摘要
目的:建立β淀粉样蛋白中Asp外消旋化的LC-MS/MS分析方法.方法:采用胰蛋白酶和Glu-C酶两步酶解β淀粉样蛋白,酶解产物LC-MS/MS分析的色谱条件:分离柱为InfinityLab Poroshell 120 EC-C18 色谱柱(2.7 μm,3.0×150 mm Agilent),流动相A为 5%乙腈-0.1%甲酸水,流动相B为 75%乙腈-水,梯度洗脱(0～30 min,0～28%B;30～30.01 min,28%～100%B;30.01～35 min,100%～100%B;35.0～35.01 min,100%～0%B;35.01～40 min,0%B),流速为 0.3 mL/min.质谱条件:采用电喷雾离子源及正离子多反应监测模式(SRM)定量.结果:在L型多肽存在下,D型多肽的质量浓度在 1～250 ng/mL内,其峰面积与多肽的质量浓度之间线性良好(r>0.999),最低定量限(LLOQ)为 1 ng/mL;LLOQ、低(LQC)、中(MQC)、高浓度(HQC)日内和日间精密度RSD值均小于 15%,加样回收率为85.3%～107.3%.建立的方法对D/(D+L)型淀粉样蛋白为 2%～50%的混合样本中Asp1,Asp23 外消旋化测定结果准确,准确度在95.35%～117%之间.联合胰蛋白酶与Glu-C成功鉴定了淀粉样蛋白中Asp7 残基的外消旋化.结论:所建立的方法可用于淀粉样蛋白中Asp残基外消旋化的分析,该方法操作简便,结果准确,灵敏度高.
Abstract
Objective:To establish a LC-MS/MS method for the determination of racemization of Asp in amyloid-β.Methods:Amyloid-β protein was digested by trypsin or combined with Glu-C enzyme.The InfinityLab Poroshell 120 EC-C18 chromatographic column(2.7 μm,3.0×150 mm Agilent)was selected for analysis with 5%acetonitrile-0.1%formic acid water as mobile phase A and 75%acetonitrile-water as mobile phase B,Gradient elution(0～30 min,0%～28%B;30～30.01 min 28%～100%B;30.01～35 min,100%～100%B;35.0～35.01 min,100%～0%B;35.01～40 min,0%B).The flow rate was 0.3 mL/min.Electrospray ion source and positive ion multiple reaction monitoring mode(SRM)were used for quantification.Results:The correlation coefficient between the ratios of peak area of D-form peptide to that of the internal standard and the amino acid concentrations were above 0.999 at 1～250 ng/mL.The lowest limit of quantification(LLOQ)was 1 ng/mL.The intra-day and inter-day precisions of measurement of QC samples at 4 Tested concentrations were less than 15%.The recoveries rate of 4 QC samples was from 85.3%to 107.3%.The established method was successfully used for the determination of racemization of Asp1 and Asp23 in the mixed samples with 2%～50%D/(D+L)amyloid-β protein,and the accuracy was between 95.35%and 117%.Racemization of Asp7 residues in amyloid-β was successfully identified in combination with trypsin and Glu-C.Conclusion:The established method is simple,accurate and sensitive and can be used for the determine of racemization of Asp in amyloid-β protein.
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