Abstract:

A robust and high throughput method was developed based on ultra performance liquid chromato-graphy and quadruple/time-of-flight mass spectrometry(UPLC-QTOFMS) for performing global metabolic profiling analysis on a large batch of human serum. Proteins in serum samples were precipitated by organic solvents. Using 38 reference standards, we compared serum preparation methods for protein precipitation by different organic solvents. Different conditions for global metabolic profiling analysis were performed and optimized on UPLC-QTOFMS. We also assayed the reproducibility, precision and stability of the method of profiling. The results indicated that methanol/acetonitrile(volume ratio 1: 9) could effectively and reproducibly precipitated human serum proteins, performing the best extracting effect on the assayed reference standards. At least four fold of pre-cold organic solvents coupled with vortex for 2 min, ultrasonic treatment for 1 min and stayed at -20℃ for 10 min were the most optimal for the extracting effect and protein precipitation. The results, according to the reproducibility, precision and stability, showed that the method was satisfactory in global metabolic profiling analysis of human serum. Furthermore, reproducibility of the method verified by principal component analysis(PCA) was consistent with the coefficient of variation(CV) analysis in the results. The method was adapted to metabolomics study, especially the large batch metabolomics study, with a high throughput of over 100 samples a day(13 min for each sample run).

Key words: Global metabolic profiling, Serum preparation method, Ultra performance liquid chromatography and quadruple/time-of-flight mass spectrometry(UPLC-QTOFMS), Ultrasonic treatment, Principal component analysis(PCA)