柱前衍生-液质联用法测定过氧化物还原酶2模拟肽中Asp外消旋化研究
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摘要
目的建立了一种柱前衍生-液质联用测定过氧化物还原酶2(Prx2)模拟肽中天冬氨酸(Asp)外消旋化的方法,测定不同p H条件下,天冬氨酸外消旋化速率常数。方法采用6.0 mol·L~(-1)盐酸水解八肽,水解液减压干燥至干,加水溶解,以L-2-氨基丁酸为内标物,GITC为柱前衍生试剂,在p H 11.0碳酸氢钠溶液中衍生化。色谱条件:分离柱为YMC-PACK ODS-AQ(4.6 mm×150 mm,5μm),流动相为5%冰醋酸溶液(A)和乙腈-甲醇(90∶10)(B),梯度洗脱(0～22 min,20%～27.5%B;22～22.01 min,27.5%～95%B;22.01～27 min,95%B;27～27.01 min,95%～20%B;27.01～30 min,20%B),流速为1 mL·min~(-1);质谱条件:采用电喷雾离子源及正离子单离子监测模式(SIM)定量,天冬氨酸GITC衍生化物m/z 523,内标GITC衍生化物m/z 493。结果 D/L-Asp质量浓度在1.010～200.2μg·m L~(-1)时,其峰面积与内标物峰面积的比值和氨基酸的质量浓度之间的线性关系良好(r> 0.999),最低定量限(LLOQ)为1.010μg·m L~(-1);LLOQ、低、中和高浓度(LQC、MQC和HQC)日内和日间精密度RSD分别为0.98%～9.7%、3.3%～10.9%,加样回收率为100.2%～108.1%。在p H 3.0～10.0,天冬氨酸外消旋化的速率常数为0～6.24×10~(-4)·d~(-1)。结论该方法准确、快速,可用于Prx2中D/L-Asp检测。
Objective To develop a liquid chromatographic-mass spectrometry(LC-MS) method to determine aspartic acid racemization in peroxiredoxin 2(Prx2) mimic peptide by precolumn derivatization. Methods The analyte was hydrolyzed with 6.0 mol·L~(-1) HCl, the extraction solution was dried completely under low pressure, and then dissolved in water mixed with L-2-aminobutyric acid as the internal standard before derivatization with 2, 3, 4, 6-tetra-O-benzoyl-β-D-glucopyranosyl isothiocyanate(GITC) as derivatization agent in ammonium bicarbonate buffer solution(pH 11.0). A YMC-PACK ODS-AQ(4.6 mm×150 mm, 5 μm) was adopted with water(containing 5% glacial acetic acid) as mobile phase A and acetonitrile: methanol =(90∶10)as mobile phase B. Gradient elution(0-22 min, 20%-27.5%B; 22-22.01 min, 27.5%-95%B; 22.01-27 min, 95%B; 27-27.01 min, 95%-20%B; 27.01-30 min, 20%B). The flow rate was 1.0 mL·min~(-1).Data were obtained with electrospray ionization and positive single ion monitoring, and the m/z of aspartic acid,L-2-aminobutyric acid derivative was 523 and 493, respectively. Results The correlation coefficient between the ratios of peak area of amino acid to that of the internal standard and the amino acid concentrations were above 0.999 at 1.010-200.2 μg·mL~(-1). The lower limit of quantification was 1.010 μg·mL~(-1). The intraday and inter-day precisions in the measurement of QC samples at 4 tested concentrations were at 0.98%-9.7%,and 3.3%-10.9%. The recoveries of 4 QC samples were from 100.2% to 108.1%. Kinetics was also studied,with racemization rate constant at 0-6.24×10~(-4)·d~(-1) between pH 3.0-10.0. Conclusion The method is rapid, accurate and can be applied to determine aspartic acid racemization in Prx2.
Objective To develop a liquid chromatographic-mass spectrometry(LC-MS) method to determine aspartic acid racemization in peroxiredoxin 2(Prx2) mimic peptide by precolumn derivatization. Methods The analyte was hydrolyzed with 6.0 mol·L~(-1) HCl, the extraction solution was dried completely under low pressure, and then dissolved in water mixed with L-2-aminobutyric acid as the internal standard before derivatization with 2, 3, 4, 6-tetra-O-benzoyl-β-D-glucopyranosyl isothiocyanate(GITC) as derivatization agent in ammonium bicarbonate buffer solution(pH 11.0). A YMC-PACK ODS-AQ(4.6 mm×150 mm, 5 μm) was adopted with water(containing 5% glacial acetic acid) as mobile phase A and acetonitrile: methanol =(90∶10)as mobile phase B. Gradient elution(0-22 min, 20%-27.5%B; 22-22.01 min, 27.5%-95%B; 22.01-27 min, 95%B; 27-27.01 min, 95%-20%B; 27.01-30 min, 20%B). The flow rate was 1.0 mL·min~(-1).Data were obtained with electrospray ionization and positive single ion monitoring, and the m/z of aspartic acid,L-2-aminobutyric acid derivative was 523 and 493, respectively. Results The correlation coefficient between the ratios of peak area of amino acid to that of the internal standard and the amino acid concentrations were above 0.999 at 1.010-200.2 μg·mL~(-1). The lower limit of quantification was 1.010 μg·mL~(-1). The intraday and inter-day precisions in the measurement of QC samples at 4 tested concentrations were at 0.98%-9.7%,and 3.3%-10.9%. The recoveries of 4 QC samples were from 100.2% to 108.1%. Kinetics was also studied,with racemization rate constant at 0-6.24×10~(-4)·d~(-1) between pH 3.0-10.0. Conclusion The method is rapid, accurate and can be applied to determine aspartic acid racemization in Prx2.
