UHPLC-MS/MS法考察FGF21-164融合蛋白在小鼠体内的药代动力学

Pharmacokinetics of FGF21-164 fusion protein in mice using UHPLC-MS/MS method

FGF21-164是通过对内源性FGF21多肽的结构修饰、偶联得到的融合蛋白,拟用于治疗肥胖引起的糖脂代谢紊乱。本文通过Skyline软件预测得到该蛋白经胰蛋白酶酶解后的候选肽段质谱信息,采用高分辨质谱法验证预测的候选肽段。通过优化超高效液相色谱-串联质谱(UHPLC-MS/MS)法分析条件,选择具有最佳质谱响应的FGF21-164特征替代肽段(YLYTDDAQQTEAHLEIR)。血清样品经磷酸缓冲液稀释后, 60℃变性并烷基化,在37℃下与胰蛋白酶孵育2 h直接酶解产生替代肽段,该肽段在质谱ESI正离子模式检测下,产生特征离子对,即三电荷的母离子m/z 689.3和单电荷的子离子m/z 738.4。色谱分离使用含0.1%甲酸的水溶液(A相)和0.1%甲酸的乙腈溶液(B相),在EclipsePlus C18柱(2.1 mm×50 mm, 1.8μm)上进行,以多反应监测模式对上述离子对监测,以外标法定量分析小鼠血清中FGF21-164融合蛋白的浓度。该方法在2.50~500μg·mL^(-1)内呈良好的线性关系(r=0.998 8),定量下限为2.50μg·mL^(-1)。该方法成功应用于FGF21-164融合蛋白在小鼠体内的药代动力学研究。动物实验经中国科学院上海药物研究所实验动物伦理委员会(批号:20180004040450)批准。药动学实验结果表明,相比于内源性FGF21, FGF21-164融合蛋白在小鼠体内的t1/2由0.5 h延长至2.6 h,有望延长该蛋白的疗效。

FGF21-164 is a fusion protein obtained by structural modification and coupling of endogenous FGF21. It is a candidate drug used in the treatment of glucose and lipid metabolic disorders caused by obesity. In this study, the candidate peptide mass spectrometry information of the protein hydrolyzed by trypsin was predicted by Skyline software and verified by high resolution mass spectrometry. The specific surrogate peptide(YLYTDDAQQTEAHLEIR) with the best mass response was selected after optimizing ultra-high performance liquid chromatography-tandem mass spectrometry(UHPLC-MS/MS). Under ESI positive ion mode, the parent ion m/z689.3 with 3 charge and the product ion m/z 738.4 with single charge can be monitored. After dilution by PBS, the serum samples were denatured under 60 ℃ and alkylated to reduce the matrix effect, then incubated with trypsin at 37 ℃ for 2 h, to obtain the surrogate peptide. The chromatographic separation was carried out on an EclipsePlus C18 column(2.1 mm×50 mm, 1.8 μm) using aqueous solution containing 0.1% formic acid(phase A) and acetonitrile solution containing 0.1% formic acid(phase B). Finally, the concentration of FGF21-164 fusion protein in mouse serum was quantitatively analyzed by external standard method by monitoring the above ion pairs using triple quadrupole mass spectrometer. This method showed a good linearity in the range of 2.50-500 μg·mL^(-1)(r =0.998 8), and was successfully applied to the pharmacokinetic study of FGF21-164 fusion protein in mice. This experiment was approved by the Experimental Animal Ethics Committee of Shanghai Institute of Materia Medica,Chinese Academy of Sciences(batch number: 20180004040450). Compared with the endogenous FGF21, the t1/2 of FGF21-164 fusion protein was prolonged from 0.5 h to 2.6 h, which is expected to prolong the therapeutic efficacy of this protein.