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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• 作者：Jun Qu ; William J. Jusko ; and Robert M. Straubinger
• 刊名：Analytical Chemistry
• 出版年：2006
• 出版时间：July 1, 2006
• 年：2006
• 卷：78
• 期：13
• 页码：4543 - 4552
• 全文大小：308K
• 年卷期：v.78,no.13(July 1, 2006)
• ISSN：1520-6882

文摘

Gene expression changes underlie important biologicaland pharmacological responses. Although mRNA expression profiling is routine, quantification of low-abundanceproteins, 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-codedaffinity tags (cICAT) was employed to reduce the complexity of the digested proteome of tissue homogenates andto improve the quantification of low-abundance proteins.Second, cICAT-treated tissue samples were analyzed ona capillary LC coupled to an ion trap MS to screen for thesubset of cICAT-peptides, derived from target proteins ofinterest, that was successfully labeled and retrieved.Third, putatively identified peptides derived from targetproteins were synthesized, cICAT-labeled, and used bothto optimize multiple reactions monitoring (MRM) analysisand to confirm chromatographic retention time andfragmentation pattern. Finally, batch quantification oftarget peptides was performed using MRM on a LC/triple-quad MS/MS using ¹²C- (control) and ¹³C (experimental)-cICAT-labeled tissue mixtures. The utility of this methodwas demonstrated by elucidating the time-course of tyrosine aminotransferase induction in the liver of ratsfollowing treatment with the corticosteroid methylprednisolone (MPL). This approach significantly improvedquantitative sensitivity, and the linear range was 10-foldgreater than published previously. An additional advantage is that archived samples may be reinterrogated toinvestigate the regulation of additional targets that becomeof interest. Stored samples were sucessfully reinterrogatedto monitor the induction of ornithine decarboxylase, whichis also an MPL-induced protein. To our knowledge, thisis the first report of an ICAT-based method that is capableof quantifying low-abundance proteins in highly complexsamples, such as tissue homogenates. The approachenables simultaneous quantification of multiple effectorproteins induced by biological or pharmacological stimuli,and the processed samples can be interrogated repeatedlyas additional targets of interest arise.