Analysis of phosphorylation sites on proteins from Saccharomyces cerevisiae by electron transfer dissociation (ETD) mass spectrometry
Analysis of phosphorylation sites on proteins from Saccharomyces cerevisiae by electron transfer dissociation (ETD) mass spectrometry
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DOI：
10.1073/pnas.0607084104
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发表时间：
2007-02-13
影响因子：
11.1
通讯作者：
Hunt, Donald F.
中科院分区：
文献类型：
--
作者：
Chi, An;Huttenhower, Curtis;Hunt, Donald F.
我们提出了一种分析酵母磷酸蛋白质组的策略，该策略使用内切赖氨酸 C 作为蛋白水解酶，用于磷酸肽富集的固定金属亲和色谱，用于磷酸肽分级和检测的 90 分钟纳流 HPLC/电喷雾电离 MS/MS 实验，气相离子/离子化学，用于肽断裂的电子转移解离， 以及用于磷蛋白鉴定和磷酸化位点分配的开放质谱搜索算法。从 30 微克（约 600 pmol）酵母总蛋白样品中，我们鉴定出了 629 种蛋白质上的 1,252 个磷酸化位点。已鉴定的磷蛋白的表达水平范围为每个细胞 < 50 至 1,200,000 个拷贝，并由参与多种细胞过程的基因编码。我们确定了一个可能代表一种或多种未表征激酶的基序的共有位点，并表明酵母激酶本身含有不成比例的大量磷酸化位点。检测到含有来自酵母蛋白 Cdc10 的肽的 pHs，表明组氨酸磷酸化在 septin 生物学中具有意想不到的作用。从不同的功能基因组学数据中，我们表明磷蛋白比普通蛋白质具有更多的相互作用，并且彼此之间的相互作用比与随机蛋白质的相互作用更多。它们也可能在较长的进化距离中得到保守。
We present a strategy for the analysis of the yeast phosphoproteome that uses endo-Lys C as the proteolytic enzyme, immobilized metal affinity chromatography for phosphopeptide enrichment, a 90-min nanoflow-HPLC/electrospray-ionization MS/MS experiment for phosphopeptide fractionation and detection, gas phase ion/ion chemistry, electron transfer dissociation for peptide fragmentation, and the Open Mass Spectrometry Search Algorithm for phosphoprotein identification and assignment of phosphorylation sites. From a 30-mu g (approximate to 600 pmol) sample of total yeast protein, we identify 1,252 phosphorylation sites on 629 proteins. Identified phosphoproteins have expression levels that range from < 50 to 1,200,000 copies per cell and are encoded by genes involved in a wide variety of cellular processes. We identify a consensus site that likely represents a motif for one or more uncharacterized kinases and show that yeast kinases, themselves, contain a disproportionately large number of phosphorylation sites. Detection of a pHis containing peptide from the yeast protein, Cdc10, suggests an unexpected role for histidine phosphorylation in septin biology. From diverse functional genomics data, we show that phosphoproteins have a higher number of interactions than an average protein and interact with each other more than with a random protein. They are also likely to be conserved across large evolutionary distances.