引文
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[2]Hall A,Karplus PA,Poole LB.Typical 2-Cys peroxiredoxins:structures,mechanisms and functions[J].FEBS J,2009,276(9):2469-2477.
[3]Fujii N,Harada K,Momose Y,et al.d-amino acid formation induced by a chiral field within a human lens protein during aging[J].B iochem Biophys Res Commun,1999,263(2):322-326.
[4]Conrad U,Fahr A,Scriba GKE.Kinetics of aspartic acid isomerization and enantiomerization in model aspartyl tripeptides under forced conditions[J].J Pharm Sci,2010,99(10):4162-4173.
[5]Takahashi O,Kirikoshi R,Manabe N.Racemization of the succinimide intermediate formed in proteins and peptides:a computational study of the mechanism catalyzedby dihydrogen phosphate ion[J].Int J Mol Sci,2016,17(10):1698-1707.
[6]Dayag S,Ken-Loon C,Michael O,et al.Molecular ageing:free radical initiated epimerization of thymopentin--a case study[J].J Chem Phys,2014,140(20):205102.
[7]Hooi MYS,Raftery MJ,Truscott RJW.Racemization of two proteins over our lifespan:deamidation of asparagine76 inγS crystallin is greater in cataract than in normal lenses across the age range[J].Invest Ophth Vis Sci,2012,53(7):3554-3561.
[8]Aki K,Okamura E.D-β-aspartyl residue exhibiting uncommon high resistance to spontaneous peptide bond cleavage[J].Sci Rep,2016,6:21594.
[9]Lu M,Shaler TA.Isomerization and autolysis at specific amino acid residues of the tau protein and its relations to Alzheimer’s disease[J].B iophys J,2014,106(2):473a-474a.
[10]Koizumi R,Ogasawara M,Sasaki N,et al.Involvement of asparatic acid racemization and isomerization in the pathogenesis of COPD[J].Eur Respir J,2013,42:P246.
[11]Rhee SG.Overview on peroxiredoxin[J].Mol Cell,2016,39(1):1-5.
[12]Kwon T,Bak Y,Park YH,et al.PeroxiredoxinⅡis essential for maintaining stemnessby redox regulation in liver cancer cells[J].Stem Cells,2016,34(5):1188-1197.
[13]Lee TH,Kim SU,Yu SL,et al.PeroxiredoxinⅡis essential for sustaining life span of erythrocytes in mice[J].Blood,2003,101(12):5033-5038.
[14]Lehtonen ST,Steffen O,Riitta KW,et al.Does the oxidative stress in chronic obstructive pulmonary disease cause thioredoxin/peroxiredoxin oxidation?[J].Antioxid Redox Sign,2008,10(4):813-819.
[15]Miyamoto T,Homma H.Detection and quantification of D-amino acid residues in peptides and proteins using acid hydrolysis[J].B BA-Protein Proteom,2017,1866(7):775-782.
[16]Péter A,Lázár L,Fül?p F,et al.High-performance liquid chromatographic enantioseparation of B-amino acids[J].J Chromatogr A,2001,926(2):229-238.
[17]Hess S.A universal HPLC-MS method to determine the stereochemistry of common and unusual amino acids[J].Methods Mol Biol,2012,828(828):63-75.
[18]Amblard M,Fehrentz JA,Martinez J,et al.Methods and protocols of modern solid phase peptide synthesis[J].Mol Biotechnol,2006,33(3):239-254.
[19]Masters PM,Bada JL,Jr ZJ.Aspartic acid racemisation in the human lens during ageing and in cataract formation[J].Nature,1977,268(5615):71-73.
[20]Brunauer LS,Clarke S.Age-dependent accumulation of protein residues which can be hydrolyzed to D-aspartic acid in human erythrocytes[J].J Biol Chem,1986,261(27):12538-12543.
[21]Ueno K,Ueda T,Sakai K,et al.Evidence for a novel racemization process of an asparaginyl residue in mouse lysozyme under physiological conditions[J].Cell Mol Life Sci,2005,62(2):199-205.