LC-MS/MS assay for protein amino acids and metabolically related compounds for large-scale screening of metabolic phenotypes
LC-MS/MS assay for protein amino acids and metabolically related compounds for large-scale screening of metabolic phenotypes
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DOI：
10.1021/ac070938b
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发表时间：
2007-11-01
影响因子：
7.4
通讯作者：
Last, Robert L.
中科院分区：
文献类型：
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作者：
Gu, Liping;Jones, A. Daniel;Last, Robert L.
开发并验证了用于大规模筛选代谢表型的改良LC-MS/MS方法。20种氨基酸和5种代谢相关的化合物在4分钟内使用多反应监测（MRM）转换每种化合物的选择性进行测量。使用离子配对试剂全氟庚酸的短C18柱和快速梯度分离允许异构体Ile和Leu的色谱分辨率和改进的色谱峰形状为赖氨酸，精氨酸和组氨酸。建立了MRM转换，即使在色谱分离不完全时，也能够区分异构体Leu和Ile以及Thr和高丝氨酸。通过向拟南芥种子提取物中加入8种稳定的同位素标记的氨基酸标准品（AA*）来测试该测定的再现性。在日内和日间试验比较中，除Gly/Gly-D-2-N-15（1）外，所有AA/AA* 峰面积比的平均变异系数均小于5%。这8种氨基酸的回收率范围为62 - 94%，基质对信号的抑制为31- 65%。种子提取物的稀释降低离子抑制早期洗脱的氨基酸，但对那些洗脱后的影响最小。这8种氨基酸的日内和日间准确度分别为标称浓度的77-131和88-133%。
A modified LC-MS/MS method for large-scale screening of metabolic phenotypes was developed and validated. Twenty amino acids and 5 metabolically related compounds are measured within 4 min using multiple reaction monitoring (MRM) transitions selective for each compound. Separation with a short C 18 column and rapid gradient using the ion-pairing reagent perfluoroheptanoic acid allows chromatographic resolution of the isomers Ile and Leu and improved chromatographic peak shapes for Lys, Arg, and His. MRM transitions were established with capability to distinguish isomers Leu from Ile and Thr from homoserine even when chromatographic resolution is incomplete. The reproducibility of the assay was tested by adding eight stable isotope-labeled amino acid standards (AA*) to extracts of Arabidopsis thaliana seeds. In intra- and interday assay comparisons, mean coefficients of variation of the peak area ratios of AA/AA* were less than 5% for all but Gly/Gly-D-2-N-15(1). Recoveries of these eight amino acids ranged from 62 to 94% and suppression of signal by the matrix were 31-65%. Dilution of seed extracts reduced ion suppression for early-eluting amino acids but had minimal effects for those eluting later. The intra- and interday accuracies for these eight amino acids were 77-131 and 88-133% of nominal concentrations, respectively.