Peptide Retention Standards and Hydrophobicity Indexes in Reversed-Phase High-Performance Liquid Chromatography of Peptides
Peptide Retention Standards and Hydrophobicity Indexes in Reversed-Phase High-Performance Liquid Chromatography of Peptides
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DOI：
10.1021/ac9016693
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发表时间：
2009-11-15
影响因子：
7.4
通讯作者：
Spicer, Vic
中科院分区：
文献类型：
--
作者：
Krokhin, Oleg V.;Spicer, Vic
多肽保留预测在蛋白质组学中的应用日益增长，这将得益于开发一个通用的多肽保留标准，以便更好地对使用各种液相色谱(LC)平台获得的层析数据进行比对。我们描述了一种为此目的设计的六肽混合物；其成员涵盖了最流行的反相多肽高效液相色谱(HPLC)模式的广泛疏水性：以三氟乙酸/甲酸为离子对修饰剂的C18吸附剂和在pH 10下的分离。我们提出了一个疏水性指数(HI)来描述有机溶剂(通常是乙腈)的浓度，在等度洗脱条件下，对任何多肽的保留因子为10。这种测量是多肽-吸附剂相互作用的基本特征，仅取决于所使用的吸附剂的类型和离子配对修饰剂。在样品中加入标准多肽混合物，可在梯度分离过程中测量所有被检测物种的HI值。除了使用多肽保留预测方案时的数据对齐和色谱运行校准外，这些从蛋白质组学实验中获得的值还可以直接用于大规模制备性分离的方法开发。
The growing utility of peptide retention prediction in proteomics would benefit from the development of a universal peptide retention standard for better alignment of chromatographic data obtained using various liquid chromatography (LC) platforms. We describe a six-peptide mixture designed for this purpose; its members cover a wide range of hydrophobicity for the most popular modes of reversed-phase peptide high-performance liquid chromatography (HPLC): C18 sorbents with trifluoroacetic/formic acid as the ion-pairing modifier and separations at pH 10. We propose a hydrophobicity index (HI) describing the concentration of organic solvent (typically acetonitrile) that yields a retention factor of 10 under isocratic elution conditions for any peptide. This measure is a fundamental characteristic of peptide-sorbent interaction, depending only on the type of sorbent and the ion-pairing modifier used. Spiking a sample with a standard peptide mixture provides a measurement of the HI values of all detected species during gradient separation. In addition to alignment of data and calibration of chromatographic runs when peptide retention prediction protocols are used, these values obtained from proteomics experiments can be utilized directly in method development for large-scale preparative separations.