This work is supported by the Genomics Research Center, Academia Sinica and the Ministry of Science and Technology of Taiwan, the Republic of China (Contract No. 104-2119-M-001-014).

참고 :이 프로토콜은 건조-방울 방법으로 제조 된 말 토트 리오스와 브라 디 키닌 단편 (1-7)의 공간적 이질성을 줄이기 위해 개발되고있다. 프로토콜은 준비 및 전처리 샘플 증착 및 건조하고, 질량 분석 데이터 분석을 포함하여 세 개의 주요 단계로 구성된다. 절차를 요약하고 아래에보다 상세히 설명된다 : 1. 준비 및 전처리 <ol><li> 샘플 플레이트 청소 <ol><li> 니트릴 장갑을 착용하고 세제와 증류수 - 탈 이온수 (DDW)으로 가볍게 샘플 판을 손 씻는다. </li><li> 메탄올 (메탄올)과 DDW와 샘플 판을 씻어. </li><li> 600 ml의 비이커에 샘플 플레이트를 삽입하고 DDW로 입력합니다. </li><li> 초음파 조 (200 W, 40 KHZ)에서 15 분 동안 DDW의 샘플 플레이트를 초음파 처리. </li><li> 비커에서 DDW를 제거하고 메탄올로 비커를 입력합니다. </li><li> 초음파 욕 (200 W, 40 KHZ)에서 15 분 동안 메탄올에있는 샘플 플레이트 초음파 처리. </li><li> 질소 가스와 함께 접시에 용매 방울을 불어 샘플 증착하기 전에 건조 샘플 플레이트를 유지합니다. </li></ol></li><li> 규제 건조 챔버 온도 참고 :. 건조 챔버는 35 X 20 X 45cm 3 (가로 x 세로 x 높이) 아크릴 챔버 그림 1은 이러한 건조 시스템의 그림을 보여줍니다. 챔버는 건조실 내부에 설치된 보정 습도계 의해 모니터링 낮은 습도 조건을 유지하기 위해 일정한 유량으로 가스 유량계를 통해 실내 온도를 질소 가스로 치환된다. 프로그래밍 항온 순환 장치를 구비 한 건조 챔버에서의 구리베이스 블록 스테인리스 샘플 판을 수용하기 위해 사용된다. 구리베이스 블록은 25 ° C에서 5 샘플 플레이트 온도를 조절 할 수있다. 공기, 구리베이스 블록 및 샘플 플레이트의 온도를 K 형 열전대에 의해 모니터링된다. <ol><li> 문을 열고 빠르게 구리에 샘플 판을 넣어베이스 블록은 문을 닫습니다. </li><li> 수동 10 시간당 표준 입방 피트 (SCFH) 내지 질소 유량을 설정하는 가스 유량계를 조정한다. </li><li> 상대 습도는 항상 25 % 이하로되도록하여, 습도계와 미세 조정 가스 유량계에 의해 건조 챔버 내의 상대 습도를 모니터링. </li><li> K 형 열전대에 의해 샘플 플레이트의 온도를 모니터링하고 샘플 플레이트는 실험 또는 제어 상온 (25 ° C)에 대한 5 ° C에 도달 할 때까지 수동으로 물 순환 장치의 온도를 조정한다. 주 : 설계된 온도에서 샘플 판을 안정화시키기 위해서, 물 순환 온도는 일반적으로 설계된 시료보다 낮은 5 ° C를 0으로 설정된다. 예를 들어, 샘플 플레이트에서 5 ° C를 유지하는 물 순환 장치의 설정 온도는 35 ° C 0의 범위이다; 25 ° C에서 샘플 플레이트를 유지하는 물 순환 장치의 설정 온도는 23-25 ​​℃의 범위이다. </li><li> 필요한 온도 및 상대 습도가 샘플 증착 이전 (표 1)에 도달되도록. 주의 : 다른 샘플 플레이트 온도가 건조 공정의 모든 매개 변수뿐만 아니라 설정 값을 표 1에 나타내었다. 참고 : 챔버 문이 오랜 시간 동안 열려있는 경우 낮은 샘플 플레이트 온도에서 시료 접시에 물 응축이 발생할 수 있습니다. 물 결로가 발생하는 경우, 문을 닫고 물 응축 밖으로 건조 될 때까지 어떠한 샘플을 입금하지 마십시오. </li></ol></li><li> 매트릭스와 분석 대상 솔루션의 준비 <ol><li> 기질 용액의 제조 <ol><li> 50 % DDW 수용액 : 50 % 아세토 니트릴 (ACN)와 0.1 M THAP 솔루션을 준비합니다. </li></ol></li><li> 분석의 준비 <ol><li> DDW 10 -4 M 말 토트 리오스 솔루션을 준비합니다. </li><li> 50 % 아세토 니트릴에 10-5 M의 브라 디 키닌 단편을 제조 (1-7) 용액(ACN) : 50 % DDW 수용액. </li></ol></li></ol></li></ol> 2. 샘플 증착 및 건조 <ol><li> 프리믹스 0.1 M의 THAP 용액 0.25 μL 및 10-4 M의 말 토트 리오스 또는 10-5 M의 브라 디 키닌 단편 (1-7) 미세 원심 튜브 용액 0.25 μL. </li><li> 3 초 동안 혼합 용액을 소용돌이. </li><li> 원심 관의 바닥에 상기 용액을 수집하기 위해 2 초 (2,000 XG)에 혼합액을 원심 분리기. </li><li> 조심스럽게, 건조실의 문을 열고 피펫으로 시료 접시에 솔루션의 0.1 μl를 입금 즉시 문을 닫습니다. </li><li> 샘플 물방울이 건조 할 때까지 기다립니다. .