JoVE Journal > Biology
Summary
Abstract
Introduction
Protocol
Results
Discussion
Disclosures
Acknowledgements
Materials
References
Automatic Translation
Please note that all translations are automatically generated. Click here for the English version.
Biology

地蒽酚作为傅立叶的基质辅助激光解吸/离子化成像矩阵变换离子回旋共振质谱仪

Published: November 26, 2013
doi:
10.3791/50733
, ,
1University of Victoria-Genome BC Proteomics Centre,University of Victoria, 2Department of Biochemistry and Microbiology,University of Victoria

Summary

蒽三酚（DT; 1,8 – 二羟基-9,10 – 二氢蒽-9 – 酮）先前已被报道作为MALDI基质为小分子的组织成像;对于使用DT的内源性脂质的对的MALDI成像协议这里所提供的组织切片由正离子的MALDI-MS在一个超高分辨率四极-FTICR仪器表面。

Abstract

质谱成像（MSI）确定的组织切片的表面上的化合物的空间定位和分布模式，主要使用MALDI（基质辅助激光解吸/电离）为基础的分析技术。需要新的矩阵为小分子的MSI，从而可以提高低分子量（MW）的化合物的分析。这些矩阵应提供增加分析物的信号，同时降低MALDI背景信号。另外，使用超高清晰度的仪器，如傅立叶变换离子回旋共振（FTICR）质谱仪，具有从矩阵信号解析分析物信号的能力，这可以部分地克服与来自MALDI背景始发相关的许多问题矩阵。在亚稳态矩阵集群由FTICR MS的强度的降低也有助于克服一些与其他乐器基质峰相关联的干扰。高分辨率文书，如FTICR质谱仪是有利的，因为他们可以同时产生许多化合物的分布模式，同时还提供信心的化学鉴定。蒽三酚（DT; 1,8 – 二羟基-9,10 – 二氢蒽-9 – 酮）先前已被报道作为MALDI基质用于组织成像。在这项工作中，一个协议的使用DT的用于MALDI成像从哺乳动物组织切片表面的内源性脂的，由正离子的MALDI-MS，对超高清晰度混合四极FTICR仪器已经提供。

Introduction

质谱成像（MSI）是一种分析技术，用于确定组织部分1,2的表面上的化合物的空间定位和分布模式。基质辅助激光解吸/电离（MALDI）微星多肽和蛋白质的分析已经使用了十多年，也有过在样品制备，检测灵敏度，空间分辨率，可重复性和数据处理方法3,4很大的改进。从组织学染色切片和MSI实验相结合的信息，病理学家能够具体化合物的分布与病理生理学有趣的功能5相关。

小分子，包括外源性药物6,7及其代谢物8-10的分布格局也被审问MALDI-MS组织成像11。脂类也许是最广泛研究共轭亚油酸SS与MALDI成像化合物，无论是在12-17的MS和MS / MS 18模式。为小分子成像中使用的MALDI MSI的已经被限制由几个因素：1）的MALDI基质本身是小分子（通常为M / Z <500），其产生大量的离子信号。这些丰富的信号可以抑制小分子分析物的电离，干扰他们的检测19,20。无溶剂基质涂料21，矩阵升华22，以及矩阵预涂MALDI MS 23，除其他外，已经开发了改进的小分子的MSI。

新的矩阵，可以提高低分子量化合物的分析是在小分子微星极大的兴趣。这些矩阵应提供增加的信号分析与降低矩阵信号。在正离子模式下，2,5 -二羟基苯甲酸（DHB）和α-氰基-4 -羟基肉桂酸（CHCA）是两种常用的MALDI MS矩阵MSI 24 </SUP>。理想的基质会形成小晶体，从而保留分析物的空间定位。 DHB容易形成较大的晶体，因此施加利用升华的基质已经开发出来，部分地克服这一问题，并允许使用该矩阵的对磷脂22,25的敏感成像。 9 -氨基吖啶已经用于质子分析物的MSI在正离子模式26和用于核苷酸和磷脂在负离子模式26-29。 2 -巯基苯并已发现，得到高效的MALDI检测脂类30的，并已被用于小鼠脑的成像神经节苷脂31。傅立叶变换的超分辨率变换离子回旋共振（FTICR）质谱仪可以通过从矩阵信号32解决分析物的信号有所缓解这个问题。利用FTICR-MS法的另一个优点是，亚稳的矩阵簇的强度是红眼版33，这也降低这些干扰27。

