Yee Soon Ling (Presenter)
University Malaysia Sabah
Bio: Dr. Ling Yee Soon obtained his PhD in Toxicology from The National Taiwan University, Taiwan. He joined Biotechnology Research Institute as a Senior Lecturer and fellow researcher under Water Research Unit based in Universiti Malaysia Sabah. His research of interests is Liquid chromatography-Mass-spectrometry based metabolomics - • in early cancer/disease detections; • heavy metal, persistent organochloride based-pesticides detection and toxicities • marine/terrestrial natural product discoveries for cancers treatments.
Authorship: Yoong Soon Yong (1), Eric Tzyy Jiann Chong(2), Hsin-Chang Chen(4), Ping-Chin Lee (2), Yee Soon Ling (1,3) *
(1)Biotechnology Research Institute, (2)Faculty of Science & Natural Resources, (3)Water Research Unit Universiti Malaysia Sabah, Jalan UMS, 88400 Kota Kinabalu, Sabah, Malaysia. 4)Genomics Bioscience & Technology Co. Ltd. 4F., No. 92, Sec. 1 Xintai 5th Rd., Xizhi Dist., New Taipei City 221, Taiwan.
| Short Abstract In high-throughput metabolomic profiling, chromatographic separation is crucial because well-performed chromatographic separation may reduce signal suppression from the complex biological matrices and improve the discoverability of low-abundance metabolites. We compared the performance of pentafluorophenyl (PFP)- and octadecylsilane (ODS)-based columns in profiling biological fluids. Total and extracted ion chromatograms demonstrated that the PFP column achieved better analyte separations than did the ODS column. Application of PFP column in metabolomes profiling extracted from urine and serum samples. There were 26 identified lipid species were significantly perturbed in overweight participants. Overall, choline-containing lipids were the most abundant perturbed lipidome, followed by sphingolipids and various phospholipids. |
Long Abstract
Untargeted profiling of biological fluids is useful for capturing and identifying the metabolic signatures of healthy and diseased individuals and simultaneously obtaining insights into their physiological status. In high-throughput metabolomic profiling, chromatographic separation is crucial because well-performed chromatographic separation may reduce signal suppression from the complex biological matrices and improve the discoverability of low-abundance metabolites. The successful acquisition of metabolites within the sample depends on the peak shape quality of the analytes. Distorted peaks often jeopardize correct peak integration and reduce analyte sensitivities and resolutions [1], which may lead to inaccurate results. Several researchers have suggested multiple factors, such as mobile phase, analytes concentration, and appropriate column selection, that affect peak quality [1]. We compared the performance of three RPLC columns, including the commonly used ODS and pentafluorophenyl (PFP) columns, in the chromatographic separation of polar and nonpolar metabolites extracted from the biological fluids of normal and overweight participants at positive and negative ionization modes.
Methodology:
This study was approved by the Ethics Review Board of University Sabah Malaysia (REF: JKEtika 1/15 (7)). Blood and urine samples were obtained from deidentified donors after informed consent was obtained. Urine samples were collected and treated as described by Contrpois et al but with a marginal modification [2]. On the other hand, serum was prepared using whole blood coagulated at a low temperature (4°C). The coagulated blood was centrifuged at 2500g for 15 min at 4°C. The supernatant was collected for extraction. The extraction protocol for serum was a modified version of the Bligh and Dyer extraction protocol [3, 4]. We compared the performance of pentafluorophenyl (PFP)- and octadecylsilane (ODS)-based columns in profiling biological fluids. Peak resolutions and consistencies were acquired using several reversed phased columns and were evaluated. Developed method was applied to analyze t metabolome perturbation in overweight compared to normal participants. All acquired data raw data were preprocessed using MZmine 2 and subjected to multivariate analyses. To inspect the contribution variables (nonpolar metabolites) and to distinguish between the groups in our PLS-DA model, variable importance in projection (VIP) was conducted with a threshold of 1.5. Subsequently, the VIP scores were cross-validated with statistical analysis by using the t test; p < 0.05 was considered significant. Nonpolar metabolites that led to clustering of both groups at different ionization modes were successfully identified.
Result
Total and extracted ion chromatograms demonstrated that the PFP column achieved better analyte separations than did the ODS column. Low relative standard deviations on peak areas and retention times (<10.2% and <0.9%, respectively) acquired using the PFP column evidenced the high reproducibility and consistency of the PFP column. The PFP column yields higher analyte peak resolutions because its stationary phase offers various interactions with analytes, including dipole–dipole, hydrogen-bonding, ion-exchange, and π–π interactions, whereas the ODS column governs the analyte separations on the basis of the hydrophobicity and silanol interactions of the analyte. In our study, a PFP column was used for profiling metabolomes extracted from urine and serum samples. This metabolomic study revealed a metabolomic difference in normal and overweight participants. In total, 26 lipid species were significantly perturbed and further identified. Choline-containing lipids were the most abundant perturbed lipidome in overweight participants, followed by sphingolipids and various phospholipids.
Conclusion
The performance of the PFP column was superior to that of the ODS columns. Metabolites in the urine sample profiled using the PFP column exhibited high resolutions (peak separations). The peak area acquired using the PFP column during intrabatch and interbatch analysis (serum) evidenced that the RSD of the PFP column was relatively lower than that of the ODS columns. This result indicated that the PFP column has higher reproducibility and enhances the discoverability of low-abundance metabolites. Through statistical analyses, we identified differences between overweight and normal participants; 26 potential lipids with significant (p < 0.05) changes were identified in samples extracted from overweight participants. Therefore, we recommend the use of a PFP column in high-throughput metabolomics to promote the development of basic biological and clinical research in the future.
References & Acknowledgements:
1. Keunchkarian, S., et al., Effect of sample solvent on the chromatographic peak shape of analytes eluted under reversed-phase liquid chromatogaphic conditions. J Chromatogr A, 2006. 1119(1-2): p. 20-8.
2. Contrepois, K., L. Jiang, and M. Snyder, Optimized Analytical Procedures for the Untargeted Metabolomic Profiling of Human Urine and Plasma by Combining Hydrophilic Interaction (HILIC) and Reverse-Phase Liquid Chromatography (RPLC)-Mass Spectrometry. Mol Cell Proteomics, 2015. 14(6): p. 1684-95.
3. Ling, Y.S., et al., NMR- and MS-based metabolomics: various organ responses following naphthalene intervention. Mol Biosyst, 2014. 10(7): p. 1918-31.
4. Jensen, S.K., Improved Bligh and Dyer extraction procedure. Lipid Tech, 2008. 20(12): p. 280-281.
ACKNOWLEDGEMENT
The authors highly appreciated the financial supports from Ministry of Higher Education, Malaysia (FRGS0428-SG-1/2015), and Universiti Malaysia Sabah (SBK0180-SG-2014 and TRGS0006-SG-2/2014).
| Description | Y/N | Source |
| Grants | yes | Centre for Research and Innovation |
| Salary | no | |
| Board Member | no | |
| Stock | no | |
| Expenses | no |
IP Royalty: no
| Planning to mention or discuss specific products or technology of the company(ies) listed above: | no |