주의 : 다른 샘플 플레이트 온도가 전형적으로 관찰 건조 시간은 표 1에 나열된 5 ° C의 시료 판 온도, 평균 건조 시간은 800 초이다 1000; 25 ° C의 샘플 플레이트 온도의 평균 건조 시간은 100 내지 150 S이고EC. </li><li> 건조 후에, 건조 챔버의 문을 연다. </li><li> 실내 온도 (25 °에 C)에 물 순환 온도를 설정합니다. 참고 : 샘플 플레이트는 건조 과정에서 실온 (25 ℃로)에서 지속적으로 유지되는 경우이 단계를 건너 뜁니다. </li><li> 상온 (25 ° C) 내지 샘플 플레이트 온도 복귀 후, 건조 챔버에서 샘플 판을 제거한다. </li><li> 5 배의 실체 현미경에서 샘플 형태를 검사하고 스냅 샷 밝은 필드 이미지를 촬영. 주 : 액정 모폴로지 같이 예상되지 않는 경우에는 같은 방법으로 새로운 샘플을 준비 할 필요가있다. 일반적인 액정 모폴로지는도 2의 상부 패널에 나타낸다. 참고 예 5 ° C 낮은 샘플 플레이트 온도와 경우에 따라서는 건조 챔버로부터 복용 ​​전 실온으로 샘플 플레이트를 예열하는 것이 중요하다. 샘플을 증착 할 때, 예 혼합 solutio을 유지하지 마십시오n은 10 초 이상 피펫의 끝에서입니다. 샘플을 증착 한 후 다시 예 혼합 용액을 사용하지 마십시오. 도 2의 상부 패널은 다양한 샘플 플레이트의 온도로 제조 된 샘플의 시야 화상을 나타낸다. </li></ol> 3. 질량 분석 데이터 분석 <ol><li> 질량 분석 데이터 수집 참고 제조 한 후, 샘플을 영상화 질량 분석법을 이용하여 분석 될 수있다. 현재 연구에서, 촬상 MS 실험은 실험실 내장 동기화 이중 극성 TOF를 이용하여 수행된다 (DP-TOF) 촬상 질량 분석계. 촬상 기능 15 상용 MALDI-TOF 질량 분석기는 또한 실험에 적합하다. 질량 분석기는 선형 추출 최적화 추출 지연을 갖는 양 이온 모드에서 동작된다. 이온의 운동 에너지는 20 kV로한다. 레이저 빔 크기는 시료 표면에 직경이 35㎛이며, 각 스폿의 스펙트럼은 AVE이고5 레이저 샷의 분노. <ol><li> MALDI 질량 분석기에 샘플 플레이트를 삽입합니다. </li><li> 단계 2.1-2.9에서 조제한 시료 질량 분광 분석 이미징 수행한다. </li><li> 결과 창에 표시된 질량 목록에서 특성 질량 피크를 선택하고 두 차원 이온 이미지를 플롯 &quot;2D&quot;을 클릭합니다. 참고 : THAP 혼합 말 토트 리오스를 들어, 특성 피크는 말 토트 리오스, 양성자 THAP을 sodiated 및 THAP을 sodiated 있습니다. THAP 혼합 키닌 단편 (1-7)의 경우, 특징적인 피크는 브라 디 키닌 단편 (1-7), 양성자 THAP를 양성자와 THAP sodiated를 포함한다. </li><li> 신호 강도의 상한 및 하한을 결정하는 팝업 창 조절 버튼을 클릭하고 &quot;사진 저장 &#39;을 클릭. 이 설정은 이온의 이미지의 콘트라스트를 정의한다. 참고 : 데이터의 모든 개인 설정에서 금이 지역과 낮은 밝기를 나타내는 널 명소가 제거된다. </li><li> 관찰하고 이온을 비교단계 2.9에서 찍은 밝은 필드 이미지와 이미지. 주 : 질량 분석, 특히 이온의 이미지 구성을 이미징 상업적기구와 함께 달성 될 수있다. 때문에 데이터 수집 및 분석 소프트웨어의 다양한, 사용자는 높은 품질의 이미지를 얻기 위해 장비 공급 업체에서 제공하는 소프트웨어의 지시를 따라야합니다. </li></ol></li><li> 데이터 분석 참고 : 샘플의 이질성이 정량적으로 분석된다. 이 데모에서는, 모든 샘플은 이온의 공간 분포를 분석하기 위해 자체 개발 한 소프트웨어에 의해 다수의 동심 영역으로 분할된다. 분석은 또한 독립형 데이터 분석 소프트웨어를 사용하여 수행 될 수있다. <ol><li> 중요하지 않은 부분을 제거하는 결과 창에 표시된 이온 이미지에서 널 명소와 금이 영역을 클릭합니다. 주 :이 절차는 이온 이미지의 필수 영역을 정의합니다. </li><li> 이온 이미지의 최 층을 찾기 위해 버튼 &quot;가장자리를 찾아&quot;을 클릭합니다. </li><li> 데이터베이스에 최 층의 이온 풍부한 정보를 저장하고 동시에 이온 이미지에서이 층을 제거하기 위해 &quot;공제&quot;를 클릭합니다. 이 최 층을 나타내는 확인란 결과 윈도우의 &quot;출력 데이터&quot;목록에 표시됩니다. </li><li> 이온 이미지의 중심이 정의 될 때까지 반복 3.2.2와 3.2.3 단계를 반복합니다. </li><li> 클릭하고 &quot;출력 데이터&quot;목록에있는 모든 확인란을 선택하고 데이터를 내보낼 &quot;내보내기&quot;를 클릭합니다. </li><li> 이온의 공간 분포 정보를 획득하기 위해 각 층의 평균 이온 풍부 계산 스프레드 시트 소프트웨어를 사용하여 보낸 데이터를 연다. </li></ol></li></ol>

