Alkaline liquid chromatography/electrospray ionization skimmer collision-induced dissociation mass spectrometry for phosphopeptide screening.
Alkaline liquid chromatography/electrospray ionization skimmer collision-induced dissociation mass spectrometry for phosphopeptide screening.
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用于磷酸肽筛选的碱性液相色谱/电喷雾电离撇渣器碰撞诱导解离质谱法。
DOI：
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发表时间：
2001
影响因子：
2
通讯作者：
R. Lehmann
中科院分区：
文献类型：
--
作者：
A. Beck;M. Deeg;K. Moeschel;E. Schmidt;E. Schleicher;W. Voelter;H. Häring;R. Lehmann
本文描述了一种利用特异标记离子信号对酶解蛋白中磷酸化肽进行快速在线鉴定的方法。在我们的研究中，我们研究了使用碱性条件以及先前描述的结合高效毛细管液相色谱（LC）和电喷雾电离质谱（ESI-MS）的选择性和敏感检测磷酸肽离子的方法。磷酸化特异性标记离子(m/z 79， PO(3)（-）和m/z 97， H(2)PO（4)(-)）在负离子模式下由撇子碰撞诱导解离（sCID）产生。在不同的碱性pH值和CID偏移量下，对该方法进行了评价和验证。碱性条件（pH 10.5）促进丝氨酸和酪氨酸磷酸化肽产生磷酸化肽特异性片段离子，并使m/z 79 (PO（3)(-)）和m/z 97 (H(2)PO（4)(-)）成为磷酸化特异性标记痕迹成为可能。请注意，在含三氟乙酸的溶剂中高效液相色谱分离会损害m/z 97 (m/z 97处的C(2)F（3)O（-）片段离子）作为磷酸化特异性标记物的使用。将优化后的方法应用于胰蛋白酶酪蛋白消化液中磷酸化肽的检测。通过（mu）LC/ESI-sCID-MS和（mu）LC/ESI-MS检测和快速鉴定预期的单磷酸化肽和四磷酸化肽。
A rapid on-line method for the identification of phosphorylated peptides in enzymatic protein digests by specific marker ion signals is described. In our study we investigated the use of alkaline conditions together with a previously described method for selective and sensitive detection of phosphopeptide ions combining high-performance capillary liquid chromatography (LC) and electrospray ionization mass spectrometry (ESI-MS). Phosphorylation-specific marker ions (m/z 79, PO(3)(-), and m/z 97, H(2)PO(4)(-)) were generated by skimmer collision-induced dissociation (sCID) in the negative-ion mode. The method was evaluated and validated for mono-phosphorylated synthetic peptides using different alkaline pH values and CID offsets. Alkaline conditions (pH 10.5) enhance the generation of phosphopeptide-specific fragment ions from serine- and tyrosine-phosphorylated peptides, and enable the use of m/z 79 (PO(3)(-)) and m/z 97 (H(2)PO(4)(-)) as phosphorylation-specific marker traces. Note that HPLC separation in trifluoroacetic acid containing solvents impairs the use of m/z 97 (C(2)F(3)O(-) fragment ion at m/z 97) as a phosphorylation-specific marker. The optimized method was applied for the detection of phosphorylated peptides in a tryptic beta-casein digest. The expected mono- and tetra-phosphorylated peptides were detected and rapidly identified by (mu)LC/ESI-sCID-MS and (mu)LC/ESI-MS analysis.
影响因子：
3.7
作者：
Katze, MG;Kwieciszewski, B;Aebersold, R
通讯作者：
Aebersold, R