Toward Analysis of Proteins in Single Cells: A Quantitative Approach Employing Isobaric Tags with MALDI Mass Spectrometry Realized with a Microfluidic Platform
Toward Analysis of Proteins in Single Cells: A Quantitative Approach Employing Isobaric Tags with MALDI Mass Spectrometry Realized with a Microfluidic Platform
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DOI：
10.1021/acs.analchem.5b03419
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发表时间：
2016-07-05
影响因子：
7.4
通讯作者：
Ros, Alexandra
中科院分区：
文献类型：
--
作者：
Yang, Mian;Nelson, Randall;Ros, Alexandra
单个细胞中的蛋白质鉴定和定量对于理解生物过程至关重要，例如参与细胞凋亡、癌症、生物标志物发现、疾病诊断、病理学或治疗的生物过程。与目前的单细胞基因组分析相比，探测单细胞的蛋白质含量由于缺乏蛋白质扩增技术而受到阻碍。在这里，我们报告了一种定量质谱方法与微流控技术相结合的发展，达到了单细胞中高丰度蛋白质的检测灵敏度。开发了具有一系列腔室和阀的微流体平台，确保了一组限定的威尔斯孔用于目标蛋白质的绝对定量，并将其与同位素标记策略结合，所述同位素标记策略采用同量异位标签用于相对和绝对定量（iTRAQ）标记。为此，我们将iTRAQ标记适应于芯片上协议。在微流控平台上同时进行的蛋白质消化和标记使得标记策略与芯片上所有必要的操作步骤兼容，包括用于MALDI-TOF分析的基质递送。我们用凋亡相关蛋白Bcl-2证明了这种方法，并定量评估了检测到的Bcl-2分子的数量。我们预计，这种方法最终将允许在单细胞水平上定量蛋白质表达。
Protein identification and quantification in individual cells is essential to understand biological processes such as those involved in cell apoptosis, cancer, biomarker discovery, disease diagnostics, pathology, or therapy. Compared with present single cell genome analysis, probing the protein content of single cells has been hampered by the lack of a protein amplification technique. Here, we report the development of a quantitative mass spectrometric approach combined with microfluidic technology reaching the detection sensitivity of high abundant proteins in single cells. A microfluidic platform with a series of chambers and valves, ensuring a set of defined wells for absolute quantification of targeted proteins, was developed and combined with isotopic labeling strategies employing isobaric tags for relative and absolute quantitation (iTRAQ)-labels. To this aim, we adapted iTRAQ labeling to an on-chip protocol. Simultaneous protein digestion and labeling performed on the microfluidic platform rendered the labeling strategy compatible with all necessary manipulation steps on-chip, including the matrix delivery for MALDI-TOF analysis. We demonstrate this approach with the apoptosis related protein Bcl-2 and quantitatively assess the number of Bcl-2 molecules detected. We anticipate that this approach will eventually allow quantification of protein expression on the single cell level.