Phosphoproteome Analysis of Rat L6 Myotubes Using Reversed-Phase C18 Prefractionation and Titanium Dioxide Enrichment
Phosphoproteome Analysis of Rat L6 Myotubes Using Reversed-Phase C18 Prefractionation and Titanium Dioxide Enrichment
复制标题
使用反相 C18 预分级和二氧化钛富集对大鼠 L6 肌管进行磷酸化蛋白质组分析
DOI：
10.1021/pr900646k
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发表时间：
2010-02-01
影响因子：
4.4
通讯作者：
Yang, Fuquan
中科院分区：
文献类型：
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作者：
Hou, Junjie;Cui, Ziyou;Yang, Fuquan
大鼠L6肌管是研究骨骼肌信号通路的重要体外模型系统。探索骨骼肌中涉及的磷酸化事件对于阐明激酶-底物关系，理解参与信号通路的调节机制以及提供对许多细胞过程的见解非常重要。在这里，我们使用基于质谱的蛋白质组学对大鼠L6肌管进行全球磷蛋白质组学分析。采用高效的磷酸化蛋白质组学策略，包括用自包装RP C18柱预分离色氨酸混合物，用TiO2富集磷酸肽，以及2D-LC (SCX/RP)-MS/MS分析，从1195个蛋白质中鉴定出2230个独特的磷酸肽，使用目标/诱饵数据库搜索策略，错误发现率低于1.0%。在确定磷酸化位点位置的确定性程度（评分值>= 19）后，使用motif - x算法从我们的数据集中获得了11个Ser基序和1个Thr基序。在肌管磷酸化蛋白组中发现了几种潜在的信号通路，如MAPK信号通路和IGF-1/胰岛素信号通路。
The rat L6 myotubes is an important in vitro model system for studying signaling pathways in skeletal muscle. Exploring phosphorylation events involved in the skeletal muscle is very significant for elucidating the kinase-substrate relationship, understanding regulatory mechanisms involved in signaling pathways and providing insights into numerous cell processes. Here, we used mass spectrometry-based proteomics to conduct global phosphoproteome profiling of rat L6 myotubes. Using an efficient phosphoproteomic strategy including prefractionation of tryptic peptide mixtures with self-packed RP C18 columns, phosphopeptide enrichment with TiO2, and 2D-LC (SCX/RP)-MS/MS analysis, a total of 2230 unique phosphopeptides from 1195 proteins were identified with a false-discovery rate of less than 1.0% using a target/decoy database searching strategy. After determining the degree of certainty of the phosphorylation site location (Ascore value >= 19), 11 Ser motifs and one Thr motif were derived from our data set using the Motif-X algorithm. Several potential signaling pathways were found in our myotubes phosphoproteome, such as the MAPK signaling pathway and the IGF-1/Insulin signaling pathway.