使用地蒽酚的（DT; 1,8 -二羟基-9,10 -二氢蒽-9 -酮）作为MALDI基质用于组织成像先前已报道34。在当前的工作中，一个详细的协议提供了一种用于对牛晶状体组织切片的表面上使用DT的内源性脂质的MSI，在正离子模式。

Protocol

1。组织切片闪光冻结问题的标本，一次采收，使用液氮，把它们运干冰上（如需要运费），并将其存储于-80℃直至组织切片。 （如果商业样品的使用，保证了样品制备以这种方式。） 剪下机关管理的大小以适合MALDI靶。修剪过的器官任何不需要的部分。在这项研究中这里所描述，小牛镜头采用组织切片之前，先前描述的方法35解封装。 从-80℃冰箱中取出整个器官和?…

Representative Results

被切片，解冻安装在ITO镀膜载玻片组织样本应完好，无明显的撕裂。对于许多组织，直接组织解冻安装到的ITO镀膜玻璃幻灯片是可以接受的。对于一些特定的组织如牛晶状体中，组织的广泛撕裂常常看到时直接解冻安装时（ 图1a）。用乙醇或甲酸在ITO玻璃载玻片的预涂（ 图1b）有助于保持组织切片的组织安装时的完整性。 矩阵两者的选择和溶剂?…

Discussion

最重要的考虑因素，成功MALDI微星是：1）组织准备; 2）基质的选择; 3）矩阵的应用，以及4）数据的解释和分析。当样品和基质被适当地制备时，MS数据采集是自动化的。从这种类型的实验的数据分析是相当费力。

适当的组织准备是成功的MALDI微星实验的关键。组织的来源和处理可以对最终的分析有很大的影响。样品必须立刻闪在液氮中冷冻并储存在-80℃，并且它们不应被存储…

Disclosures

The authors have nothing to disclose.

Acknowledgements

作者要感谢加拿大基因组和基因组不列颠哥伦比亚省为平台的资金和支持。我们也感谢博士卡罗尔E.帕克的稿件和编辑援助的严格审查。 CHL也感谢不列​​颠哥伦比亚省蛋白质组学网络支持。

Materials

Name of Reagent/Material Company Catalog Number Comments
Rat Liver Pel-Freez Biologicals 56023-2
Bovine Calf Lens Pel-Freez Biologicals 57114-2 Sample should be decapsulated29 before use
Dithranol (DT) Sigma-Aldrich 10608 MALDI Matrix
α-Cyano-4-hydroxy-cinnamic Acid (CHCA) Sigma-Aldrich 70990 MALDI Matrix
2,5-Dihydroxybenzoic Acid (DHB) Sigma-Aldrich 85707 MALDI Matrix
Reserpine Sigma-Aldrich 83580
Terfenadine Sigma-Aldrich T9652
Formic Acid Sigma-Aldrich 14265
Ammonium Formate Sigma-Aldrich 14266
Ammonium Hydroxide Sigma-Aldrich 320145
Trifluoroacetic Acid (TFA) Sigma-Aldrich 302031
Water Sigma-Aldrich 39253
Methanol Sigma-Aldrich 34860
Acetonitrile Sigma-Aldrich 34967
Ethyl Acetate Sigma-Aldrich 34972
Isopropanol Sigma-Aldrich 34965
Chloroform Sigma-Aldrich 366927
Acetone Sigma-Aldrich 34850
Ethanol Commercial Alcohols 95%
ES Tuning Mix Agilent Technologies G2431A
ITO Coated Glass Slides Hudson Surface Technology PSI1207000 Ensure that samples are placed on the electrically conductive side
Wite-Out Shake-N-Squeeze Correction Pen Bic WOSQP11
Airbrush Sprayer Iwata Eclipse HP-CS
ImagePrep Bruker 249500-LS
MALDI adapter Bruker 235380
DOWNLOAD MATERIALS LIST

