Fast proteolytic digestion coupled with organelle enrichment for proteomic analysis of rat liver
Fast proteolytic digestion coupled with organelle enrichment for proteomic analysis of rat liver
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DOI：
10.1021/pr034110r
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发表时间：
2004-05-01
影响因子：
4.4
通讯作者：
Novotny, MV
中科院分区：
文献类型：
--
作者：
Arnold, RJ;Hrncirova, P;Novotny, MV
使用不稳定酸的表面活性剂作为尿素变性的替代方法，可以在同一天进行蛋白质分解消化，并快速清除细胞裂解产物样品。将大鼠肝组织匀浆分离成富含细胞核、线粒体、微粒体(剩余细胞器)和胞浆的四个组分。用不稳定酸的表面活性剂(ALS)变性后，用胰酶消化2 h，用纳米流反相高效液相色谱分离，然后在三维离子陷阱中进行串联质谱仪的质量分析。细胞器富集物和全细胞裂解物样品的ALS变性结果与相同样品经还原、烷基化和尿素变性处理的等量样品的结果相当。每种方法都得到了相似数量的多肽(尿素694个，肌萎缩侧索硬化症674个)和蛋白质(尿素225个，肌萎缩侧索硬化症229个)的鉴定，大致相同的蛋白质鉴定(47%重叠)。正如预期的那样，与整个细胞裂解液相比，细胞器浓缩能够鉴定更多的蛋白质(尿素多66%，肌萎缩侧索硬化症多60%)。随着细胞器的丰富，与整个细胞裂解液相比，尿素和ALS的序列覆盖率相等或增加的蛋白质数量分别增加了73%和67%。关于许多蛋白质的亚细胞位置的额外信息是通过细胞器浓缩获得的。虽然细胞器浓缩是用自下而上的蛋白质组学方法来证明的，但它应该很容易服从于自上而下的蛋白质组学方法。
The use of an acid-labile surfactant as an alternative to urea denaturation allows for same-day proteolytic digestion and fast cleanup of cellular lysate samples. Homogenized rat liver tissue was separated into four fractions enriched in nuclei, mitochondria, microsomes (remaining organelles), and cytosol. Each subcellular fraction was then subjected to proteolytic digestion with trypsin for 2 h after denaturing with an acid-labile surfactant (ALS), separated by nanoflow reversed phase HPLC, and mass analyzed by tandem mass spectrometry in a 3-D ion trap. The results obtained from ALS denaturation for both organelle enrichment and whole cell lysate samples were comparable to those obtained from aliquots of the same samples treated by reduction, alkylation, and urea denaturation. Each method resulted in a similar number of peptides (694 for urea, 674 for ALS) and proteins (225 for urea, 229 for ALS) identified, with generally the same proteins (47% overlap) identified. As expected, organelle enrichment enabled the identification of more proteins (66% more with urea, 60% more with ALS) compared to a whole cell lysate. With organelle enrichment, the number of proteins with equal or increased sequence coverage went up by 73% with urea and 67% with ALS compared to the whole cell lysate. Additional information regarding the subcellular location of many proteins is obtained by organelle enrichment. While organelle enrichment is demonstrated with a bottom-up proteomics approach, it should be easily amenable to top-down proteomics approaches.