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Detergent powder</td><td>Alconox</td><td>242985</td><td></td></tr><tr><td>Methanol</td><td>Merck</td><td>106009</td><td></td></tr><tr><td>Acetonitrile</td><td>Merck</td><td>100003</td><td></td></tr><tr><td>2,5-dihydroxybenzoic acid (DHB)</td><td>Alfa Aesar</td><td>A11459</td><td></td></tr><tr><td>sialyl-lewis A (SLe&lt;sup&gt;A&lt;/sup&gt;)</td><td>Sigma-Aldrich</td><td>S1782</td><td></td></tr><tr><td>Maltoheptaose</td><td>Sigma-Aldrich</td><td>M7753</td><td></td></tr><tr><td>Pipette tips</td><td>Mettler Toledo</td><td>17005091</td><td></td></tr><tr><td>Microcentrifuge tube</td><td>Axygen</td><td>MCT-150-C</td><td></td></tr><tr><td>&lt;strong&gt;Equipment&lt;/strong&gt;</td><td></td><td></td><td></td></tr><tr><td>Milli-Q water purification system</td><td>Millipore</td><td>ZMQS6VFT1</td><td></td></tr><tr><td>Powder-free nitrile gloves</td><td>Microflex</td><td>SU-690</td><td></td></tr><tr><td>600 mL beaker</td><td>Duran</td><td>2110648</td><td></td></tr><tr><td>Ultrasonic cleaner</td><td>Delta</td><td>DC300H</td><td></td></tr><tr><td>Hygrometer</td><td>Wisewind</td><td>5330</td><td></td></tr><tr><td>Nitrogen gas flowmeter</td><td>Dwyer</td><td>RMA-6-SSV</td><td></td></tr><tr><td>K-type thermocouples</td><td>Digitron</td><td>311-1670</td><td></td></tr><tr><td>Vortex mixer</td><td>Scientific Industries</td><td>&amp;nbsp;SI-0236</td><td></td></tr><tr><td>Mini centrifuge</td><td>Select BioProducts</td><td>Force Mini&amp;nbsp;</td><td></td></tr><tr><td>Pipette</td><td>Rainin</td><td>pipet-lite XLS</td><td></td></tr><tr><td>Stereomicroscope</td><td>Olympus</td><td>SZX16</td><td></td></tr><tr><td>Temperature controllable drying chamber</td><td>This lab</td><td></td><td></td></tr><tr><td>Ultraflex II TOF/TOF mass spectrometer</td><td>Bruker Daltonics</td><td></td><td></td></tr><tr><td>MTP 384 target plate polished steel BC</td><td>Bruker Daltonics</td><td>8280781</td><td></td></tr><tr><td>Flexcontrol Version 3.4</td><td>Bruker Daltonics</td><td></td><td>Control software</td></tr><tr><td>Fleximaging Version 2.1</td><td>Bruker Daltonics</td><td></td><td>Imaging software</td></tr><tr><td>Flexanalysis Version 3.4</td><td>Bruker Daltonics</td><td></td><td>Analysis software</td></tr></tbody></table>

an efficient sample preparation method to enhance carbohydrate ion signals in matrix-assisted laser desorption/ionization mass spectrometry

샘플 준비는 탄수화물의 질량 분석 (MS) 분석에 중요 한 과정 이다. 매트릭스 보조 레이저 탈 착 또는 이온화 (MALDI) MS 선택 탄수화물 분석에서의 방법 이지만, 가난한 이온 신호 및 데이터 재현성 탄수화물 샘플의 심각한 문제가 계속. 탄수화물의 정량 분석을 위해 뛰어난 데이터 품질을 제공 하는 효과적인 분석 프로토콜은 필요 합니다. 이 비디오 신호 강도 향상 하 여 MALDI MS에서 탄수화물의 데이터 변화를 최소화 샘플 준비 프로토콜을 보여 줍니다. 건조와 샘플 방울의 결정 화, 후 크리스탈 형태학 대량 spectrometric 분석 전에 메탄올에 의해 개혁 이다. 탄수화물 신호에 향상은 MALDI 이미징 질량 분석 (IMS)으로 시험 된다. 실험 결과 크리스탈 개혁 결정 구조를 조정 하 고 재분배 탄수화물 analytes 보여 줍니다. 기존의 MALDI-MS, 메탄올 보여줍니다 훨씬 더 나은 신호 강도, 이온 이미지 배포 및 데이터 안정성과 개혁 탄수화물 크리스탈 형태학의 말린된 방울 준비 방법 비교 때문에 여기에서 설명 하는 프로토콜 샘플 구성에 변화를 포함 하지 않는, 그들은 일반적으로 다양 한 탄수화물과 행렬에 적용 됩니다.