References

  1. Chaurand, P., Stoeckli, M., Caprioli, R. M. Direct Profiling of Proteins in Biological Tissue Sections by MALDI Mass Spectrometry. Anal. Chem. 71, 5263-5270 (1999).
  2. Caprioli, R. M., Farmer, T. B., Gile, J. Molecular Imaging of Biological Samples. Localization of Peptides and Proteins Using MALDI-TOF MS. Anal. Chem. 69, 4751-4760 (1997).
  3. Amstalden van Hove, E. R., Smith, D. F., Heeren, R. M. A. A concise review of mass spectrometry imaging. J. Chromatogr. A. 1217, 3946-3954 (2010).
  4. Norris, J. L., Caprioli, R. M. Analysis of Tissue Specimens by Matrix-Assisted Laser Desorption/Ionization Imaging Mass Spectrometry in Biological and Clinical Research. Chem. Rev. Feb 11, (2013).
  5. Walch, A., Rauser, S., Deininger, S. -. O., Höfler, H. MALDI imaging mass spectrometry for direct tissue analysis: a new frontier for molecular histology. Histochem. Cell Biol. 130, 421-434 (2008).
  6. Hsieh, Y., et al. Matrix-assisted laser desorption/ionization imaging mass spectrometry for direct measurement of clozapine in rat brain tissue. Rapid Commun. Mass Spectrom. 20, 965-972 (2006).
  7. Trim, P. J., et al. Matrix-assisted laser desorption/ionization-ion mobility separation-mass spectrometry imaging of vinblastine in whole body tissue sections. Anal. Chem. 80, 8628-8634 (2008).
  8. Khatib-Shahidi, S., Andersson, M., Herman, J. L., Gillespie, T. A., Caprioli, R. M. Direct molecular analysis of whole-body animal tissue sections by imaging MALDI mass spectrometry. Anal. Chem. 78, 6448-6456 (2006).
  9. Atkinson, S. J., Loadman, P. M., Sutton, C., Patterson, L. H., Clench, M. R. Examination of the distribution of the bioreductive drug AQ4N and its active metabolite AQ4 in solid tumours by imaging matrix-assisted laser desorption/ionisation mass spectrometry. Rapid Commun. Mass Spectrom. 21, 1271-1276 (2007).
  10. Drexler, D. M., et al. Utility of imaging mass spectrometry (IMS) by matrix-assisted laser desorption ionization (MALDI) on an ion trap mass spectrometer in the analysis of drugs and metabolites in biological tissues. J. Pharmacol. Toxicol. Methods. 55, 279-288 (2007).
  11. Prideaux, B., Stoeckli, M. Mass spectrometry imaging for drug distribution studies. J. Proteomics. 75, 4999-5013 (2012).
  12. Sugiura, Y., Setou, M. Imaging Mass Spectrometry for Visualization of Drug and Endogenous Metabolite Distribution: Toward In Situ Pharmacometabolomes. J. Neuroimmune Pharmacol. 5, 31-43 (2009).
  13. Garrett, T. J., Yost, R. A. Analysis of intact tissue by intermediate-pressure MALDI on a linear ion trap mass spectrometer. Anal. Chem. 78, 2465-2469 (2006).
  14. Woods, A. S., Jackson, S. N. Brain tissue lipidomics: direct probing using matrix-assisted laser desorption/ionization mass spectrometry. AAPS J. 8, 391-395 (2006).
  15. Cha, S., Yeung, E. S. Colloidal graphite-assisted laser desorption/ionization mass spectrometry and MSn of small molecules. 1. Imaging of cerebrosides directly from rat brain tissue. Anal. Chem. 79, 2373-2385 (2007).
  16. Burnum, K. E., et al. Spatial and temporal alterations of phospholipids determined by mass spectrometry during mouse embryo implantation. J. Lipid Res. 50, 2290-2298 (2009).
  17. Veloso, A., et al. Anatomical distribution of lipids in human brain cortex by imaging mass spectrometry. J. Am. Soc. Mass Spectrom. 22, 329-338 (2011).
