Combination of Improved 18O Incorporation and Multiple Reaction Monitoring: A Universal Strategy for Absolute Quantitative Verification of Serum Candidate Biomarkers of Liver Cancer Yan Zhao,§ Wei Jia,§ Wei Sun,§ Wenhai Jin,† Lihai Guo,† Junying Wei,§ Wantao Ying,§ Yangjun Zhang,§ Yongming Xie,† Ying Jiang,§ Fuchu He,§ and Xiaohong Qian*,§ State Key Laboratory of Proteomics, Beijing Proteome Research Center, Beijing Institute of Radiation Medicine, No.33 Life Science Park Road, Changping District, Beijing 102206, P. R. China, and AB SCIEX Asia Pacific Application Support Center, Shanghai, 200233, P. R. China Received December 24, 2009
Abstract: 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 * To whom correspondence may be addressed: Xiaohong Qian, e-mail:
[email protected]; phone: 8610-80705055; fax: 8610-80705155. § State Key Laboratory of Proteomics, Beijing Proteome Research Center. † AB SCIEX Asia Pacific Application Support Center. 10.1021/pr9011969
2010 American Chemical Society
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. Keywords: MRM • 18O-Labeling • Absolute quantification • Biomarker verification • Serum • Liver cancer • Vitronectin • Clusterin
Introduction Biomarkers play important roles in diagnosis, treatment, progression, and prognosis of cancer,1 the discovery of which have attracted extensive attentions in the medical field.2-4 The advent of proteomics technology including two-dimensional polyacrylamide gel electrophoresis, mass spectrometry, and protein microarray have led to a proliferation of numerous biomarker candidates by global detection and quantitation of proteins, which greatly accelerate the discovery process.5 However, before the results can be applied to clinical management, these biomarker candidates need further validation to address their sensitivity, specificity, reproducibility, and accuracy.6 A general pipeline to obtain ultimate biomarkers for clinical use includes four phases: discovery, qualification, verification, and clinical validation.7,8 Although hundreds of candidates can be discovered, further verification of these candidates in more cases of samples has become the ratelimiting step in a biomarker pipeline,9 because the conventional verification methods, such as Western blotting and enzymelinked immunosorbent assay (ELISA), are not suitable for largescale analysis due to poor throughput and problems associated with obtaining monoclonal antibodies.10 Take the study of Piersma et al.11 as an example, among the 136 secretome candidate biomarkers yielded by comparative analysis of MEF/ Toff cells expressing IGF1R and stimulated with IGF1, only 2 of these were able to be further verified by ELISA assays. In recent years, stable isotope dilution-multiple reaction monitoring-mass spectrometry (SID-MRM-MS) was introduced to alleviate the bottleneck in biomarker development.12-16 In Journal of Proteome Research 2010, 9, 3319–3327 3319 Published on Web 04/27/2010
technical notes this method, a signature peptide from a target protein is first selected, and then the signature peptide is absolutely quantified against a spiked known amount of stable isotope-labeled signature peptide.17-19 Finally, the protein concentration is calculated from the stoichiometry of the signature peptide to the protein. Using this method, Christine et al.20 quantified 47 major plasma proteins within a single chromatography run, and Hasmik et al.21 extended the detection limit to 1-10 ng/mL after the depletion of high-abundance proteins and separation by strong cation exchange chromatography. The significant advantages, which are high throughput, multiplexing capabilities, high sensitivity, and independence of antibodies, make SID-MRM-MS an effective alternative method for biomarker candidate verification. Although SID-MRM-MS has received more and more attention,22-24 the broad application of this method has been prevented due to the inconvenience and high consumption of stable isotope-labeled peptides as the internal standards for the quantitation experiments. The standard Fmoc chemistry method can produce labeled peptides using isotope-incorporated amino acids as a raw material, but these amino acids are expensive and the reaction is time-consuming for largescale preparation. Alternatively, the Qconcat strategy,25 as well as concatemers of Q peptides (QCAT)26 and peptide-concatenated standards (PCS),27 has been proposed to produce labeled peptides through expression of a synthetic gene (derived from labeled peptides) that encodes a recombinant protein in medium containing the isotopic amino acids. A large number of labeled peptides can be generated simultaneously by this approach. However, it is difficult to control the individual internal standard concentrations to the approximate concentrations of endogenous analytes, resulting in exceeding the maximum linear dynamic range in the MRM assay. Other than the above two methods, chemical modification can also be applied to incorporate an isotopic label into peptides. Leroi et al.28 labeled peptides conveniently and flexibly using the mTRAQ reagent to absolutely quantify endogenous levels of a potential cancer marker in cancerous and normal endometrial tissues. However, the analysis was inconvenient because endogenous analytes were also labeled. We introduced the 18O incorporation method to prepare internal standard peptides for the widespread use of SID-MRMMS. 18O-labeling has been routinely applied as an easy and economical labeling method in comparative proteomic studies.29-32 Compared with other isotope incorporation methods, trypsincatalyzed 18O-labeling is suitable for a wide variety of analyses and uses relatively moderate reaction conditions to avoid changing the physicochemical characteristics of the peptides. These features show the convenience and distinct advantages of the 18O isotope incorporation for internal standard preparation. However, there are still doubts about the peptide labeling efficiency and stability as well as the specificity of the MS signals, which will affect the accuracy of the absolute quantification. Thus, in our study, we made the following optimizations of the 18O-labeling method to meet the requirements for absolute quantification. First, a satisfactory labeling efficiency in a short reaction time (1 h) was obtained by the addition of RapiGest SF and microwave heating to enhance peptide dispersion. Second, the labeled peptide stability was verified over a week, since loss of the isotope label was prevented through reduction and alkylation of trypsin. Finally, the MRM parameters were optimized to eliminate cross talk between the labeled and unlabeled peptide signals. 3320
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Zhao et al. On the basis of the work mentioned above, we developed a fast absolute quantification strategy using 18O-labeled peptides as internal standards to verify biomarker candidates in undepleted serum. Method validation experiments demonstrated that excellent linear responses (r2 > 0.99) were obtained with an attomole level of quantitation (