SLeA 의 대표적인 SEM 이미지 DHB DD를 사용 하 여 준비와 혼합 하 고 recrystallization 메서드는 그림 1에 나와 있습니다. 전형적인 DHB 형태학 DD 메서드가 준비 테두리 샘플 관광 명소의 중심에 미세 결정 구조에서 큰 바늘 모양의 결정 이다. 이러한 바늘 모양 결정의 일반적인 길이 ~ 100 µ m. MeOH에 의해 recrystallization, 후 샘플 더 큰 지역 정밀한 조각 ?...

Watch this Scientific Journal Video about An Efficient Sample Preparation Method to Enhance Carbohydrate Ion Signals in Matrix-assisted Laser Desorption/Ionization Mass Spectrometry at JoVE.com

An Efficient Sample Preparation Method to Enhance Carbohydrate Ion Signals in Matrix-assisted Laser Desorption/Ionization Mass Spectrometry

샘플 준비 과정 동안 개혁 결정 구조에 의해 MALDI 질량 분석에서 탄수화물 이온 신호를 향상을 위한 프로토콜은 설명 했다.

매트릭스 보조 레이저 탈 착/이온화 질량 분석에서 탄수화물 이온 신호를 강화 하는 효율적인 샘플 준비 방법

Sample preparation is a critical process in mass spectrometry (MS) analysis of carbohydrates. Although matrix-assisted laser desorption/ionization (MALDI) ...

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Research

JoVE Journal

Chemistry

7.0K 조회수.  Academia Sinica. 샘플 준비 과정 동안 개혁 결정 구조에 의해 MALDI 질량 분석에서 탄수화물 이온 신호를 향상을 위한 프로토콜은 설명 했다.

비디오: An Efficient Sample Preparation Method to Enhance Carbohydrate Ion Signals in Matrix-assisted Laser Desorption/Ionization Mass Spectrometry

Preparation of Homogeneous MALDI Samples for Quantitative Applications

This protocol demonstrates a simple sample preparation to reduce spatial heterogeneity in ion signals during matrix-assisted laser desorption/ionization (MALDI) mass spectrometry. The heterogeneity of ion signals is a severe problem in MALDI, which results in poor data reproducibility and makes MALDI unsuitable for quantitative analysis. By regulating sample plate temperature during sample preparation, thermal-induced hydrodynamic flows inside droplets of sample solution are able to reduce the heterogeneity problem. A room-temperature sample preparation chamber equipped with a temperature-regulated copper base block that holds MALDI sample plates facilitates precise control of the sample drying condition. After drying of sample droplets, the temperature of sample plates is returned to room temperature and removed from the chamber for subsequent mass spectrometric analysis. The areas of samples are examined with MALDI-imaging mass spectrometry to obtain the spatial distribution of all components in the sample. In comparison with the conventional dried-droplet method that prepares samples under ambient conditions without temperature control, the samples prepared with the method demonstrated herein show significantly better spatial distribution and signal intensity. According to observations using carbohydrate and peptide samples, decreasing substrate temperature while maintaining the surroundings at ambient temperature during the drying process can effectively reduce the heterogeneity of ion signals. This method is generally applicable to various combinations of samples and matrices.

A protocol for reducing spatial heterogeneities of ion signals in MALDI mass spectrometry by regulating substrate temperature during sample drying processes is demonstrated.

