He Wei-Tao, Liang Bo-Cheng, Shi Zhen-Yu, Li Xu-Yun, Li Chun-Wen, Shi Xiao-Lin
The Second Clinical Medical College, Zhejiang Chinese Medical University, Hangzhou, 310005 China.
Department of Diagnostics of Traditional Chinese Medicine, College of Basic Medical Science, Zhejiang Chinese Medical University, Hangzhou, 310005 China.
Springerplus. 2016 May 21;5(1):679. doi: 10.1186/s40064-016-2276-4. eCollection 2016.
The present study aimed at investigating the weak cation magnetic separation technology and matrix-assisted laser desorption ionization-time of flight-mass spectrometry (MALDI-TOF-MS) in screening serum protein markers of osteopenia from ten postmenopausal women and ten postmenopausal women without osteopenia as control group, to find a new method for screening biomarkers and establishing a diagnostic model for primary type I osteoporosis. Serum samples were collected from postmenopausal women with osteopenia and postmenopausal women with normal bone mass. Proteins were extracted from serum samples by weak cation exchange magnetic beads technology, and mass spectra acquisition was done by MALDI-TOF-MS. The visualization and comparison of data sets, statistical peak evaluation, model recognition, and discovery of biomarker candidates were handled by the proteinchip data analysis system software(ZJU-PDAS). The diagnostic models were established using genetic arithmetic based support vector machine (SVM). The SVM result with the highest Youden Index was selected as the model. Combinatorial Peaks having the highest accuracy in distinguishing different samples were selected as potential biomarker. From the two group serum samples, a total of 133 differential features were selected. Ten features with significant intensity differences were screened. In the pair-wise comparisons, processing of MALDI-TOF spectra resulted in the identification of ten differential features between postmenopausal women with osteopenia and postmenopausal women with normal bone mass. The difference of features by Youden index showed that the highest features had a mass to charge ratio of 1699 and 3038 Da. A diagnosis model was established with these two peaks as the candidate marker, and the specificity of the model is 100 %, the sensitivity was 90 % by leave-one-out cross validation test. The two groups of specimens in SVM results on the scatter plot could be clearly distinguished. The peak with m/z 3038 in the SVM model was suggested as Secretin by TagIdent tool. To provide further validation, the secretin levels in serum were analyzed using enzyme-linked immunosorbent assays that is a competitive inhibition enzyme immunoassay technique for the in vitro quantitative measurement of secretin in human serum.
本研究旨在探讨弱阳离子磁分离技术和基质辅助激光解吸电离飞行时间质谱（MALDI-TOF-MS），以筛选10名绝经后骨质疏松症女性和10名无骨质疏松症的绝经后女性作为对照组的血清蛋白标志物，寻找一种筛选生物标志物的新方法，并建立原发性I型骨质疏松症的诊断模型。收集绝经后骨质疏松症女性和骨量正常的绝经后女性的血清样本。采用弱阳离子交换磁珠技术从血清样本中提取蛋白质，并用MALDI-TOF-MS进行质谱采集。数据集的可视化和比较、统计峰评估、模型识别以及候选生物标志物的发现均由蛋白质芯片数据分析系统软件（ZJU-PDAS）处理。使用基于遗传算法的支持向量机（SVM）建立诊断模型。选择约登指数最高的SVM结果作为模型。选择在区分不同样本时准确率最高的组合峰作为潜在生物标志物。从两组血清样本中，共选择了133个差异特征。筛选出10个强度差异显著的特征。在两两比较中，MALDI-TOF光谱分析结果显示，绝经后骨质疏松症女性和骨量正常的绝经后女性之间有10个差异特征。约登指数特征差异显示，最高特征的质荷比为1699和3038 Da。以这两个峰作为候选标志物建立诊断模型，经留一法交叉验证试验，模型的特异性为100%，敏感性为90%。SVM结果在散点图上的两组标本能够清晰区分。SVM模型中质荷比为3038的峰经TagIdent工具提示为促胰液素。为进一步验证，采用酶联免疫吸附测定法分析血清中促胰液素水平，该方法是一种用于体外定量测定人血清中促胰液素的竞争性抑制酶免疫测定技术。