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F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=.">.</a> Surface Tension. in Physicochemical Hydrodynamics : An Introduction. , 305-361 (1994).</li><li>Lai, Y. -. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Reducing+Spatial+Heterogeneity+of+MALDI+Samples+with+Marangoni+Flows+During+Sample+Preparation.">Reducing Spatial Heterogeneity of MALDI Samples with Marangoni Flows During Sample Preparation.</a> J. Am. Soc. Mass Spectrom. 27, 1314-1321 (2016).</li><li>Hsiao, C. -. H., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Comprehensive+Molecular+Imaging+of+Photolabile+Surface+Samples+with+Synchronized+Dual-Polarity+Time-of-Flight+Mass+Spectrometry.">Comprehensive Molecular Imaging of Photolabile Surface Samples with Synchronized Dual-Polarity Time-of-Flight Mass Spectrometry.</a> Rapid Commun. Mass Spectrom. 25, 834-842 (2011).</li><li>Vorm, O., Roepstorff, P., Mann, M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Improved+Resolution+and+Very+High-Sensitivity+in+MALDI+TOF+of+Matrix+Surfaces+Made+by+Fast+Evaporation.">Improved Resolution and Very High-Sensitivity in MALDI TOF of Matrix Surfaces Made by Fast Evaporation.</a> Anal. Chem. 66, 3281-3287 (1994).</li><li>Gabriel, S. J., Schwarzinger, C., Schwarzinger, B., Panne, U., Weidner, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Matrix+Segregation+as+the+Major+Cause+for+Sample+Inhomogeneity+in+MALDI+Dried+Droplet+Spots.">Matrix Segregation as the Major Cause for Sample Inhomogeneity in MALDI Dried Droplet Spots.</a> J. Am. Soc. Mass Spectrom. 25, 1356-1363 (2014).</li><li>Mampallil, D., Eral, H. B., van den Ende, D., Mugele, F. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Control+of+Evaporating+Complex+Fluids+through+Electrowetting.">Control of Evaporating Complex Fluids through Electrowetting.</a> Soft Matter. 8, 10614-10617 (2012).</li></ol>