양적 응용 프로그램에 대한 균일 MALDI 시료의 제조

This protocol demonstrates a simple sample preparation to reduce spatial heterogeneity in ion signals during matrix-assisted laser desorption/ionization ...

연구

생화학

Matrix-assisted Laser Desorption/Ionization Time of Flight (MALDI-TOF) Mass Spectrometric Analysis of Intact Proteins Larger than 100 kDa

Effectively determining masses of proteins is critical to many biological studies (e.g. for structural biology investigations). Accurate mass determination allows one to evaluate the correctness of protein primary sequences, the presence of mutations and/or post-translational modifications, the possible protein degradation, the sample homogeneity, and the degree of isotope incorporation in case of labelling (e.g. 13C labelling).
Electrospray ionization (ESI) mass spectrometry (MS) is widely used for mass determination of denatured proteins, but its efficiency is affected by the composition of the sample buffer. In particular, the presence of salts, detergents, and contaminants severely undermines the effectiveness of protein analysis by ESI-MS. Matrix-assisted laser desorption/ionization (MALDI) MS is an attractive alternative, due to its salt tolerance and the simplicity of data acquisition and interpretation. Moreover, the mass determination of large heterogeneous proteins (bigger than 100 kDa) is easier by MALDI-MS due to the absence of overlapping high charge state distributions which are present in ESI spectra.
Here we present an accessible approach for analyzing proteins larger than 100 kDa by MALDI-time of flight (TOF). We illustrate the advantages of using a mixture of two matrices (i.e. 2,5-dihydroxybenzoic acid and &#945;-cyano-4-hydroxycinnamic acid) and the utility of the thin layer method as approach for sample deposition. We also discuss the critical role of the matrix and solvent purity, of the standards used for calibration, of the laser energy, and of the acquisition time. Overall, we provide information necessary to a novice for analyzing intact proteins larger than 100 kDa by MALDI-MS.

Matrix-assisted Laser Desorption/Ionization Time of Flight MALDI-TOF Mass Spectrometric Analysis of Intact Proteins Larger than 100 kDa

Accurate mass measurement represents an important step during the investigation of proteins. Matrix-assisted laser desorption/ionization (MALDI) mass spectrometry (MS) can be used for such determination and its key advantage is the tolerance to salts, detergents and contaminants. Here we illustrate an accessible approach for the analysis of proteins larger than 100 kDa by MALDI-MS.

비행의 레이저 탈착 / 이온화 시간 본래대로 단백질의 (MALDI-TOF) 질량 분광 분석 매트릭스를 이용한 큰 100 kDa의

Effectively determining masses of proteins is critical to many biological studies (e.g. for structural biology investigations). Accurate mass ...

화학

Sheathless Capillary Electrophoresis&#8211;Mass Spectrometry for Metabolic Profiling of Biological Samples

In metabolomics, a wide range of analytical techniques is used for the global profiling of (endogenous) metabolites in complex samples. In this paper, a protocol is presented for the analysis of anionic and cationic metabolites in biological samples by capillary electrophoresis&#8211;mass spectrometry (CE-MS). CE is well-suited for the analysis of highly polar and charged metabolites as compounds are separated on the basis of their charge-to-size ratio. A recently developed sheathless interfacing design, i.e., a porous tip interface, is used for coupling CE to electrospray ionization (ESI) MS. This interfacing approach allows the effective use of the intrinsically low-flow property of CE in combination with MS, resulting in nanomolar detection limits for a broad range of polar metabolite classes. The protocol presented here is based on employing a bare fused-silica capillary with a porous tip emitter at low-pH separation conditions for the analysis of a broad array of metabolite classes in biological samples. It is demonstrated that the same sheathless CE-MS method can be used for the profiling of cationic metabolites, including amino acids, nucleosides and small peptides, or anionic metabolites, including sugar phosphates, nucleotides and organic acids, by only switching the MS detection and separation voltage polarity. Highly information-rich metabolic profiles in various biological samples, such as urine, cerebrospinal fluid and extracts of the glioblastoma cell line, can be obtained by this protocol in less than 1 hr of CE-MS analysis.