  18. Tanaka, H., et al. Distribution of phospholipid molecular species in autogenous access grafts for hemodialysis analyzed using imaging mass spectrometry. Anal. Bioanalyt. Chem. 400, 1873-1880 (2011).
  19. Lou, X., van Dongen, J. L., Vekemans, J. A., Meijer, E. W. Matrix suppression and analyte suppression effects of quaternary ammonium salts in matrix-assisted laser desorption/ionization time-of-flight mass spectrometry: an investigation of suppression mechanism. Rapid Comm. Mass Spectrom. 23, 3077-3082 (2009).
  20. Knochenmuss, R., Karbach, V., Wiesli, U., Breuker, K., Zenobi, R. The matrix suppression effect in matrix-assisted laser desorption/ionization: application to negative ions and further characteristics. Rapid Commun. Mass Spectrom. 12, 529-534 (1998).
  21. Puolitaival, S. M., Burnum, K. E., Cornett, D. S., Caprioli, R. M. Solvent-free matrix dry-coating for MALDI imaging of phospholipids. J. Am. Soc. Mass Spectrom. 19, 882-886 (2008).
  22. Hankin, J. A., Barkley, R. M., Murphy, R. C. Sublimation as a Method of Matrix Application for Mass Spectrometric Imaging. J. Am. Soc. Mass Spectrom. 19, 1646-1652 (2007).
  23. Grove, K. J., Frappier, S. L., Caprioli, R. M. Matrix pre-coated MALDI MS targets for small molecule imaging in tissues. J. Am. Soc. Mass Spectrom. 22, 192-195 (2011).
  24. Fuchs, B., Süss, R., Schiller, J. An update of MALDI-TOF mass spectrometry in lipid research. Prog. Lipid Res. 49, 450-475 (2010).
  25. Murphy, R. C., Hankin, J. A., Barkley, R. M., Zemski Berry, K. A. MALDI imaging of lipids after matrix sublimation/deposition. Biochim. Biophys. Acta. 1811, 970-975 (2011).
  26. Vermillion-Salsbury, R. L., Hercules, D. M. 9-Aminoacridine as a matrix for negative mode matrix-assisted laser desorption/ionization. Rapid Commun. Mass Spectrom. 16, 1575-1581 (2002).
  27. Hu, C., et al. Analytical strategies in lipidomics and applications in disease biomarker discovery. J. Chromatogr. B Analyt. Technol. Biomed. Life Sci. 877, 2836-2846 (2009).
  28. Miura, D., et al. Ultrahighly sensitive in situ metabolomic imaging for visualizing spatiotemporal metabolic behaviors. Anal. Chem. 82, 9789-9796 (2010).
  29. Cerruti, C. D., Benabdellah, F., Laprevote, O., Touboul, D., Brunelle, A. MALDI Imaging and Structural Analysis of Rat Brain Lipid Negative Ions with 9-Aminoacridine Matrix. Anal. Chem. 84, 2164-2171 (2012).
  30. Astigarraga, E., et al. Profiling and Imaging of Lipids on Brain and Liver Tissue by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometry Using 2-Mercaptobenzothiazole as a Matrix. Anal. Chem. 80, 9105-9114 (2008).
  31. Whitehead, S. N., et al. Imaging mass spectrometry detection of gangliosides species in the mouse brain following transient focal cerebral ischemia and long-term recovery. PloS one. 6, e20808 (2011).
  32. Cornett, D. S., Frappier, S. L., Caprioli, R. M. MALDI-FTICR imaging mass spectrometry of drugs and metabolites in tissue. Anal. Chem. 80, 5648-5653 (2008).
  33. Deininger, S. O., et al. Normalization in MALDI-TOF imaging datasets of proteins: practical considerations. Anal. Bioanalyt. Chem. 401, 167-181 (2011).
  34. Le, C. H., Han, J., Borchers, C. H. Dithranol as a MALDI matrix for tissue imaging of lipids by Fourier transform ion cyclotron resonance mass spectrometry. Anal. Chem. 84, 8391-8398 (2012).
  35. Han, J., Schey, K. L. MALDI Tissue Imaging of Ocular Lens α-Crystallin. Invest. Ophthalmol. Vis. Sci. 47, 2990-2996 (2006).