The authors declare no competing financial interest.

이전 이론적 예측에 기초하여, 방울 내에서의 온도 - 유도 된 유체 역학적 흐름이 외측으로 용매 증발에 의한 모세관 흐름을 극복 할 수있다. 온도가 증가 액적 내에 그라디언트 때 분자의 내부 재순환 등의 효율이 향상된다. 주위 온도에서 주변을 유지하면서 5 ℃ 하에서 샘플 플레이트의 온도를 유지하는 경우, 예측 결과에 따라, 상기 액적 내의 재순환 흐름의 평균 속도는 외측 모세관 흐름보다 ...

Mass spectrometry (MS) is one of the most important analytical techniques for analyzing the molecular compositions of complex samples. Among all the ionization methods used in MS, matrix-assisted laser desorption/ionization (MALDI) is the most sensitive and widely used method in bioanalytical applications.1 In comparison to other ionization techniques, MALDI has the highest sensitivity and high tolerance to salt contaminants. Such analytical properties make MALDI the first choice for carbohydrate analysis and many proteomics applications. However, sample preparation is a crucial step for obtaining high quality data in MALDI-MS.
The most commonly used sample preparation method for MALDI-MS is the dried-droplet method, in which sample droplets are deposited on a surface and dried under ambient conditions. This drying method is simple and generally effective.2-5 However, a common problem in the dried-droplet method is that the resultant analyte/matrix crystals normally distribute irregularly. In many cases, the crystals aggregate at the periphery of sample areas, resulting in the so-called ring-stain formation.6-8 The heterogeneous crystal morphologies affect the spatial distribution of analyte molecules, which results in severe fluctuation in ion signal over sample areas. Such severe signal fluctuations and poor data reproducibility are known as the "sweet spot" problem in MALDI-MS.9 Thus, there is a great need for reducing spatial heterogeneities in MALDI-MS dried droplet applications.
Hydrodynamic flows in the sample droplet play an important role in determining the spatial distribution of samples prepared with the dried-droplet method.10-12 It was found that the evaporation of solvent induces outward capillary flows within droplets, which are responsible for the ring-stain formation.7,10 In contrast, recirculation flows induced by tangential surface-tension gradients may counterbalance the outward capillary flows.13 If the recirculation flow speeds are higher than that of the outward capillary flows, the samples can be efficiently redistributed to reduce the heterogeneity problem.14
In this work, we demonstrate a detailed protocol for preparing samples with a simple drying chamber to induce efficient recirculation flows during droplet drying processes. Droplet drying conditions are precisely controlled, including the temperatures of the sample plate and its surroundings, and the relative humidity within the chamber. The model analytes include maltotriose and bradykinin chain (1-7). The matrix used for the demonstration is 2,4,6-trihydroxyacetophenone (THAP). The samples are examined with time-of-flight (TOF) MS, and the data are analyzed quantitatively to show the reduction of heterogeneity.