A protocol for metabolic profiling of biological samples by capillary electrophoresis&#8211;mass spectrometry using a sheathless porous tip interface design is presented.

생물 시료의 대사 프로파일 링에 대한 쉬스 모세관 전기 영동 - 질량 분석

In metabolomics, a wide range of analytical techniques is used for the global profiling of (endogenous) metabolites in complex samples. In this paper, a ...

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 ...

생물학

Low Molecular Weight Protein Enrichment on Mesoporous Silica Thin Films for Biomarker Discovery

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the monitoring of therapeutic efficiency.1-3 The circulating low molecular weight proteome (LMWP) composed of small proteins shed from tissues and cells or peptide fragments derived from the proteolytic degradation of larger proteins, has been associated with the pathological condition in patients and likely reflects the state of disease.4,5 Despite these potential clinical applications, the use of Mass Spectrometry (MS) to profile the LMWP from biological fluids has proven to be very challenging due to the large dynamic range of protein and peptide concentrations in serum.6 Without sample pre-treatment, some of the more highly abundant proteins obscure the detection of low-abundance species in serum/plasma. Current proteomic-based approaches, such as two-dimensional polyacrylamide gel-electrophoresis (2D-PAGE) and shotgun proteomics methods are labor-intensive, low throughput and offer limited suitability for clinical applications.7-9 Therefore, a more effective strategy is needed to isolate LMWP from blood and allow the high throughput screening of clinical samples. 
Here, we present a fast, efficient and reliable multi-fractionation system based on mesoporous silica chips to specifically target and enrich LMWP.10,11 Mesoporous silica (MPS) thin films with tunable features at the nanoscale were fabricated using the triblock copolymer template pathway. Using different polymer templates and polymer concentrations in the precursor solution, various pore size distributions, pore structures, connectivity and surface properties were determined and applied for selective recovery of low mass proteins. The selective parsing of the enriched peptides into different subclasses according to their physicochemical properties will enhance the efficiency of recovery and detection of low abundance species. In combination with mass spectrometry and statistic analysis, we demonstrated the correlation between the nanophase characteristics of the mesoporous silica thin films and the specificity and efficacy of low mass proteome harvesting. The results presented herein reveal the potential of the nanotechnology-based technology to provide a powerful alternative to conventional methods for LMWP harvesting from complex biological fluids. Because of the ability to tune the material properties, the capability for low-cost production, the simplicity and rapidity of sample collection, and the greatly reduced sample requirements for analysis, this novel nanotechnology will substantially impact the field of proteomic biomarker research and clinical proteomic assessment.

We developed a technology based on mesoporous silica thin film for the selective recovery of low molecular weight proteins and peptides from human serum. The physico-chemical properties of our mesoporous chips were finely tuned to provide substantial control in peptide enrichment and consequently profile the serum proteome for diagnostic purposes.

Biomarker 디스커버리를위한 Mesoporous 실리카 박막의 낮은 분자량의 단백질 농축

The identification of circulating biomarkers holds great potential for non invasive approaches in early diagnosis and prognosis, as well as for the ...

생체공학

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 ...

Characterization of Synthetic Polymers via Matrix Assisted Laser Desorption Ionization Time of Flight (MALDI-TOF) Mass Spectrometry

There are many techniques that can be employed in the characterization of synthetic homopolymers, but few provide as useful of information for end group analysis as matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS). This tutorial demonstrates methods for optimization of the sample preparation, spectral acquisition, and data analysis of synthetic polymers using MALDI-TOF MS. Critical parameters during sample preparation include the selection of the matrix, identification of an appropriate cationization salt, and tuning the relative proportions of the matrix, cation, and analyte. The acquisition parameters, such as mode (linear or reflector), polarization (positive or negative), acceleration voltage, and delay time, are also important. Given some knowledge of the chemistry involved to synthesize the polymer and optimizing both the data acquisition parameters and the sample preparation conditions, spectra should be obtained with sufficient resolution and mass accuracy to enable the unambiguous determination of the end groups of most homopolymers (masses below 10,000) in addition to the repeat unit mass and the overall molecular weight distribution. Though demonstrated on a limited set of polymers, these general techniques are applicable to a much wider range of synthetic polymers for determining mass distributions, though end group determination is only possible for homopolymers with narrow dispersity.

