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首页> 外文期刊>Analytical Sciences >Titania as a Chemo-affinity Support for the Column-switching HPLC Analysis of Phosphopeptides: Application to the Characterization of Phosphorylation Sites in Proteins by Combination with Protease Digestion and Electrospray Ionization Mass Spectrometry
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Titania as a Chemo-affinity Support for the Column-switching HPLC Analysis of Phosphopeptides: Application to the Characterization of Phosphorylation Sites in Proteins by Combination with Protease Digestion and Electrospray Ionization Mass Spectrometry

机译：二氧化钛作为磷酸肽的柱切换HPLC分析的化学亲和性支持物：通过与蛋白酶消化和电喷雾电离质谱联用在蛋白质的磷酸化位点表征中的应用

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摘要

A method for the determination of phosphorylation sites in phosphoproteins based on column-switching high-performance liquid chromatography (HPLC) has been developed. The HPLC system consisted of a titania precolumn for the selective adsorption of phosphopeptides, an anion-exchange analytical column and a UV detector (215 nm). Rabbit muscle phosphorylase a (RPa) and porcine stomach pepsin (PSP) were tested as model phosphoproteins. After protease digestion, the resulting phosphopeptides were successfully isolated by column-switching HPLC. The phosphopeptide fractions were analyzed by electrospray ionization mass spectrometry with a positive or negative ion mode after purification by reversed-phase HPLC. Pseudo-molecular ion peaks corresponding to Gln-Ile-Ser(p)-Val-Arg (MW 681.7) and Glu-Ala-Thr-Ser(p)-Gln-Glu-Leu (MW 856.8) were detected from the tryptic digest of RPa and chymotryptic digest of PSP, respectively, which agreed with the theoretically expected phosphopeptide fragments.
机译：已经开发了一种基于柱切换高效液相色谱法（HPLC）的磷蛋白中磷酸化位点的测定方法。 HPLC系统由用于选择性吸附磷酸肽的二氧化钛预柱，阴离子交换分析柱和UV检测器（215 nm）组成。测试了兔肌肉磷酸化酶a（RPa）和猪胃胃蛋白酶（PSP）作为模型磷蛋白。蛋白酶消化后，通过柱切换HPLC成功分离了所得的磷酸肽。通过反相HPLC纯化后，通过电喷雾电离质谱以正或负离子模式分析磷酸肽级分。从胰蛋白酶消化物中检测到对应于Gln-Ile-Ser（p）-Val-Arg（MW 681.7）和Glu-Ala-Thr-Ser（p）-Gln-Glu-Leu（MW 856.8）的伪分子离子峰PSP的RPa和胰凝乳蛋白酶消化产物分别与理论上预期的磷酸肽片段一致。

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