Urinary amino acid analysis: a comparison of iTRAQ-LC-MS/MS, GC-MS, and amino acid analyzer.
Urinary amino acid analysis: a comparison of iTRAQ-LC-MS/MS, GC-MS, and amino acid analyzer.
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DOI：
10.1016/j.jchromb.2009.05.019
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发表时间：
2009-07-01
期刊：
影响因子：
--
通讯作者：
Oefner PJ
中科院分区：
文献类型：
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作者：
Kaspar H;Dettmer K;Chan Q;Daniels S;Nimkar S;Daviglus ML;Stamler J;Elliott P;Oefner PJ
尿氨基酸分析通常通过阳离子交换色谱法进行，然后用茚三酮进行柱后衍生化和UV检测。该方法缺乏通量和特异性。两个最近推出的稳定同位素比质谱法有望克服这些缺点。使用两组设盲的尿液重复样本和经认证的氨基酸标准品，我们将氯甲酸丙酯和iTRAQ®衍生氨基酸的气相色谱/质谱（GC-MS）和液相色谱-串联质谱（LC-MS/MS）的精密度和准确度分别与常规氨基酸分析进行了比较。GC-MS方法建立在用氯甲酸丙酯直接衍生稀释尿液中的氨基酸、GC分离和使用稳定同位素标记标准品对衍生物进行质谱定量的基础上。LC-MS/MS方法需要预先进行尿蛋白沉淀，然后用iTRAQ®标签标记尿液和标准氨基酸，所述iTRAQ®标签含有通过MS/MS片段化可区分的不同可裂解报告离子。通过氨基酸分析仪、GC-MS和iTRAQ®-LC-MS/MS分析33份一式两份和一式三份尿液标本测定的20种氨基酸计算的技术误差百分比（%TE）的平均值和标准差分别为7.27±5.22、21.18±10.94和18.34±14.67。在第二批144份尿液标本中测定的13种氨基酸的相应值分别为8.39±5.35、6.23±3.84和35.37±29.42。GC-MS和iTRAQ®-LC-MS/MS都适用于高通量氨基酸分析，前者目前提供更高的重现性和完全自动化的样品预处理，而后者涵盖更多的氨基酸和相关胺。
Urinary amino acid analysis is typically done by cation-exchange chromatography followed by post-column derivatization with ninhydrin and UV detection. This method lacks throughput and specificity. Two recently introduced stable isotope ratio mass spectrometric methods promise to overcome those shortcomings. Using two blinded sets of urine replicates and a certified amino acid standard, we compared the precision and accuracy of gas chromatography/mass spectrometry (GC-MS) and liquid chromatography-tandem mass spectrometry (LC-MS/MS) of propyl chloroformate and iTRAQ® derivatized amino acids, respectively, to conventional amino acid analysis. The GC-MS method builds on the direct derivatization of amino acids in diluted urine with propyl chloroformate, GC separation and mass spectrometric quantification of derivatives using stable isotope labeled standards. The LC-MS/MS method requires prior urinary protein precipitation followed by labeling of urinary and standard amino acids with iTRAQ® tags containing different cleavable reporter ions distinguishable by MS/MS fragmentation. Means and standard deviations of percent technical error (%TE) computed for 20 amino acids determined by amino acid analyzer, GC-MS, and iTRAQ®-LC-MS/MS analyses of 33 duplicate and triplicate urine specimens were 7.27±5.22, 21.18±10.94, and 18.34±14.67, respectively. Corresponding values for 13 amino acids determined in a second batch of 144 urine specimens measured in duplicate or triplicate were 8.39±5.35, 6.23±3.84, and 35.37±29.42. Both GC-MS and iTRAQ®-LC-MS/MS are suited for high-throughput amino acid analysis, with the former offering at present higher reproducibility and completely automated sample pretreatment, while the latter covers more amino acids and related amines.