本文利用反相高效制备液相色谱对酪蛋白磷酸肽（CPPs）进行初步分离，根据色谱图峰形收集得到4个组分，用分析型反相高效液相色谱进一步分离纯化组分F2，得到亚组分P3，低温减压浓缩回收溶剂，冷冻干燥得到CPPs单体P3粉末。通过已知CPPs单体P3的氨基酸序列人工固相化学合成CPPs单体P3，经质谱和高效液相色谱鉴定，合成CPPs单体P3的分子量与天然单体分子量一致且纯度为98.13%。天然与合成CPPs单体P3的液相保留时间一致，相同浓度条件下合成单体的紫外吸收显著高于天然单体，可能与空间结构差异有关。经红外光谱和圆二色光谱分析，天然与合成CPPs单体P3均含有α-螺旋、β-转角、β-折叠和无规卷曲特征结构，主要以β-折叠-反平行式和无规卷曲形式存在，但不同二级结构形式比例存在差异。天然与合成CPPs单体P3的α-螺旋、β-转角和无规则卷曲比例存在显著差异。天然与合成CPPs单体P3浓度从2 mg/mL上升到4 mg/mL时，天然单体P3不同形式二级结构变化较小，主要由α-螺旋和无规则卷曲向β-折叠-反平行式转变，合成单体P3不同形式二级结构变化显著，主要由α-螺旋和β-转角向β-折叠-反平行式和无规则卷曲转变。

In this paper, the preliminary purification of casein phosphopeptides(CPPs) was investigated using preparative reverse phase high performance liquid chromatography. Four components were collected according to the peak shape of the chromatogram. The F2 component was further separated and purified by analytical RP-HPLC to obtain sub-component P3 The casein phosphopeptides active monomers P3 powder was collected by reduced pressure distillation and lyophilization to remove the solvent. The casein phosphopeptide monomers P3 were synthetically synthesized based on the amino acid sequence of P3. The molecular weight of synthesized P3 was uniform with the natural monomer and the puritywas 98.13%, which was determined by Mass Spectrometry and High Performance Liquid Chromatography. The retention time of the natural and synthetic casein phosphopeptides monomer P3 was consistent, and the ultraviolet absorption of the synthesized monomer was much higher than that of the natural monomer, which could be related to the diversity of protein tertiary structure. There were α-helix, β-turn, β-sheet and random coil in the secondary structure of the natural and synthetic CPPs monomer P3 according to the analysis of Fourier Transform Infrared Spectroscopy (FT-IR) and circular dichroism. The synthesized and natural monomers were mainly in the form of antiparallel and random coil, but there were difference in the ratio of the α-helix, β-turn and random curl of natural and synthetic CPPs monomer P3. The second structural change of natural monomer P3 changed small with the concentration of monomers from 2 mg/mL up to 4 mg/mL, which the α-helix and random coil were transformed to the antiparallel. The second structural of synthesized monomer P3 had a significant variation, which mainly transformed the α-helix and β-turn to the antiparallel and random coil.

国家自然科学基金资助项目（31601474）；广东省自然科学基金项目（2016A030310442）；广东省教育厅科研项目（平台）（2013gjhz0003）