<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>2,4,6-trihydroxyacetophenone (THAP)</td><td>Sigma-Aldrich</td><td>T64602&amp;nbsp;</td><td></td></tr><tr><td>Bradykinin fragment (1-7)</td><td>Sigma-Aldrich</td><td>B1651</td><td></td></tr><tr><td>Maltotriose</td><td>Sigma-Aldrich</td><td>47884</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>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>Synchronized dual-polarity time-of-flight imaging mass spectrometer (DP-TOF IMS)</td><td>this lab</td><td></td><td></td></tr><tr><td>MALDI-TOF stainless steel sample target</td><td>this lab</td><td></td><td></td></tr></tbody></table>

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.

밝은 필드 화상과 시료 판 (5)의 온도가 25 ° C로 제조 토트 리오스 및 브라 디 키닌 단편 (1-7)의 MS 이미지는도 1에 도시되어있다. sodiated 말 토트 리오스, 이온 신호를 중심으로 채워의 경우 샘플 영역의 주변에이를 25 ° C의 샘플 플레이트의 온도로 제조되는 경우. 5 ° C에 샘플 플레이트의 온도를 감소시킴으로써, 신호는 균일하게 전체 샘플 면적에 걸쳐 채워. 5 ...

Watch this Scientific Journal Video about Preparation of Homogeneous MALDI Samples for Quantitative Applications at JoVE.com

Preparation of Homogeneous MALDI Samples for Quantitative Applications

샘플 건조 공정 중에 기판의 온도를 조절하여 MALDI 질량 분석기에서 이온 이질성 신호 공간을 감소시키는 프로토콜이 설명된다.

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 ...

preparation-of-homogeneous-maldi-samples-for-quantitative-applications

Universidad San Sebastian

Research

JoVE Journal

Biochemistry

8.7K 조회수.  Academia Sinica.  샘플 건조 공정 중에 기판의 온도를 조절하여 MALDI 질량 분석기에서 이온 이질성 신호 공간을 감소시키는 프로토콜이 설명된다. 

비디오: Preparation of Homogeneous MALDI Samples for Quantitative Applications

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 ...

연구

화학

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 ...

생화학

Peptide and Protein Quantification Using Automated Immuno-MALDI (iMALDI)

Mass spectrometry (MS) is one of the most commonly used technologies for quantifying proteins in complex samples, with excellent assay specificity as a result of the direct detection of the mass-to-charge ratio of each target molecule. However, MS-based proteomics, like most other analytical techniques, has a bias towards measuring high-abundance analytes, so it is challenging to achieve detection limits of low ng/mL or pg/mL in complex samples, and this is the concentration range for many disease-relevant proteins in biofluids such as human plasma. To assist in the detection of low-abundance analytes, immuno-enrichment has been integrated into the assay to concentrate and purify the analyte before MS measurement, significantly improving assay sensitivity. In this work, the immuno- Matrix-Assisted Laser Desorption/Ionization (iMALDI) technology is presented for the quantification of proteins and peptides in biofluids, based on immuno-enrichment on beads, followed by MALDI-MS measurement without prior elution. The anti-peptide antibodies are functionalized on magnetic beads, and incubated with samples. After washing, the beads are directly transferred onto a MALDI target plate, and the signals are measured by a MALDI-Time of Flight (MALDI-TOF) instrument after the matrix solution has been applied to the beads. The sample preparation procedure is simplified compared to other immuno-MS assays, and the MALDI measurement is fast. The whole sample preparation is automated with a liquid handling system, with improved assay reproducibility and higher throughput. In this article, the iMALDI assay is used for determining the peptide angiotensin I (Ang I) concentration in plasma, which is used clinically as readout of plasma renin activity for the screening of primary aldosteronism (PA).

Peptide and Protein Quantification Using Automated Immuno-MALDI iMALDI

A protocol for the protein quantification in complex biological fluids using automated immuno-MALDI (iMALDI) technology is presented.

