A proteomic approach to identify phosphoproteins encoded by cDNA libraries.
A proteomic approach to identify phosphoproteins encoded by cDNA libraries.
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一种鉴定 cDNA 文库编码的磷蛋白的蛋白质组学方法。
DOI：
10.1016/j.ab.2004.03.033
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发表时间：
2004
期刊：
影响因子：
--
通讯作者：
Miller,DavidJ
中科院分区：
文献类型：
--
作者：
Shi,Xudong;BeltonJr,RobertJ;Burkin,HeatherR;Vieira,AnaP;Miller,DavidJ
我们报道了一种将固定化金属亲和层析（IMAC）与噬菌体展示cDNA文库筛选相结合的大规模快速分析组织或细胞中磷酸化蛋白的方法。我们将睾丸cDNA文库表达为噬菌体上的融合蛋白，并使用IMAC富集编码磷酸化蛋白的序列。选择的克隆进行聚合酶链反应扩增和测序。大部分被测序的克隆（80%）编码已知的蛋白质，以前被鉴定为磷蛋白。磷酸化酪氨酸抗体的免疫印迹证实，在噬菌体上表达时，一些选定的序列编码酪氨酸磷酸化蛋白。该方法的一个优点是可以快速识别由cDNA文库编码的磷酸化蛋白，该文库可以识别体内可能磷酸化的蛋白。当这种方法与有限的酶消化和串联质谱技术相结合时，可以确定蛋白质中的特定磷酸化位点。该技术可用于蛋白质组学研究，有效地检测磷酸化蛋白，避免耗时和昂贵的肽测序。
We report a method for large-scale rapid analysis of phosphoproteins in tissues or cells by combining immobilized metal affinity chromatography (IMAC) with phage display cDNA library screening. We expressed a testis cDNA library as fusion proteins on phage and, using IMAC, enriched for sequences encoding phosphoproteins. Selected clones were polymerase chain reaction amplified and sequenced. The majority of the clones sequenced (80%) encoded known proteins previously identified as phosphoproteins. Immunoblotting with phosphotyrosine antibodies confirmed that some of the selected sequences encoded tyrosine phosphorylated proteins when expressed on phage. An advantage of this method is the rapid identification of phosphoproteins encoded by a cDNA library, which can identify proteins that are potentially phosphorylated in vivo. When this method is combined with limited enzymatic digestion and tandem mass spectrometric techniques, the specific phosphorylation site in a protein can be identified. This technique can be used in proteomics studies to effectively detect phosphorylated proteins and avoid time-consuming and expensive peptide sequencing.
噬菌体 T7 的蛋白激酶仅诱导受感染细胞中少量蛋白质的磷酸化。
影响因子：
3.7
作者：
E. S. Robertson;Allen W. Nicholson
通讯作者：
Allen W. Nicholson
DOI：
10.1006/bbrc.2001.6275
发表时间：
2002-01-25
影响因子：
3.1
作者：
Conrads, TP;Issaq, HJ;Veenstra, TD
通讯作者：
Veenstra, TD
铁(III)离子亲和色谱模型研究。
影响因子：
4.1
作者：
G. Muszyǹska;G. Dobrowolska;A. Medin;P. Ekman;J. Porath
通讯作者：
J. Porath