Analysis of single mammalian cell lysates by mass spectrometry
Analysis of single mammalian cell lysates by mass spectrometry
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DOI：
10.1021/ja9627499
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发表时间：
1996-11-20
影响因子：
15
通讯作者：
Whittal, RM
中科院分区：
文献类型：
--
作者：
Li, L;Golding, RE;Whittal, RM
单细胞分析可以在个体基础上提供对细胞的详细化学性质的洞察，这允许探索细胞的异质性及其与其整体生物功能的关系。1-4然而，由于“样品”体积小，分析物的痕量，和复杂的细胞基质，单细胞的化学分析仍然是艰巨的。微柱分离方法的最新进展，如毛细管电泳（CE）和开管液相色谱，随着小型化和高灵敏度检测方案的到来，导致了在单细胞中检测细胞组分的方式。5-7其中，电化学和荧光检测系统提供了最灵敏的方法，虽然从这些技术推导出的化学身份的信息往往是非常有限的。另一方面，质谱法（MS）可以提供细胞水平的化学分析，具有无与伦比的化学特异性。实际上，存在关于单个大生物细胞中的细胞组分的分析的少数报告。8还报告了使用电喷雾电离质谱结合CE对来自小群体（即5-20个细胞）的人红细胞的细胞蛋白进行分析。9然而，对单个小体积细胞的分析并不成功。分析这些小细胞的主要障碍在于质谱方法可以提供的有限灵敏度以及用于将细胞呈递到质谱仪的特殊样品处理技术的要求。我们提出了一种高灵敏度检测小体积样品的质谱方法。显示了来自总细胞体积为87 fL的单个红细胞的细胞蛋白的分析。分析这种微小细胞的成功表明，这种方法也适用于其他重要哺乳动物细胞的分析。我们注意到，在最近的一次会议上，我们介绍了这项工作，Hofstadler等人。描述了最近的进展，包括检测血红蛋白从单个红细胞的CE MS。10我们的方法涉及使用基质辅助激光解吸电离（MALDI）11从细胞组分产生离子。在常规操作中，选择合适的基质与分析物以> 500：1的比例混合。通常使用微量移液管将约1 μL溶液混合物置于直径约2 mm的样品探针上。单细胞分析的成功在很大程度上归功于一种新的微点样品沉积方法的发展，而不是传统的1 μL样品输送方法。微点MALDI的思想是减少质谱仪中相对于激光解吸位点和离子接受体积的样品呈现表面，以提高进样效率。先前的报告描述了直接在样品探针上使用化学蚀刻或激光钻孔的皮升体积孔12，13以及将小体积样品沉积到预涂有基质的纤维素膜上14，这些报告显示出提高的灵敏度。在我们的方法中，如图1所示，用于样品输送的熔融石英毛细管和辅助毛细管连接到可移动的xy载物台并连接到一次性注射器。使用甲硅烷基化流体使这些毛细管失活以减少样品吸附。在样品加载过程中，将样品瓶放置在样品保持器上，并移动xy载物台以使毛细管接触样品溶液。然后在显微镜观察下使用注射器抽取样品的测量体积，该体积由毛细管中样品塞的长度确定。
Single-cell analysis can provide an insight to the detailed chemistry of a cell on individual bases, which allows exploration of the heterogeneity of the cells and the relationship to their overall biological functionality. 1-4 However, because of the small “sample” volume, trace quantity of analyte, and complex cellular matrix, the chemical analysis of single cells remains formidable. Recent advances in microcolumn separation methods, such as capillary electrophoresis (CE) and open-tubular liquid chromatography, with the arrival of miniaturized and highly sensitive detection schemes have led the way to detecting cellular components in single cells. 5-7 Among them, electrochemical and fluorescence detection systems provide the most sensitive approaches, although information on chemical identity deduced from these techniques is often very limited. On the other hand, mass spectrometry (MS) can potentially provide chemical analysis at the cellular level with the benefit of unsurpassed chemical specificity. Indeed, a few reports on the analysis of cellular components in single large biological cells exist. 8 The analysis of cellular proteins from small populations (ie, 5-20 cells) of human erythrocytes using electrospray ionization MS combined with CE is also reported. 9 However, the analysis of single small-volume cells has not been as successful. The major obstacles to analyzing these small cells lie in the limited sensitivity that a mass spectrometric method can provide and the requirement of special sample handling techniques for cell presentation to the mass spectrometer. We present a mass spectrometric approach for highly sensitive detection of small-volume samples. The analysis of cellular proteins from a single erythrocyte with a total cellular volume of 87 fL is shown. The success in analyzing such a tiny cell suggests that this method is applicable to the analysis of other important mammalian cells as well. We note that, in a recent conference where we presented this work, Hofstadler et al. described recent progress in CE MS including the detection of hemoglobin from single erythrocytes. 10 Our method involves the use of matrix-assisted laser desorption ionization (MALDI) 11 for the generation of ions from the cellular components. In conventional operation, a suitable matrix is chosen to mix with the analyte in a ratio of> 500: 1. About 1 μL of the solution mixture is typically placed on a sample probe of about 2 mm diameter using a micropipet. The success of single-cell analysis is largely attributed to the development of a novel microspot sample deposition method as opposed to the conventional 1 μL sample delivery method. The idea of microspot MALDI is to reduce the sample presentation surface with respect to the laser desorption site and ion acceptance volume in the mass spectrometer to improve the sampling efficiency. Previous reports describing the use of chemical etching or laser drilling of a picoliter volume hole directly on the sample probe12, 13 as well as depositing a small volume sample onto cellulose membranes precoated with matrix14 show improved sensitivity. In our approach, as shown in Figure 1, a fused silica capillary for sample delivery and an auxiliary capillary are attached to a movable xy stage and connected to a disposable syringe. These capillaries are deactivated using a silylation fluid to reduce sample adsorption. During sample loading, the sample vial is placed on a sample holder and the xy stage is moved to allow the capillary to contact the sample solution. A measured volume of a sample, which is determined by the length of the sample plug in the capillary, is then withdrawn using the syringe under microscopic observation …