Imaging Mass Spectrometry for Oxidized Lipidomics in Acute Lung Injury

描述（申请人提供）：尽管氧化脂肪酸和氧化磷脂是急性肺损伤（ALI）中的关键信号分子（和/或生物标志物），但它们异常的多样性沿着生化方法的局限性，阻碍了对它们的系统研究。我们最近描述了一种结合液相色谱-电喷雾电离-质谱（LC-ESI- MS）的氧化脂质组学新方法。我们现在建议开发一种新的使能技术的成像质谱（IMS）的氧化脂质组学，将解决的空间限制的问题，脂质的过氧化反应在肺的区域，并最终接近个人肺细胞的限制。因此，本提案的总体目标是：开发氧化脂质组学的IMS，并确定氧化剂（产生H2 O2的葡萄糖氧化酶）介导的ALI后完整小鼠肺中氧化、可氧化和不可氧化磷脂（特别是阴离子心磷脂（CL）和磷脂酰丝氨酸（PS））的空间分布。为此，我们将利用多种方法来提高分辨率：a）基质辅助激光解吸电离（MALDI）-IMS-TOF-TOF（~ 50 mm）; B）傅立叶变换离子回旋共振质谱的高质量分辨能力和测量精度（~ 20 mm分辨率，Bruker Daltonics SolariX，Billerica，MA）;和c）纳米尺度基质的高空间分辨能力与MALDI-后电离迁移率-正交飞行时间MS（~ 10 mm Ionwerks，Inc，NIH NIDA）和过采样-步进（<10 mm）相结合。连续切片将用于：a）通过LC-ESI-MS定量测量氧化脂质组学;和B）荧光免疫组织化学，其将与IMS光谱共同配准，以揭示ALI中肺的结构和功能。线粒体靶向抗氧化剂的使用将作为该技术的验证，并将这种成像的应用扩展到代谢组学和分子药理学。该提案将为氧化脂质组学建立一种新的使能技术（IMS），该技术将：a）解决肺中过氧化的空间限制问题，否则无法容易地检查; B）同时提供数百个信号的全景快照; c）提供关于关键磷脂氧化的信息（CL和PS）是细胞死亡的早期信号分子（凋亡）和炎症（凋亡细胞的清除）;和d）用于代谢组学模式以揭示关于小分子保护剂在ALI中的处置的分子药理学信息。

DESCRIPTION (provided by applicant): Although oxygenated fatty acids and oxidized phospholipids are critical signaling molecules (and/or biomarkers) in acute lung injury (ALI), their extraordinary diversity along with limitations in biochemical methodologies have prohibited their systematic study. We recently described new methodology of oxidative lipidomics that combines the use of liquid chromatography-electrospray ionization-mass spectrometry (LC-ESI- MS). We now propose to develop a new enabling technology of imaging mass spectrometry (IMS) for oxidative lipidomics that will resolve issues of spatial confinements of peroxidation reactions in lipids in regions of the lung and ultimately near limits of individual pulmonary cells. Accordingly, the overall aim of this proposal is to: develop IMS of oxidative lipidomics and define spatial distribution of oxidized, oxidizable and non-oxidizable phospholipids - particularly of anionic cardiolipin (CL) and phosphatidylserine (PS) - in lungs of intact mice after oxidant (H2O2-generating glucose oxidase)-mediated ALI. To this end, we will utilize multiple approaches of increasing power in resolution: a) Matrix assisted laser desorption ionization (MALDI)-IMS-TOF-TOF(~50mm); b) the high mass resolving power and measurement accuracy of Fourier Transform Ion Cyclotron Resonance MS (~20mm resolution, Bruker Daltonics SolariX, Billerica, MA); and c) high spatial resolving power of nano-scale matrices combined with MALDI-post-ionization mobility-orthogonal Time of Flight MS (~10mm Ionwerks, Inc, NIH NIDA) and oversampling-stepping (<10mm). Serial sections will be used for: a) quantitative measures of oxidative lipidomics via LC-ESI-MS; and b) fluorescence immunohistochemistry that will be co-registered with IMS spectra to reveal structure and function of lung in ALI. The use of mitochondrial targeted antioxidants will serve as a validation of this technology and expand application of such imaging to metabolomics and molecular pharmacology. This proposal will establish a new enabling technology (IMS) for oxidative lipidomics that will: a) resolve issues of spatial confinements of peroxidation in lung that cannot be readily examined otherwise; b) provide a panoramic snap-shot of hundreds of signals simultaneously; c) be informative about oxidation of critical phospholipids (CL and PS) that are early signaling molecules in cell death (apoptosis) and inflammation (clearance of apoptotic cells); and d) be used in a metabolomic mode to reveal molecular pharmacological information on disposition of small molecule protectants in ALI.