Determination of Aspartic Acid Racemization in Amyloid-β by UPLC-MS/MS
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.
NETL
NSTL
万方数据