Characterization of Synthetic Polymers via Matrix Assisted Laser Desorption Ionization Time of Flight MALDI-TOF Mass Spectrometry

A protocol for the matrix-assisted laser desorption ionization time of flight mass spectrometry (MALDI-TOF MS) characterization of synthetic polymers is described including the optimization of sample preparation, spectral acquisition, and data analysis.

매트릭스를 통해 합성 고분자의 특성화 지원 레이저 탈 착 이온화 시간의 비행 (TOF MALDI) 질량 분석

There are many techniques that can be employed in the characterization of synthetic homopolymers, but few provide as useful of information for end group ...

Using a Cyclic Ion Mobility Spectrometer for Tandem Ion Mobility Experiments

Accurate characterization of chemical structures is important to understand their underlying biological mechanisms and functional properties. Mass spectrometry (MS) is a popular tool but is not always sufficient to completely unveil all structural features. For example, although carbohydrates are biologically relevant, their characterization is complicated by numerous levels of isomerism. Ion mobility spectrometry (IMS) is an interesting complement because it is sensitive to ion conformations and, thus, to isomerism. 
Furthermore, recent advances have significantly improved the technique: the last generation of Cyclic IMS instruments offers additional capabilities compared to linear IMS instruments, such as an increased resolving power or the possibility to perform tandem ion mobility (IMS/IMS) experiments. During IMS/IMS, an ion is selected based on its ion mobility, fragmented, and reanalyzed to obtain ion mobility information about its fragments. Recent work showed that the mobility profiles of the fragments contained in such IMS/IMS data can act as a fingerprint of a particular glycan and can be used in a molecular networking strategy to organize glycomics datasets in a structurally relevant way. 
The goal of this protocol is thus to describe how to generate IMS/IMS data, from sample preparation to the final Collision Cross Section (CCS) calibration of the ion mobility dimension that yields reproducible spectra. Taking the example of one representative glycan, this protocol will show how to build an IMS/IMS control sequence on a Cyclic IMS instrument, how to account for this control sequence to translate IMS arrival time into drift time (i.e., the effective separation time applied to the ions), and how to extract the relevant mobility information from the raw data. This protocol is designed to clearly explain the critical points of an IMS/IMS experiment and thus help new Cyclic IMS users perform straightforward and reproducible acquisitions.

Ion mobility spectrometry (IMS) is an interesting complement to mass spectrometry for the characterization of biomolecules, notably because it is sensitive to isomerism. This protocol describes a tandem IMS (IMS/IMS) experiment, which allows the isolation of a molecule and the generation of the mobility profiles of its fragments.

탠덤 이온 이동성 실험을 위한 순환 이온 이동성 분광계 사용

Accurate characterization of chemical structures is important to understand their underlying biological mechanisms and functional properties. Mass ...

