Combination of Improved 18O Incorporation and Multiple Reaction Monitoring: A Universal Strategy for Absolute Quantitative Verification of Serum Candidate Biomarkers of Liver Cancer

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• 作者：Yan Zhao ; Wei Jia ; Wei Sun ; Wenhai Jin ; Lihai Guo ; Junying Wei ; Wantao Ying ; Yangjun Zhang ; Yongming Xie ; Ying Jiang ; Fuchu He ; Xiaohong Qian
• 刊名：Journal of Proteome Research
• 出版年：2010
• 出版时间：June 4, 2010
• 年：2010
• 卷：9
• 期：6
• 页码：3319-3327
• 全文大小：283K
• 年卷期：v.9,no.6(June 4, 2010)
• ISSN：1535-3907

文摘

Stable isotope dilution-multiple reaction monitoring-mass spectrometry (SID-MRM-MS), which is an alternative to immunoassay methods such as ELISA and Western blotting, has been used to alleviate the bottlenecks of high-throughput verification of biomarker candidates recently. However, the inconvenience and high isotope consumption required to obtain stably labeled peptide impedes the broad application of this method. In our study, the ¹⁸O-labeling method was introduced to generate stable isotope-labeled peptides instead of the Fmoc chemical synthesis and Qconcat recombinant protein synthesis methods. To make ¹⁸O-labeling suitable for absolute quantification, we have added the following procedures: (1) RapiGest SF and microwave heating were added to increase the labeling efficiency; (2) trypsin was deactivated completely by chemical modification using tris(2-carboxyethyl)phosphine (TCEP) and iodoacetamide (IAA) to prevent back-exchange of ¹⁸O to ¹⁶O, and (3) MRM parameters were optimized to maximize specificity and better distinguish between ¹⁸O-labeled and unlabeled peptides. As a result, the ¹⁸O-labeled peptides can be prepared in less than 1 h with satisfactory efficiency (>97%) and remained stable for 1 week, compared to traditional protocols that require 5 h for labeling with poor stability. Excellent separation of ¹⁸O-labeled and unlabeled peptides was achieved by the MRM-MS spectrum. Finally, through the combined improvement in ¹⁸O-labeling with multiple reaction monitoring, an absolute quantification strategy was developed to quantitatively verify hepatocellular carcinoma-related biomarker candidates, namely, vitronectin and clusterin, in undepleted serum samples. Sample preparation and capillary-HPLC analysis were optimized for high-throughput applications. The reliability of this strategy was further evaluated by method validation, with accuracy (%RE) and precision (%RSD) of less than 20% and good linearity (r² > 0.99), and clinical validation, which were consistent with previously reported results. In summary, our strategy can promote broader application of SID-MRM-MS for biomarkers from discovery to verification regarding the significant advantages of the convenient and flexible generation of internal standards, the reduction in the sample labeling steps, and the simple transition.