差异蛋白质组学发现了大量肿瘤蛋白质候选标志物，如何从中验证出可用于临床诊疗的标志物至关重要。多反应监测质谱(MRM-MS)作为一种新的标志物验证方法，具有高灵敏和高准确等优势，但通常每次进样只能验证1例样本。我们曾发展蛋白质组分析新方法检测到当时最大规模的早期结直肠癌蛋白质候选标志物群，首次在国际上提出了阵列MRM-MS的概念，将MRM-MS一次进样验证样本数拓展到4例。本项目拟在之前的工作基础上，采用多种同位素试剂组合标记建立更高通量的阵列MRM-MS方法，一次进样实现近40例样本的同时定性和定量验证，由于采用无色谱分馏效应的同位素标记试剂确保了不同样本中相同目标肽标记后色谱流出时间相同，保证了定量验证的准确性，并由于同时利用了MRM和串级质谱两种信息确保了目标肽定性验证的可靠性。将所建立的阵列MRM-MS新方法验证一批结直肠癌蛋白质候选标志物，并为其他重大疾病的候选标志物验证提供利器。

Differential proteomics has already found a large number of candidate biomarkers of diseases. How to verify these biomarkers is of crucial importance for clinical diagnosis and therapy. Multiple reaction monitoring mass spectrometry (MRM-MS) technique has the advantages of high sensitivity, high accuracy in verification of candidate biomarkers. However, in general MRM analysis, only one sample can be quantified in a single experiment, which restricts its application. In our previous study, we have reported the largest candidate protein biomarker dataset for early colorectal cancer (CRC) through differential proteomics research at that time. Then, we put forward the concept of a new high throughput MRM-MS (Hyperplex-MRM-MS) technology to verification these potential biomarkers for the first time, which expanded the samples to 4 cases MRM-MS in single experiment. On the basis of previous work, this project will combine iTRAQ technique (8-plex) and dimethyl technique (3-5 plex) in order to create Hyperplex-MRM-MS technique with higher throughput. We therefore can qualify and quantify nearly 40 cases of samples in a single experiment at the same time. Because of the isotope labeling reagents are without chromatographic fractionation effect, we can ensure each target peptide in different samples share the same retention time of chromatographic flow. This feature guarantees the accuracy of quantitative validation. Additionally, because of using the information of MRM and tandem mass spectrometry at the same time, we can ensure the both the identification and quantitation reliability of each target peptides. Finally, the high throughput Hyperplex MRM-MS technique will be applied into the verification a batch of candidate biomarkers of CRC, and serves as a sharp sword in biomarkers verification for other diseases.