Utility of cleavable isotope-coded affinity-tagged reagents for quantification of low-copy proteins induced by methylprednisolone using liquid chromatography/tandem mass spectrometry.
Utility of cleavable isotope-coded affinity-tagged reagents for quantification of low-copy proteins induced by methylprednisolone using liquid chromatography/tandem mass spectrometry.
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DOI：
10.1021/ac0521697
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发表时间：
2006-07
影响因子：
7.4
通讯作者：
J. Qu;W. Jusko;R. Straubinger
中科院分区：
文献类型：
--
作者：
J. Qu;W. Jusko;R. Straubinger
基因表达变化是重要的生物学和药理学反应的基础。虽然mRNA表达谱是常规的，但低丰度蛋白（通常代表反应的关键效应因子）的定量仍然具有挑战性。开发了一种新的策略，用于高度复杂的生物基质中低丰度蛋白质的敏感和准确定量。首先，利用可切割同位素编码亲和标签（cICAT）的半胱氨酸特异性，降低组织匀浆物消化蛋白质组的复杂性，提高低丰度蛋白质的定量。其次，用离子阱质谱联用毛细管LC分析经cicat处理的组织样本，筛选从感兴趣的靶蛋白衍生的cicat肽亚群，并成功标记和检索。第三，合成从目标蛋白衍生的推定鉴定肽，并进行cicat标记，用于优化多反应监测（MRM）分析，并确定色谱保留时间和碎片化模式。最后，使用（12）C-（对照）和（13）C（实验）- cicat标记的组织混合物，在LC/三重质谱/质谱上使用MRM对目标肽进行批量定量。该方法的实用性通过阐明糖皮质类固醇甲基强的松龙（MPL）治疗后大鼠肝脏中酪氨酸转氨酶诱导的时间过程得到证实。该方法显著提高了定量灵敏度，线性范围比先前发表的大10倍。另一个优点是，存档的样本可以被重新询问，以调查其他目标的规则，成为感兴趣的。储存的样品被成功地重新询问，以监测鸟氨酸脱羧酶的诱导，这也是一种mpl诱导的蛋白质。据我们所知，这是基于icat的方法的第一篇报道，该方法能够定量高度复杂样品（如组织匀浆）中的低丰度蛋白质。该方法能够同时定量由生物或药理学刺激诱导的多种效应蛋白，并且处理后的样品可以反复询问其他感兴趣的靶标。
Gene expression changes underlie important biological and pharmacological responses. Although mRNA expression profiling is routine, quantification of low-abundance proteins, which typically represent key effectors of responses, remains challenging. A novel strategy was developed for sensitive and accurate quantification of low-abundance proteins in highly complex biological matrixes. First, the cysteine specificity of cleavable isotope-coded affinity tags (cICAT) was employed to reduce the complexity of the digested proteome of tissue homogenates and to improve the quantification of low-abundance proteins. Second, cICAT-treated tissue samples were analyzed on a capillary LC coupled to an ion trap MS to screen for the subset of cICAT-peptides, derived from target proteins of interest, that was successfully labeled and retrieved. Third, putatively identified peptides derived from target proteins were synthesized, cICAT-labeled, and used both to optimize multiple reactions monitoring (MRM) analysis and to confirm chromatographic retention time and fragmentation pattern. Finally, batch quantification of target peptides was performed using MRM on a LC/triple-quad MS/MS using (12)C- (control) and (13)C (experimental)-cICAT-labeled tissue mixtures. The utility of this method was demonstrated by elucidating the time-course of tyrosine aminotransferase induction in the liver of rats following treatment with the corticosteroid methylprednisolone (MPL). This approach significantly improved quantitative sensitivity, and the linear range was 10-fold greater than published previously. An additional advantage is that archived samples may be reinterrogated to investigate the regulation of additional targets that become of interest. Stored samples were sucessfully reinterrogated to monitor the induction of ornithine decarboxylase, which is also an MPL-induced protein. To our knowledge, this is the first report of an ICAT-based method that is capable of quantifying low-abundance proteins in highly complex samples, such as tissue homogenates. The approach enables simultaneous quantification of multiple effector proteins induced by biological or pharmacological stimuli, and the processed samples can be interrogated repeatedly as additional targets of interest arise.