Subcellular Microdissection for the Identification of Organelle Proteins

显微病理学定义了许多人类疾病，但大多数组织学观察到的亚细胞结构的化学组成在很大程度上仍未表征。目前，没有一种方法可以在不破坏细胞的情况下分离出感兴趣的亚细胞区域，从而扭曲自然发生的蛋白质相互作用。我们建议结合原位标记方案和随后的显微解剖与质谱直接分析亚细胞复合物和细胞器。为此，我们正在改进表达微解剖（xMD），以便使用与质谱分析兼容的优化方案分离光学暗靶（化学，抗体或金属基染色），以允许无监督的热塑性捕获亚细胞结构。与激光捕获显微解剖相反，xMD中的薄膜不含染料，因此热塑性熔化和粘附所需的热量来自染色的细胞器。xMD与显微方法兼容，避免组织均质化。将xMD技术与随后的液相色谱耦合纳米喷雾质谱（LC-MS/MS）相结合，为鉴定亚细胞成分的组成和表征其在细胞中的生理功能提供了一种新颖而独特的分析方法。对于xMD，将一层由0.5-2微米热熔胶（EVA）组成的透明复合薄膜放在柔性衬底（12.5微米厚的PET）上，置于免疫组织化学染色的福尔马林固定石蜡包埋或冷冻大鼠脑组织（5-10微米厚）的顶部。EVA层是由含有不同百分比（4 - 10% w/v） EVA的甲苯溶液旋转镀膜制成的，而柔性底膜则利用毛细力暂时固定在刚性玻璃基板上。全光谱氙灯在载玻片上的光吸收产生的热量熔化了染色区域的薄膜，并提供了对目标的选择性粘附。当膜被提起时，目标染色细胞器被捕获用于随后的质谱分析。值得注意的是，捕获的分辨率不依赖于系统的光学分辨率，而是由染色的空间范围和辐照组件的热性能决定的。采用自制的xMD样机，从10微米的福尔马林固定石蜡包埋的大鼠脑组织中解剖60- 80%的神经元核。用组蛋白免疫组化染色或苏木精化学染色的组织取得了同样的成功。核转移到EVA膜上的选择性取决于多个可控变量，但一致的结果表明，xMD方法在无监督模式下分离亚细胞器是可行的。转移细胞核的扫描电镜图像显示，有内质网碎片随部分细胞核转移。我们测试了多种溶剂溶解和消化方法来分析dab过氧化物酶免疫组织化学染色蛋白。三氟乙醇-碳酸氢铵膜上胰蛋白酶消化方案被确定为最成功的微解剖样品。在纳米喷雾-高分辨率轨道阱仪器上采用LC-MS/MS对所得色氨酸进行分析。通过自动搜索（Mascot）将MS/MS光谱与肽序列匹配，然后通过mass筛和人工检测对细胞核中的蛋白质进行精简排序。为了进行比较，采用常规方法（蔗糖密度梯度方案）从新鲜大鼠脑组织中分离细胞核。新鲜的、未染色的细胞核的可测量浓度的结果表明，在膜上捕获的蛋白质的数量相当于每平方约150纳克。从这个数量中，350个蛋白质可以在常规分离的细胞核中用两个或更多的肽鉴定，288个蛋白质来自血红素染色、福尔马林固定石蜡包埋的组织，对应于229个蛋白质的重叠（xMD集的80%）。xMD分离的肽在精氨酸和赖氨酸c -末端富集20%，可能是固定过程中甲酰化导致赖氨酸被阻断的结果。xMD样品中某些特征性核蛋白的缺失可能是同样现象的结果。基因本体隔室分析表明，xMD方法产生的核蛋白百分比与传统的分离方法相当。计划定量测量细胞核与其他细胞器的蛋白质特征。综上所述，免疫组织化学和化学引导下的无监督xMD联合LC-MS/MS在DAB和血红素染色的大鼠脑神经元核上的实验证明了其重复性和可行性，并且核转移的特异性较好。多个捕获膜可以使用改进的甲醇-氯仿蛋白质沉淀步骤组合，以提高肽的回收率。肽的恢复足以用于随后的质谱蛋白质组学。数据表明，利用xMD进行亚细胞细胞器富集和随后的蛋白质组学分析是可以实现的。

Microscopic pathology defines many human diseases, but the chemical composition of most histologically observed subcellular structures remains largely uncharacterized. Currently, there is no method for isolating subcellular areas of interest without disrupting the cells and thus distorting naturally occurring protein interactions. We propose to combine in situ labeling schemes and subsequent microdissection with mass spectrometry to directly analyze subcellular complexes and organelles. To this end, we are improving expression microdissection (xMD) in order to isolate optically dark targets (chemical, antibody, or metal-based stains) using optimized protocols compatible with mass spectrometric analysis to allow unsupervised thermoplastic capture of subcellular structures. In contrast to Laser Capture Microdissection, the film in xMD does not contain a dye and consequently the heat required for thermoplastic melting and adhesion is derived from the stained organelle. xMD is compatible with microscopic methodologies and avoids tissue homogenization. Combining the xMD technique with subsequent liquid chromatography coupled nano-spray mass spectrometry (LC-MS/MS) provides a novel and unique analytical method to identify the composition of subcellular components and characterize their physiological functions in cells. For xMD, a clear composite thin film consisting of 0.5-2 microns of a hot-melt adhesive (EVA) on a flexible substrate (12.5 micron thick PET) is placed on the top of immunohistochemically stained formalin-fixed paraffin- embedded or frozen rat brain tissue(5-10 micron thick). The EVA layer was made by spin-coating from toluene solutions with varying percentages (4 to 10% w/v) of EVA while the flexible backing film was temporarily held in place on a rigid glass substrate using capillary forces. The heat generated by optical