Academy of Integrative Medicine, Fujian University of Traditional Chinese Medicine, Fuzhou, Fujian, PR China.
Electrophoresis. 2010 Oct;31(20):3400-6. doi: 10.1002/elps.201000194.
In this article, an approach has been developed for the analysis of some small peptides with similar pI values by CE-ESI-MS based on the online concentration strategy of dynamic pH junction. The factors affected on the separation, detection and online enrichment, such as BGE, injection pressure, sheath flow liquid and separation voltage have been investigated in detail. Under the optimum conditions, i.e. using 0.5 mol/L formic acid (pH 2.15) as the BGE, preparing the sample in 50 mM ammonium acetate solution (pH 7.5), 50 mbar of injection pressure for 300 s, using 7.5 mM of acetic acid in methanol-water (80% v/v) solution as the sheath flow liquid and 20 kV as the separation voltage, four peptides with similar pI values, such as L-Ala-L-Ala (pI = 5.57), L-Leu-D-Leu (pI = 5.52), Gly-D-Phe (pI = 5.52) and Gly-Gly-L-Leu (pI = 5.52) achieved baseline separation within 18.3 min with detection limits in the range of 0.2-2.0 nmol/L. RSDs of peak migration time and peak area were in the range of 1.45-3.57 and 4.93-6.32%, respectively. This method has been applied to the analysis of the four peptides in the spiked urine sample with satisfactory results.
本文提出了一种基于动态 pH 连接的在线浓缩策略，用于分析等电点（pI）相近的小肽的毛细管电泳-电喷雾质谱（CE-ESI-MS）分析方法。详细考察了影响分离、检测和在线富集的因素，如背景电解质（BGE）、进样压力、鞘流液和分离电压等。在最优实验条件下，采用 0.5 mol/L 甲酸（pH 2.15）作为 BGE，50 mM 乙酸铵溶液（pH 7.5）作为样品制备液，进样压力 50 mbar 持续 300 s，7.5 mM 乙酸的甲醇-水（80% v/v）溶液作为鞘流液，分离电压 20 kV，实现了四种 pI 值相近的小肽（L-Ala-L-Ala，pI = 5.57；L-Leu-D-Leu，pI = 5.52；Gly-D-Phe，pI = 5.52；Gly-Gly-L-Leu，pI = 5.52）的基线分离，检测限在 0.2-2.0 nmol/L 范围内。峰迁移时间和峰面积的相对标准偏差（RSD）分别在 1.45%-3.57%和 4.93%-6.32%范围内。该方法已成功应用于加标尿液样品中四种小肽的分析，结果令人满意。
