酪蛋白多肽的制备和色谱分离方法

Preparation and high performance liquid chromatographic separation of casein peptides

为了得到低成本的多肽,本文利用胰蛋白酶对酪蛋白进行了充分的酶解。采用分析级反相高效液相色谱-电喷雾质谱联用技术(RP-HPLC/ESI-MS)分析了酶解产物各组分的组成,并通过改变流动相的梯度洗脱程序,优化了分析级色谱条件以充分分离相对含量较高的多肽组分;将优化的分析级色谱条件直接放大到制备级RP-HPLC中,在程序控制下通过紫外吸收信号结合ESI-MS信号共同引导实现了多肽的全自动化分离和收集。整个过程方便快捷,经过这样一个单一的分离步骤,得到了多个纯度较高的多肽。除此之外,本文还考察了流动相的酸碱性、柱上样量等因素对该体系制备级分离的影响,并对一次分离中分辨率不好的亲水性多肽混合物进行了二次分离,得到了多个新的多肽。本文建立的多肽制备方法为多肽和多肽材料的广泛应用提供了一种选择。

A preparative reversed-phase high performance liquid chromatography-electrospray ionization mass spectrometry （RP-HPLC/ESI-MS） method to obtain low cost casein peptides was developed.Casein was hydrolyzed by trypsin and the hydrolysate was analyzed by RP-HPLC/ESI-MS firstly.The gradient elution program in an analytical column was optimized to achieve a better separation.The effect of pH of the mobile phase on the separation was also investigated.Then,the optimal analytical conditions were applied in a preparative column directly.In each loading of the hydrolysate,several pure peptide fractions were collected automatically by the inductions of ultraviolet absorbance and mass spectrometry signals together.The process is simple and effective.The influence of the loading amount in a preparative column was investigated intensively.For the collection consisting of hydrophilic peptides that were poorly separated in the primary process,secondary separation was performed.By changing the gradient elution,several different pure peptides were obtained.