Journal
JOURNAL OF PROTEOME RESEARCH
Volume 9, Issue 6, Pages 3319-3327Publisher
AMER CHEMICAL SOC
DOI: 10.1021/pr9011969
Keywords
MRM; O-18-Labeling; Absolute quantification; Biomarker verification; Serum; Liver cancer; Vitronectin; Clusterin
Categories
Funding
- National Natural Science Foundation of China [20635010, 20735005, 30900258]
- National Key Program for Basic Research of China [2006CB910803, 2010CB912700]
- National High Technology Research and Development Program of China [2006AA02A308]
- Chinese State Key Project Specialized for Infectious Disease [2009ZX10004-103, 2008ZX10002-016]
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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 O-18-labeling method was introduced to generate stable isotope-labeled peptides instead of the Fmoc chemical synthesis and Qconcat recombinant protein synthesis methods. To make O-18-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 O-18 to O-16, and (3) MRM parameters were optimized to maximize specificity and better distinguish between O-18-labeled and unlabeled peptides. As a result, the O-18-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 O-18-labeled and unlabeled peptides was achieved by the MRM-MS spectrum. Finally, through the combined improvement in O-18-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 (r(2) > 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.
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