目的: 建立了一种同时测定硫酸鱼精蛋白中主成分纯度和含量的HPLC方法,并采用LC-MS法对肽段氨基酸序列进行鉴定。方法: HPLC方法采用的色谱柱为ThermoHypersil GOLD色谱柱(250 mm×4.6 mm,5 μm),流动相A为0.1 mol·L^-1磷酸二氢钠溶液(用磷酸调节pH至1.8),流动相B为乙腈-0.1 mol·L^-1磷酸二氢钠溶液(用磷酸调节pH至1.8)(6.5∶93.5),梯度洗脱,流速1.0 mL·min^-1,柱温55 ℃,紫外检测波长为214 nm,进样量100 μL。LC-MS法先采用馏分收集方式对各肽段进行提取富集,然后液质联用采用Waters ACQUITY UPLC Peptide BEH C₁₈色谱柱(15 cm×0.21 cm,1.7 μm),流速为0.2 mL·min^-1,流动相A为0.1%甲酸溶液,流动相B为5%乙腈-0.1%甲酸溶液,梯度洗脱;Thermo Q-Exactive Plus,ESI离子源,阳离子检测模式,雾化电压为3 800 V,离子传输管温度为300 ℃,扫描范围m/z 200~2 000。结果: 建立的HPLC法对2家企业提供的鲑科和鲱科来源的硫酸鱼精蛋白进行有效鉴别,鲑科来源硫酸鱼精蛋白含量测定结果为94.4%~96.2%,鲱科来源硫酸鱼精蛋白含量测定结果为88.4%~92.3%;鲑科来源硫酸鱼精蛋白纯度测定结果均为96%,鲱科来源硫酸鱼精蛋白纯度测定结果为66%~69%。在Uniprot蛋白数据库检索并获取鱼精蛋白目标肽段的氨基酸序列,采用ThermoBioPharmaFinder v3.0软件对图谱碎片离子峰和目标序列理论碎片离子进行匹配,得到鲑科来源4条主要肽段和鲱科来源3条主要肽段的氨基酸序列鉴定结果。结论: 本文建立的HPLC方法可用于硫酸鱼精蛋白的鉴别、含量测定和纯度分析,LC-MS法可对鲑科和鲱科来源的硫酸鱼精蛋白肽段序列进行鉴定。

Objective: To establish a high performance liquid chromatography (HPLC) method for the simultaneous determination of principal components in protamine sulfate, and to employ LC-MS for the identification for the amino acids sequences of main peptides. Methods: The ThermoHypersil GOLD column (250 mm×4.6 mm, 5 μm) was selected for analysis with a gradient program of mobile phase A[0.1 mol·L^-1 sodium dihydrogen phosphate solution(adjusted to pH 1.8 with phosphoric acid)]and B[acetonitrile-0.1 mol·L^-1 sodium dihydrogen phosphate solution(adjusted to pH 1.8 with phosphoric acid) (6.5∶93.5, v/v)].The column temperature was 55 ℃. The UV detection wavelength was 214 nm and the flow rate was 1.0 mL·min^-1. The injection volume was 100 μL. The LC-MS method adopted distillate collection method to extract and enrich each peptide, Waters ACQUITY UPLC Peptide BEH C₁₈ column (15 cm×0.21 cm, 1.7 μm) was selected for liquid-mass spectrometry method, flow rate was 0.2 mL·min^-1, the mobile phase A was 0.1% formic acid solution, mobile phase B was 5% acetonitrile-0.1% formic acid solution, gradient elution. Thermo Q-Exactive Plus, ESI ion source, cation detection mode, atomization voltage was 3 800 V, ion transfer tube temperature was 300 ℃, scanning range of m/z 200-2 000. Results: The HPLC method established in this paper could identify the protamine sulfate from salmon and herring provided by the two companies. The content of protamine sulfate was 94.4%-96.2% from salmon, and 88.4%-92.3% from herring, protamine sulfate purity determination result was 96% from salmon, and 66%-69% from herring. The amino acid sequence of the target peptide of protamine was retrieved and obtained from Uniprot protein database, and analyzed by ThermoBioPharmaFinder v3.0 software to map fragments ion peaks were matched with theoretical fragment ions of the target sequence, and the amino acid sequence identification results of 4 main peptides from salmon and 3 main peptides from herring were obtained. Conclusion: The method established in this paper can be used for the identification, content determination and purity analysis of protamine sulfate. The LC-MS method can be used for the identification of protamine sulfatepeptide sequences from salmon and herring.