Villén Judit, Gygi Steven P
Department of Cell Biology, Harvard Medical School, Boston, MA 02115, USA.
Nat Protoc. 2008;3(10):1630-8. doi: 10.1038/nprot.2008.150.
The success in profiling the phosphoproteome by mass spectrometry-based proteomics has been intimately related to the availability of methods that selectively enrich for phosphopeptides. To this end, we describe a protocol that combines two sequential enrichment steps. First, strong cation exchange (SCX) chromatography separates peptides by solution charge. Phosphate groups contribute to solution charge by adding a negative charge at pH 2.7. Therefore, at that pH, phosphopeptides are expected to elute earlier than their nonphosphorylated homologs. Second, immobilized metal affinity chromatography (IMAC) takes advantage of phosphate's affinity for metal ions such as Fe(3+) to uniformly enrich for phosphopeptides from the previously collected SCX fractions. We have successfully employed the SCX/IMAC enrichment strategy in the exploration of phosphoproteomes from several systems including mouse liver and Drosophila embryos characterizing over 5,500 and 13,000 phosphorylation events, respectively. The SCX/IMAC enrichment protocol requires 2 days, and the entire procedure from cells to a phosphorylation data set can be completed in less than 10 days.
通过基于质谱的蛋白质组学对磷酸化蛋白质组进行分析的成功，与选择性富集磷酸肽的方法的可用性密切相关。为此，我们描述了一种结合两个连续富集步骤的方案。首先，强阳离子交换（SCX）色谱通过溶液电荷分离肽段。磷酸基团通过在pH 2.7时添加负电荷来影响溶液电荷。因此，在该pH值下，磷酸肽预计比其未磷酸化的同源物更早洗脱。其次，固定化金属亲和色谱（IMAC）利用磷酸盐对金属离子（如Fe(3+)）的亲和力，从先前收集的SCX馏分中均匀富集磷酸肽。我们已成功将SCX/IMAC富集策略应用于多个系统的磷酸化蛋白质组研究，包括小鼠肝脏和果蝇胚胎，分别鉴定出超过5500个和13000个磷酸化事件。SCX/IMAC富集方案需要2天时间，从细胞到磷酸化数据集的整个过程可以在不到10天内完成。
