Quantitative mitochondrial phosphoproteomics using iTRAQ on an LTQ-Orbitrap with high energy collision dissociation.
Quantitative mitochondrial phosphoproteomics using iTRAQ on an LTQ-Orbitrap with high energy collision dissociation.
复制标题
在具有高能碰撞解离的 LTQ-Orbitrap 上使用 iTRAQ 进行定量线粒体磷酸蛋白质组学。
DOI：
10.1021/pr900387b
复制
发表时间：
2009-10
影响因子：
4.4
通讯作者：
Balaban, Robert S.
中科院分区：
文献类型：
--
作者：
Boja, Emily S.;Phillips, Darci;French, Stephanie A.;Harris, Robert A.;Balaban, Robert S.
在具有 HCD 功能的 LTQ-Orbitrap 上使用 iTRAQ 标记和质谱分析，我们评估了生理扰动时线粒体中蛋白质磷酸化的相对变化。作为参考反应，我们通过丙酮酸脱氢酶激酶/丙酮酸脱氢酶磷酸酶响应二氯乙酸、断电和 Ca2+ 的磷酸化/去磷酸化来监测丙酮酸脱氢酶 (PDH) 活性的充分表征的调节。对猪心脏 PDH-E1α 亚基磷酸肽的相对定量揭示了三个丝氨酸位点（Ser231、Ser292 和 Ser299）的去磷酸化。如之前报道的，Ser292（即抑制位点）的 DCA 去磷酸化与 PDH 活性的激活相关，这与我们的去能数据一致。钙还可使 PDH 去磷酸化（即激活），从而证实钙对 PDP 的激活。通过这种方法，我们成功监测了线粒体蛋白的其他磷酸化位点，包括腺嘌呤核苷酸转位酶、苹果酸脱氢酶和线粒体肌酸激酶等。其中四种蛋白在这些生理刺激下表现出磷酸化变化：（1）BCKDH-E1α亚基用DCA增加了Ser337的磷酸化， 断电； (2)钙离子作用使凋亡诱导因子Ser345位点磷酸化升高； (3) ATP 合酶 F1 复合体 α 亚基和 (4) 丝裂素在断电后在 Ser65 和 Ser264 处去磷酸化。该筛选验证了 iTRAQ/HCD 技术作为线粒体蛋白磷酸化功能定量的方法，并提供了通过磷酸化调节线粒体的见解。
Using iTRAQ labeling and mass spectrometry on an LTQ-Orbitrap with HCD capability, we assessed relative changes in protein phosphorylation in the mitochondria upon physiological perturbation. As a reference reaction, we monitored the well-characterized regulation of pyruvate dehydrogenase (PDH) activity via phosphorylation/dephosphorylation by pyruvate dehydrogenase kinase/pyruvate dehydrogenase phosphatase in response to dichloroacetate, de-energization and Ca2+. Relative quantification of phosphopeptides of PDH-E1α subunit from porcine heart revealed dephosphorylation at three serine sites (Ser231, Ser292 and Ser299). Dephosphorylation at Ser292 (i.e., the inhibitory site) with DCA correlated with an activation of PDH activity as previously reported, consistent with our de-energization data. Calcium also dephosphorylated (i.e., activated) PDH thus confirming calcium activation of PDP. With this approach, we successfully monitored other phosphorylation sites of mitochondrial proteins including adenine nucleotide translocase, malate dehydrogenase and mitochondrial creatine kinase, etc. Among them four proteins exhibited phosphorylation changes with these physiological stimuli: (1) BCKDH-E1α subunit increased phosphorylation at Ser337 with DCA and de-energization; (2) apoptosis-inducing factor phosphorylation was elevated at Ser345 with calcium; (3) ATP synthase F1 complex α subunit and (4) mitofilin dephosphorylated at Ser65 and Ser264 upon de-energization. This screening validated the iTRAQ/HCD technology as a method for functional quantitation of mitochondrial protein phosphorylation as well as providing insights into the regulation of mitochondria via phosphorylation.
登录
查看更多内容
影响因子：
10.8
作者：
Feng, Jianhua;Zhu, Min;Zaugg, Michael
通讯作者：
Zaugg, Michael
影响因子：
4.8
作者：
Schieke, Stefan M.;Phillips, Darci;Finkel, Toren
通讯作者：
Finkel, Toren
影响因子：
7
作者：
Ross, PL;Huang, YLN;Pappin, DJ
通讯作者：
Pappin, DJ
影响因子：
4.4
作者：
Aponte, Angel M.;Phillips, Darci;Hopper, Rachel K.;Johnson, D. Thor;Harris, Robert A.;Blinova, Ksenia;Boja, Emily S.;French, Stephanie;Balaban, Robert S.
通讯作者：
Balaban, Robert S.
影响因子：
7
作者：
Collins, Mark O.;Yu, Lu;Choudhary, Jyoti S.
通讯作者：
Choudhary, Jyoti S.