OLIgo Mass Profiling (OLIMP) of Extracellular Polysaccharides

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is that they contain complex sugar moieties in form of glycoproteins, proteoglycans, and/or polysaccharides. Examples include the extracellular matrix of humans and animal cells consisting mainly of fibrillar proteins and proteoglycans or the polysaccharide based cell walls of plants and fungi, and the proteoglycan/glycolipid based cell walls of bacteria. All these glycostructures play vital roles in cell-to-cell and cell-to-environment communication and signalling.
An extraordinary complex example of an extracellular matrix is present in the walls of higher plant cells. Their wall is made almost entirely of sugars, up to 75% dry weight, and consists of the most abundant biopolymers present on this planet. Therefore, research is conducted how to utilize these materials best as a carbon-neutral renewable resource to replace petrochemicals derived from fossil fuel. The main challenge for fuel conversion remains the recalcitrance of walls to enzymatic or chemical degradation due to the unique glycostructures present in this unique biocomposite.
Here, we present a method for the rapid and sensitive analysis of plant cell wall glycostructures. This method OLIgo Mass Profiling (OLIMP) is based the enzymatic release of oligosaccharides from wall materials facilitating specific glycosylhydrolases and subsequent analysis of the solubilized oligosaccharide mixtures using matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF/MS)1 (Figure 1). OLIMP requires walls of only 5000 cells for a complete analysis, can be performed on the tissue itself2, and is amenable to high-throughput analyses3. While the absolute amount of the solubilized oligosaccharides cannot be determined by OLIMP the relative abundance of the various oligosaccharide ions can be delineated from the mass spectra giving insights about the substitution-pattern of the native polysaccharide present in the wall.
OLIMP can be used to analyze a wide variety of wall polymers, limited only by the availability of specific enzymes4. For example, for the analysis of polymers present in the plant cell wall enzymes are available to analyse the hemicelluloses xyloglucan using a xyloglucanase5, 11, 12, 13, xylan using an endo-&beta;-(1-4)-xylanase 6,7, or for pectic polysaccharides using a combination of a polygalacturonase and a methylesterase 8. Furthermore, using the same principles of OLIMP glycosylhydrolase and even glycosyltransferase activities can be monitored and determined 9.

OLIgo Mass Profiling OLIMP of Extracellular Polysaccharides

A rapid way is described to gain insights into the structure of polysaccharides in an extracellular matrix. The method takes advantage of the specificity of glycosylhydrolases and the sensitivity of mass spectrometry allowing minute amounts of materials to be analyzed. This technique is adaptable to be used directly on tissue itself.

세포외 다당류의 OLIgo 질량 프로파일 (OLIMP)

The direct contact of cells to the environment is mediated in many organisms by an extracellular matrix. One common aspect of extracellular matrices is ...

Dithranol as a Matrix for Matrix Assisted Laser Desorption/Ionization Imaging on a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer

Mass spectrometry imaging (MSI) determines the spatial localization and distribution patterns of compounds on the surface of a tissue section, mainly using MALDI (matrix&#160;assisted laser desorption/ionization)-based analytical techniques. New matrices for small-molecule MSI, which can improve the analysis of low-molecular weight (MW) compounds, are needed. These matrices should provide increased analyte signals while decreasing MALDI background signals. In addition, the use of ultrahigh-resolution instruments, such as Fourier transform ion cyclotron resonance (FTICR) mass spectrometers, has&#160;the ability to resolve analyte signals from matrix signals, and this can partially overcome many problems associated with the background originating from the MALDI matrix. The reduction in the intensities of the metastable matrix clusters by FTICR MS can also help to overcome some of the interferences associated with matrix peaks on other instruments. High-resolution instruments such as the FTICR mass spectrometers are advantageous as they can produce distribution patterns of many compounds simultaneously while still providing confidence in chemical identifications. Dithranol (DT; 1,8-dihydroxy-9,10-dihydroanthracen-9-one) has previously been reported as a MALDI matrix for tissue imaging. In this work, a protocol for the use of DT for MALDI imaging of endogenous lipids from the surfaces of mammalian tissue sections, by positive-ion MALDI-MS, on an ultrahigh-resolution hybrid quadrupole FTICR instrument has been provided.

Dithranol (DT; 1,8-dihydroxy-9,10-dihydroanthracen-9-one) has previously been reported as a MALDI matrix for tissue imaging of small molecules; protocols for the use of DT for the MALDI imaging of endogenous lipids on the surface of tissue sections&#160;by positive-ion MALDI-MS on an ultrahigh-resolution quadrupole-FTICR instrument are provided here.

푸리에에 매트릭스 보조 레이저 탈착 / 이온화 이미징 매트릭스 디 트라 놀은 이온 사이클로트론 공명 질량 분석기를 변환

Mass spectrometry imaging (MSI) determines the spatial localization and distribution patterns of compounds on the surface of a tissue section, mainly ...