  36. Schwartz, S. A., Reyzer, M. L., Caprioli, R. M. Direct tissue analysis using matrix-assisted laser desorption/ionization mass spectrometry: practical aspects of sample preparation. J. Mass Spectrom. 38, 699-708 (2003).
  37. Chen, Y., et al. Imaging MALDI mass spectrometry of sphingolipids using an oscillating capillary nebulizer matrix application system. Meth. Mol. Biology. 656, 131-146 (2010).
  38. Han, J., et al. Towards high throughput metabolomics using ultrahigh field Fourier transform ion cyclotron resonance mass spectrometry. Metabolomics. 4, 128-140 (2008).
  39. Smith, C. A., et al. METLIN: a metabolite mass spectral database. Ther. Drug Monit. 27, 747-751 (2005).
  40. Wishart, D. S., et al. HMDB: a knowledgebase for the human metabolome. Nucleic Acids Res. 37, D603-D610 (2009).
  41. Hoteling, A. J., Erb, W. J., Tyson, R. J., Owens, K. G. Exploring the importance of the relative solubility of matrix and analyte in MALDI sample preparation using HPLC. Anal. Chem. 76, 5157-5164 (2004).
  42. Hoteling, A. J., Mourey, T. H., Owens, K. G. Importance of solubility in the sample preparation of poly(ethylene terephthalate. for MALDI TOFMS. Anal. Chem. 77, 750-756 (2005).
  43. Shroff, R., Rulísek, L., Doubsky, J., Svatos, A. Acid-base-driven matrix-assisted mass spectrometry for targeted metabolomics. Proc. Nat. Acad. Sci. U.S.A. 106, 10092-10096 (2009).
  44. Eikel, D., et al. Liquid extraction surface analysis mass spectrometry (LESA-MS) as a novel profiling tool for drug distribution and metabolism analysis: the terfenadine example. Rapid Comm. Mass Spectrom. 25, 3587-3596 (2011).
  45. Sadeghi, M., Vertes, A. Crystallite size dependence of volatilization in matrix-assisted laser desorption ionization. Appl. Surf. Sci. 127 – 129, 226-234 (1998).
  46. O’Connor, P. B., Costello, C. E. Internal Calibration on Adjacent Samples (InCAS) with Fourier Transform Mass Spectrometry. Anal. Chem. 72, 5881-5885 (2000).
  47. Jing, L., Amster, I. J. An improved calibration method for the matrix-assisted laser desorption/ionization-Fourier transform ion cyclotron resononance analysis of 15N-metabolically- labeled proteome digests using a mass difference approach. Eur. J. Mass Spectrom. 18, 269-277 (2012).
  48. Zhang, L. -. K., Rempel, D., Pramanik, B. N., Gross, M. L. Accurate mass measurements by Fourier transform mass spectrometry. Mass Spectrom. Rev. 24, 286-309 (2005).
  49. Clemis, E. J., et al. Quantitation of spatially-localized proteins in tissue samples using MALDI-MRM imaging. Anal. Chem. 84, 3514-3522 (2012).
  50. Schwamborn, K., Caprioli, R. M. Molecular imaging by mass spectrometry–looking beyond classical histology. Nat. Rev. Cancer. 10, 639-646 (2010).
  51. Oppenheimer, S. R., Mi, D., Sanders, M. E., Caprioli, R. M. Molecular analysis of tumor margins by MALDI mass spectrometry in renal carcinoma. J. Proteome Res. 9, 2182-2190 (2010).

Tags

MALDI ImagingDithranol MatrixFTICR Mass SpectrometryLow-molecular Weight CompoundsEndogenous Lipids
check_url/50733?article_type=t

Play Video
PDF
DOI
DOWNLOAD MATERIALS LIST
Cite This Article
Le, C. H., Han, J., Borchers, C. H. Dithranol as a Matrix for Matrix Assisted Laser Desorption/Ionization Imaging on a Fourier Transform Ion Cyclotron Resonance Mass Spectrometer. J. Vis. Exp. (81), e50733, doi:10.3791/50733 (2013).
Download Citation
Reprints and Permissions

View Video

✖

To prove you're not a robot, please enter the text in the image below

CAPTCHA Image
Play CAPTCHA Audio
Refresh Image