absorption of a full spectrum xenon lamp over the slide melts the film in stained areas and provides selective adhesion to the targets. When the film is lifted, the targeted stained organelles are captured for subsequent mass spectrometric analysis. Significantly, the resolution for capture is not dependent on the optical resolution of the system, but is instead determined by the spatial extent of the stain and the thermal properties of the irradiated assembly. An in-house prototype xMD instrument was used to dissect 60-80 % of neuronal nuclei from formalin fixed paraffin embedded rat brain tissue (10 micron). Equal success was achieved with either histone-immunohisto- chemical- or hematoxylin-chemical stained tissues. The selectivity of transfer of nuclei onto the EVA film was dependent upon multiple controllable variables, but consistent results demonstrated that the xMD method is feasible for isolation of subcellular organelles in an unsupervised mode. Scanning electron microscopic images of the transferred nuclei suggest that there are accompanying fragments of endoplasmic reticulum transferred with some of the nuclei. We tested multiple solvent solubilization and digestion methods for the analysis of DAB-peroxidase immunohistochemically stained proteins. A trifluoroethanol-ammonium bicarbonate on-film trypsin digestion protocol was determined to be most successful for microdissected samples. The resulting tryptic peptides were analyzed using LC-MS/MS on nanospray-high resolution orbitrap instruments. MS/MS spectra were matched to peptide sequences using automated searching (Mascot), and then a parsimonious list of proteins in nuclei assigned using MassSieve and manual inspection. For comparison purposes, nuclei were isolated from fresh rat brain tissue using conventional methods (sucrose density gradient protocol). Results from measurable concentrations of fresh, unstained nuclei indicate that the amount of protein captured on-film corresponds to approximately 150 nanograms per sq. cm. From that quantity, 350 proteins could be identified with two or more peptides in the conventionally isolated nuclei, and 288 proteins from the hemotoxylin stained, formalin fixed paraffin embedded tissue, corresponding to an overlap of 229 proteins (80% of the xMD set). The xMD isolated peptides were 20% enriched in arginine vs. lysine C-termini, probably a result of blocked lysines due to formylation during fixation. The absence of certain characteristic nuclear proteins in the xMD sample may be the result of the same phenomenon. Gene ontology compartment analysis shows that the xMD method yielded a comparable percentage of nuclear proteins as the conventional fractionation method. Quantitative measures of proteins characteristic of nuclei vs. other organelles are planned. In summary, immunohistochemical and chemical guided unsupervised xMD combined with LC-MS/MS has proven to be reproducible and feasible by experiments on DAB- and hemotoxylin stained neuronal nuclei of rat brain, and the specificity of nuclear transfer is good. Multiple capture films can be combined using a modified methanol-chloroform protein precipitation step to improve peptide recovery. The recovery of peptides is sufficient for subsequent mass spectrometric proteomics. Data suggests subcellular organelle enrichment using xMD with subsequent proteomic analysis is achievable.