Liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometric characterization of protein kinase C phosphorylation
Liquid chromatography-Fourier transform ion cyclotron resonance mass spectrometric characterization of protein kinase C phosphorylation
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DOI：
10.1021/pr030004d
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发表时间：
2003-07-01
影响因子：
4.4
通讯作者：
Marshall, AG
中科院分区：
文献类型：
--
作者：
Chalmers, MJ;Quinn, JP;Marshall, AG
描述了一种用于磷酸肽鉴定的通风柱、毛细管液相色谱 (LC) 微电喷雾电离 (ESI) 傅里叶变换离子回旋共振 (FT-ICR (9.4 T)) 质谱 (MS) 方法。构建了能够在两个独立控制的 ESI 发射器之间快速（大约 200 ms）切换的双 ESI 源。双 ESI 源与线性八极离子阱中的外部离子积累相结合，可以在 LC 过程中对每个质谱进行内部校准。蛋白激酶 C (PKC) 的 LC ESI FT-ICR 正离子 IVIS 显示了四种先前未鉴定的磷酸化肽（一种位于 PKCalpha 内，一种位于 PKCdelta 内，两种位于 PKCzeta 内）。内部校准将 LC IVIS 谱图的质量准确度从绝对平均值（47 个肽离子）的 11.5 ppm 提高到 1.5 ppm。通过随后的负离子直接输注纳米电喷雾观察到另外五个（八个已知的）PKC 磷酸化激活位点，在正离子实验中未检测到。该方法的扩展使得在每次其他质量测量之前能够对 ICR 细胞中的所有离子进行红外多光子解离，揭示了磷酸化肽离子中 H3PO4 的诊断中性损失。精确质量 MS 和 MS/MS 的结合提供了一种强大的新工具，用于识别肽中磷酸化的存在和位点，而无需额外的湿化学衍生化。
A vented column, capillary liquid chromatography (LC) microelectrospray ionization (ESI) Fourier transform ion cyclotron resonance (FT-ICR (9.4 T)) mass spectrometry (MS) approach to phosphopeptide identification is described. A dual-ESI source capable of rapid (similar to200 ms) switching between two independently controlled ESI emitters was constructed. The dual-ESI source, combined with external ion accumulation in a linear octopole ion trap, allowed for internal calibration of every mass spectrum during LC. LC ESI FT-ICR positive-ion IVIS of protein kinase C (PKC) revealed four previously unidentified phosphorylated peptides (one within PKCalpha, one within PKCdelta, and two within PKCzeta). Internal calibration improved the mass accuracy for LC IVIS spectra from an absolute mean (47 peptide ions) of 11.5 ppm to 1.5 ppm. Five additional (out of eight known) activating sites of PKC phosphorylation, not detected in positive-ion experiments, were observed by subsequent negative-ion direct infusion nanoelectrospray. Extension of the method to enable infrared multiphoton dissociation of all ions in the ICR cell prior to every other mass measurement revealed the diagnostic neutral loss of H3PO4 from phosphorylated peptide ions. The combination of accurate-mass MS and MS/MS offers a powerful new tool for identifying the presence and site(s) of phosphorylation in peptides, without the need for additional wet chemical derivatization.