健壮phosphoproteomic酪氨酸磷酸化位点的分析人类T细胞使用固定化金属亲和色谱法和串联质谱分析。
文章的细节
-
引用
复制到剪贴板 -
布里尔LM,所罗门AR、Ficarro某人Mukherji M,彼得斯Stettler-Gill M, EC
健壮phosphoproteomic酪氨酸磷酸化位点的分析人类T细胞使用固定化金属亲和色谱法和串联质谱分析。
肛门化学。2004年5月15日,76 (10):2763 - 72。
- PubMed ID
-
15144186 (在PubMed]
- 文摘
-
蛋白质酪氨酸磷酸化途径很难分析和高等真核生物细胞信号至关重要。方法分析酪氨酸磷酸化,认为这里是多个蛋白质酪氨酸磷酸化位点单一的任务分析,近日报道(a·r·所罗门;Ficarro s b;布里尔,l . m .;Brinker, a;问:t Phung;Ericson c;萨奥尔,k;布洛克,a;角,d . m .; Schultz, P. G.; Peters, E. C. Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 443-448). The technology platform included the use of immunoprecipitation, immobilized metal affinity chromatography (IMAC), liquid chromatography, and tandem mass spectrometry. In the present report, we show that when using complex mixtures of peptides from human cells, methylation improved the selectivity of IMAC for phosphopeptides and eliminated the acidic bias that occurred with unmethylated peptides. The IMAC procedure was significantly improved by desalting methylated peptides, followed by gradient elution of the peptides to a larger IMAC column. These improvements resulted in assignment of approximately 3-fold more tyrosine phosphorylation sites, from human cell lysates, than the previous methodology. Nearly 70 tyrosine-phosphorylated peptides from proteins in human T cells were assigned in single analyses. These proteins had unknown functions or were associated with a plethora of fundamental cellular processes. This robust technology platform should be broadly applicable to profiling the dynamics of tyrosine phosphorylation.
DrugBank数据引用了这篇文章
- 多肽
-
的名字 UniProt ID t细胞表面糖蛋白CD3ε链 P07766 细节 Microtubule-associated蛋白1 P78559 细节 酪氨酸受体激酶zap - 70 P43403 细节 酪氨酸受体激酶ITK /啧啧 Q08881 细节 t细胞表面糖蛋白CD3三角洲链 P04234 细节 t细胞表面糖蛋白CD3ζ链 P20963 细节 Microtubule-associated蛋白4 P27816 细节 Receptor-type酪氨酸受体磷酸酶β P23467 细节 SRSF蛋白激酶2 P78362 细节 Afadin P55196 细节 碳酸氢钠转运蛋白3 Q9Y6M7 细节 断点集群地区蛋白质 P11274 细节 核仁的和coiled-body磷蛋白质1 Q14978 细节 TRAF2 NCK-interacting蛋白激酶 Q9UKE5 细节 丝氨酸/ threonine-protein激酶LATS1 O95835 细节 Receptor-interacting丝氨酸/ threonine-protein激酶1 Q13546 细节 1-phosphatidylinositol 4, 5-bisphosphate磷酸二酯酶gamma-2 P16885 细节