Imaging Mass Spectrometry for Oxidized Lipidomics in Acute Lung Injury

描述（由申请人提供）：虽然氧化脂肪酸和氧化磷脂是急性肺损伤（ALI）的关键信号分子（和/或生物标志物），但它们的异常多样性以及生物化学方法的局限性阻碍了它们的系统研究。我们最近描述了氧化脂组学的新方法，该方法结合了液相色谱-电喷雾电离-质谱（LC-ESI- MS）的使用。我们现在建议开发一种新的氧化脂质组学成像质谱（IMS）技术，该技术将解决肺区域脂质过氧化反应的空间限制问题，并最终接近单个肺细胞的极限。因此，本研究的总体目标是：建立氧化脂质组学的IMS，并确定氧化、氧化和非氧化磷脂——特别是阴离子心磷脂（CL）和磷脂酰丝氨酸（PS）——在氧化剂（生成h2o2的葡萄糖氧化酶）介导的ALI后完整小鼠肺部的空间分布。为此，我们将采用多种方法来提高分辨率：a)矩阵辅助激光解吸电离(MALDI)-IMS-TOF-TOF(~50mm)；b)傅里叶变换离子回旋共振质谱的高质量分辨率和测量精度（~20mm分辨率，Bruker Daltonics SolariX, Billerica， MA）；c)结合maldi -电离后迁移率-正交飞行时间质谱（~10mm Ionwerks, Inc, NIH NIDA）和过采样-步进（<10mm）的纳米尺度矩阵的高空间分辨能力。连续切片将用于：a)通过LC-ESI-MS定量测量氧化脂质组学；b)荧光免疫组织化学，将与IMS光谱共同注册，以揭示ALI患者肺的结构和功能。线粒体靶向抗氧化剂的使用将作为这项技术的验证，并将这种成像技术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），它将：a)解决肺部过氧化的空间限制问题，否则无法轻易检查；B)同时提供数百个信号的全景快照；c)提供关于关键磷脂（CL和PS）氧化的信息，这些磷脂是细胞死亡（凋亡）和炎症（凋亡细胞清除）的早期信号分子；d)在代谢组学模式中用于揭示ALI中小分子保护剂配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.