Microscale Phosphoproteome Analysis of 10 000 Cells from Human Cancer Cell Lines
Microscale Phosphoproteome Analysis of 10 000 Cells from Human Cancer Cell Lines
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DOI：
10.1021/ac201093g
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发表时间：
2011-10-15
影响因子：
7.4
通讯作者：
Ishihama, Yasushi
中科院分区：
文献类型：
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作者：
Masuda, Takeshi;Sugiyama, Naoyuki;Ishihama, Yasushi
我们开发了一种小型化的LC-MS系统，该系统具有高回收率的磷酸多肽浓缩方案，可以对10(4)个细胞进行磷酸蛋白质组分析。在浓缩方案中，关键步骤是在缓冲液中加入脱氧胆酸钠和月桂酰肌氨酸钠，用于蛋白质提取和消化，并省略在磷酸肽浓缩之前的任何后续脱盐/脱表面活性剂步骤。通过羟基酸修饰的金属氧化物层析(HAMMOC)富集的磷肽直接进样到小型液晶柱上，使用氮气压力驱动池，而不是切换阀式进样器。直径为25微米的微型分析柱的灵敏度比传统的100微米直径的柱提高了3.6倍。总体而言，我们的分析系统在LC-MS响应方面提供了大约80倍的平均改进，我们基于对10(4)个HeLa细胞(大约1微克蛋白质)的一次分析，基于995个独特的磷酸化多肽确定了1011个独特的磷酸化位点。这是迄今为止报道的用于哺乳动物细胞体内磷酸化的蛋白质组范围分析的最灵敏的磷酸蛋白质组学系统。
We developed a miniaturized LC-MS system with a high-recovery phosphopeptide enrichment protocol that allows phosphoproteome analysis of 10(4) cells. In the enrichment protocol, the key step is to add sodium deoxycholate and sodium lauroyl sarcosinate to the buffer solution for protein extraction and digestion and to omit any subsequent desalt/desurfactant step before phosphopeptide enrichment. The phosphopeptides enriched by hydroxy acid-modified metal oxide chromatography (HAMMOC) are directly injected onto a miniaturized LC column using a nitrogen-pressure-driven cell, instead of switching valve-type injectors. The miniaturized analytical column of 25 mu m diameter provided a 3.6-fold improvement in sensitivity over the conventional 100 mu m diameter column. Overall, our analytical system provided approximately 80-fold improvement on average in the LC-MS response, and we identified 1011 unique phosphorylated sites based on 995 unique phosphopeptides from a single analysis of 10(4) HeLa cells (approximately 1 mu g of proteins). This is the most sensitive phosphoproteomics system that has so far been reported for proteome-wide analysis of in vivo phosphorylation in mammalian cells.