Mass spectrometry-based methods for phosphorylation site mapping of hyperphosphorylated proteins applied to Net1, a regulator of exit from mitosis in yeast
Mass spectrometry-based methods for phosphorylation site mapping of hyperphosphorylated proteins applied to Net1, a regulator of exit from mitosis in yeast
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DOI：
10.1074/mcp.m100032-mcp200
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发表时间：
2002-03-01
影响因子：
7
通讯作者：
Carr, SA
中科院分区：
文献类型：
--
作者：
Chen, SL;Huddleston, MJ;Carr, SA
在酿酒酵母的后期之前，CDC14蛋白磷酸酶被隔离在核仁中，并被萌芽酵母中RREE复合体的一种成分Net1抑制。在后期，Rate复合体解体，允许CDC14迁移到细胞核和细胞质，在那里它催化退出有丝分裂。CDC14的释放机制似乎涉及到Polo-like激酶CDC5，它能够通过Net1的磷酸化在体外促进重组Net1(.)CDC14复合体的解离。我们报道了19个丝氨酸或苏氨酸突变为丙氨酸的重组Net1(Net1N)和突变的Net1N等位基因(Net1N-19M)的磷酸化位点定位。使用了各种基于色谱和质谱学的策略，包括固定化金属亲和层析、碱性磷酸酶处理、基质辅助激光解吸后源衰变以及基于多维电喷雾质谱学的方法。没有一种方法能够识别这些蛋白质胰酶消化中的所有磷酸肽。最值得注意的是，磷酸化残基附近的碱性残基的存在显著阻碍了碱性磷酸酶对磷酸部分的水解能力。蛋白质组学研究的一个主要目标是确定所有蛋白质及其相互作用和翻译后修饰状态。然而，任何单一方法都无法识别高度磷酸化的Net1N中的所有位点，这引发了人们对使用现有技术绘制整个蛋白质组中的磷酸化位点图的可行性的严重担忧。
Prior to anaphase in Saccharomyces cerevisiae, Cdc14 protein phosphatase is sequestered within the nucleolus and inhibited by Net1, a component of the RENT complex in budding yeast. During anaphase the RENT complex disassembles, allowing Cdc14 to migrate to the nucleus and cytoplasm where it catalyzes exit from mitosis. The mechanism of Cdc14 release appears to involve the polo-like kinase Cdc5, which is capable of promoting the dissociation of a recombinant Net1(.)Cdc14 complex in vitro by phosphorylation of Net1. We report here the phosphorylation site mapping of recombinant Net1 (Net1N) and a mutant Net1N allele (Net1N-19m) with 19 serines or threonines mutated to alanine. A variety of chromatographic and mass spectrometric-based strategies were used, including immobilized metal-affinity chromatography, alkaline phosphatase treatment, matrix-assisted laser-desorption post-source decay, and a multidimensional electrospray mass spectrometry-based approach. No one approach was able to identify all phosphopeptides in the tryptic digests of these proteins. Most notably, the presence of a basic residue near the phosphorylated residue significantly hampered the ability of alkaline phosphatase to hydrolyze the phosphate moiety. A major goal of research in proteomics is to identify all proteins and their interactions and post-translational modification states. The failure of any single method to identify all sites in highly phosphorylated Net1N, however, raises significant concerns about how feasible it is to map phosphorylation sites throughout the proteome using existing technologies.