Ahmadzadeh Hossein, Johnson Ryan D, Thompson LaDora, Arriaga Edgar A
Department of Chemistry, University of Minnesota, 207 Pleasant Street SE, Minneapolis, Minnesota 55455, USA.
Anal Chem. 2004 Jan 15;76(2):315-21. doi: 10.1021/ac034809g.
Muscle is a highly heterogeneous tissue. Practical approaches to sample selectively small regions of muscle cross sections would help to effectively utilize analytical techniques on muscle studies while taking into account tissue heterogeneity. In this report, semimembranosus muscle tissue cross sections were directly sampled and analyzed by capillary electrophoresis (CE) with laser-induced fluorescence detection (LIF). Prior to CE-LIF analysis, a small region in the muscle cross section was stained with 10-nonyl acridine orange (NAO) which is a mitochondrion-selective fluorescent probe known to form a stable complex with cardiolipin, a phospholipid found only in mitochondria. By micromanipulation, the injection end of the capillary was brought into contact with the tissue exhibiting fluorescently labeled mitochondria. Sampling from a region similar in size to the cross section of a single fiber was carried out by applying 11 kPa of negative pressure for 3 s. When an electric field of -200V/cm was applied, fluorescently labeled mitochondria electromigrated and were individually detected by postcolumn LIF detection. For each sample, the electropherogram displays a migration time window with a collection of narrow peaks. The collection of individual peak measurements is represented as a distribution of individual intensities related to cardiolipin content of mitochondria and a distribution of individual electrophoretic mobilities. Positioning the capillary injection end was sufficiently spatially accurate to deplete mitochondria in the sampled region upon repetitive injections. Treatment of a muscle cross section with a protease (trypsin) prior to mitochondria sampling resulted in a higher number of detected mitochondria, suggesting that one of the effects of this enzyme is a partial digestion of the muscles myofibrils, which eases the release of interfibrillar mitochondria entangled within these fibers. The protease treatment also resulted in changes to the electrophoretic mobility distribution of individual mitochondria, which may imply that partial digestion of proteins bound to the mitochondria contributes to the alteration in the electrophoretic mobility of mitochondria. The ability to sample a region as small as a single muscle fiber cross section and its direct CE-LIF analysis opens exciting possibilities for the direct analysis of muscle biopsies and mapping the mitochondrial electrophoretic properties in highly heterogeneous tissues.
肌肉是一种高度异质性的组织。在考虑组织异质性的同时，选择性地对肌肉横截面的小区域进行采样的实用方法将有助于在肌肉研究中有效利用分析技术。在本报告中，采用激光诱导荧光检测（LIF）的毛细管电泳（CE）对半膜肌组织横截面进行直接采样和分析。在进行CE-LIF分析之前，用10-壬基吖啶橙（NAO）对肌肉横截面中的一个小区域进行染色，NAO是一种线粒体选择性荧光探针，已知它能与心磷脂形成稳定的复合物，心磷脂是一种仅存在于线粒体中的磷脂。通过显微操作，使毛细管的进样端与显示有荧光标记线粒体的组织接触。通过施加11 kPa的负压持续3 s，从与单根纤维横截面大小相似的区域进行采样。当施加-200V/cm的电场时，荧光标记的线粒体发生电迁移，并通过柱后LIF检测进行逐个检测。对于每个样品，电泳图显示出一个迁移时间窗口以及一系列窄峰。单个峰测量值的集合表示为与线粒体心磷脂含量相关的单个强度分布以及单个电泳迁移率分布。毛细管进样端的定位在空间上足够精确，以便在重复进样时耗尽采样区域内的线粒体。在线粒体采样之前用蛋白酶（胰蛋白酶）处理肌肉横截面会导致检测到的线粒体数量增加，这表明该酶的作用之一是对肌肉肌原纤维进行部分消化，从而使缠绕在这些纤维内的肌原纤维间线粒体更容易释放出来。蛋白酶处理还导致单个线粒体的电泳迁移率分布发生变化，这可能意味着与线粒体结合的蛋白质的部分消化导致了线粒体电泳迁移率的改变。能够对小至单根肌肉纤维横截面的区域进行采样并对其进行直接的CE-LIF分析，为直接分析肌肉活检样本以及绘制高度异质性组织中线粒体的电泳特性开辟了令人兴奋的可能性。