펩 티 드 및 단백질 정량화를 사용 하 여 자동 면역-MALDI (iMALDI)

Mass spectrometry (MS) is one of the most commonly used technologies for quantifying proteins in complex samples, with excellent assay specificity as a ...

Using the Open-Source MALDI TOF-MS IDBac Pipeline for Analysis of Microbial Protein and Specialized Metabolite Data

In order to visualize the relationship between bacterial phylogeny and specialized metabolite production of bacterial colonies growing on nutrient agar, we developed IDBac&#8212;a low-cost and high-throughput matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) bioinformatics pipeline. IDBac software is designed for non-experts, is freely available, and capable of analyzing a few to thousands of bacterial colonies. Here, we present procedures for the preparation of bacterial colonies for MALDI-TOF MS analysis, MS instrument operation, and data processing and visualization in IDBac. In particular, we instruct users how to cluster bacteria into dendrograms based on protein MS fingerprints and interactively create Metabolite Association Networks (MANs) from specialized metabolite data.

IDBac is an open-source mass spectrometry-based bioinformatics pipeline that integrates data from both intact protein and specialized metabolite spectra, collected on cell material scraped from bacterial colonies. The pipeline allows researchers to rapidly organize hundreds to thousands of bacterial colonies into putative taxonomic groups, and further differentiate them based on specialized metabolite production.

미생물 단백질 및 특수 대사 산물 데이터 분석을 위한 오픈 소스 MALDI TOF-MS IDBac 파이프라인 사용

In order to visualize the relationship between bacterial phylogeny and specialized metabolite production of bacterial colonies growing on nutrient agar, ...

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 ...

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

Sample preparation is a critical process in mass spectrometry (MS) analysis of carbohydrates. Although matrix-assisted laser desorption/ionization (MALDI) MS is the method of choice in carbohydrate analysis, poor ion signal and data reproducibility of carbohydrate samples continue to be severe problems. For quantitative analysis of carbohydrates, an effective analytical protocol providing superior data quality is necessary. This video demonstrates sample preparation protocols to improve signal intensity and minimize data variation of carbohydrates in MALDI-MS. After drying and crystallization of sample droplets, the crystal morphology is reformed by methanol before mass spectrometric analysis. The enhancement in carbohydrate signal is examined with MALDI imaging mass spectrometry (IMS). Experimental results show that crystal reformation adjusts crystalline structures and redistributes carbohydrate analytes. In comparison with the dried droplet preparation method in conventional MALDI-MS, reforming carbohydrate crystal morphologies with methanol shows significantly better signal intensity, ion image distribution, and data stability. Since the protocols demonstrated herein do not involve changes in sample composition, they are generally applicable to various carbohydrates and matrixes.

A protocol for enhancing carbohydrate ion signals in MALDI mass spectrometry by reforming crystalline structures during sample preparation processes is demonstrated.

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) ...

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 ...

Sample Preparation for Metabolic Profiling using MALDI Mass Spectrometry Imaging

Metabolomics, the study to identify and quantify small molecules and metabolites present in an experimental sample, has emerged as an important tool to investigate the biological activities during development and diseases. Metabolomics approaches&#160;are widely employed in the study of cancer, nutrition/diet, diabetes, and other physiological and pathological conditions involving metabolic processes. An advantageous tool that aids in metabolomic profiling advocated in this paper is matrix-assisted laser desorption/ionization mass spectrometry imaging (MALDI MSI). Its ability to detect metabolites in situ without labeling, structural modifications, or other specialized reagents, such as those used in immunostaining, makes MALDI MSI a unique tool in advancing methodologies relevant in the field of metabolomics. An appropriate sample preparation process is critical to yield optimal results and will be the focus of this paper.

The goal of this protocol is to provide detailed guidance on the sample preparation when planning for experiments using MALDI MSI to maximize metabolic and molecular detection in biological samples.

MALDI 질량 분광화상 영상을 이용한 대사 프로파일링을 위한 샘플 준비

Metabolomics, the study to identify and quantify small molecules and metabolites present in an experimental sample, has emerged as an important tool to ...