Anion-exchange chromatography of phosphopeptides: weak anion exchange versus strong anion exchange and anion-exchange chromatography versus electrostatic repulsion-hydrophilic interaction chromatography.
Anion-exchange chromatography of phosphopeptides: weak anion exchange versus strong anion exchange and anion-exchange chromatography versus electrostatic repulsion-hydrophilic interaction chromatography.
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DOI：
10.1021/ac504420c
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发表时间：
2015
影响因子：
7.4
通讯作者：
Mechtler, Karl
中科院分区：
文献类型：
--
作者：
Alpert, Andrew J.;Hudecz, Otto;Mechtler, Karl
大多数磷酸蛋白质组学实验依赖于在在线液相色谱-质谱分析之前对胰酶消化进行预分馏。本研究比较了静电斥力-亲水作用层析(ERLIC)和阴离子交换层析(AEX)的潜力和局限性。当pH高于5时，磷酸肽的每个残基有两个负电荷，并且在AEX中保持良好。然而，含有一个或两个磷酸基团的多肽不能从含有多个天冬氨酸或谷氨酸残基的多肽中分离出来，这干扰了磷酸多肽的鉴定。在pH值为2时，磷酸残基只有一个负电荷，而天冬氨酸和谷氨酸不带电荷。这有利于从未修饰的酸性多肽中分离出磷酸肽。在这些条件下，除非亲水作用以ERLIC模式叠加，否则由于静电斥力，单一的磷酸化肽将被微弱地保留。将弱阴离子交换(WAX)和强阴离子交换(SAX)柱与两种多肽标准和HeLa细胞胰酶消化进行了比较。SAX柱在pH=6时的保留率高于蜡柱。然而，在pH为6时，SAX检测到的磷酸肽仅为ERLIC在pH 2时的60%左右。在一次ERLIC操作中，共鉴定出12个 467磷酸肽，其中4233个含有多个磷酸盐。我们得出结论，在ERLIC模式下，在低pH条件下进行磷酸肽的层析是最好的。在此条件下，SAX和蜡质材料的性能相当。数据已保存在标识符为PXD001333的ProteomeXchange中。
Most phosphoproteomics experiments rely on prefractionation of tryptic digests before online liquid chromatography-mass spectrometry. This study compares the potential and limitations of electrostatic repulsion–hydrophilic interaction chromatography (ERLIC) and anion-exchange chromatography (AEX). At a pH higher than 5, phosphopeptides have two negative charges per residue and are well-retained in AEX. However, peptides with one or two phosphate groups are not separated from peptides with multiple Asp or Glu residues, interfering with the identification of phosphopeptides. At a pH of 2, phosphate residues have just a single negative charge but Asp and Glu are uncharged. This facilitates the separation of phosphopeptides from unmodified acidic peptides. Singly phosphorylated peptides are retained weakly under these conditions, due to electrostatic repulsion, unless hydrophilic interaction is superimposed in the ERLIC mode. Weak anion-exchange (WAX) and strong anion-exchange (SAX) columns were compared, with both peptide standards and a HeLa cell tryptic digest. The SAX column exhibited greater retention at pH 6 than did the WAX column. However, only about 60% as many phosphopeptides were identified with SAX at pH 6 than via ERLIC at pH 2. In one ERLIC run, 12 467 phosphopeptides were identified, including 4233 with more than one phosphate. We conclude that chromatography of phosphopeptides is best performed at low pH in the ERLIC mode. Under those conditions, the performances of the SAX and WAX materials were comparable. The data have been deposited with the ProteomeXchange with identifier PXD001333.
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影响因子：
4.4
作者：
Chien, Ko-yi;Liu, Hsiao-Ching;Goshe, Michael B.
通讯作者：
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影响因子：
7.4
作者：
Alpert, Andrew J.
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DOI：
10.1016/j.jchromb.2010.11.004
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影响因子：
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作者：
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