Abstract
Peptide biomarkers in plasma or serum are subject to proteolytic degradation caused by intrinsic peptidase activities, resulting in a potential barrier in translating a discovered biomarker into clinical application. This chapter describes a method using time-course MALDI-TOF MS analysis to investigate the stability of a plasma peptide biomarker under a variety of preanalytical situations. A synthesized peptide with the same primary sequence as a potential endogenous biomarker is spiked into a blood sample, and the sample is incubated over time at r.t. (25 ± 1°C) or other preanalytical situations. At a specific period of incubation time, the sample is quenched with the addition of acid with or without an internal control peptide. The spiked peptides in the sample are extracted with one of three procedures for highly soluble, moderately soluble, or essentially insoluble peptides. The peptide samples are then analyzed using MALDI-TOF MS. The abundance changes of the peptide biomarker are monitored by time-course changes of the mass spectra. These changes over-time are measured and fitted to a first-order degradation reaction so that stability of the peptide biomarker (half-life) can be calculated. Kinetics analysis of both parent and shorter (daughter) peptides are also possible by fitting to a sequential multiple-step reaction (SMSR) model. This optimized method facilitates evaluation of biomarker stability, and helps to define sample handling and analytical processing steps that contribute to instability of measured peptide biomarker(s).
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Notes
- 1.
For Research Use Only, Not For Clinic Diagnostics. BD P800 tube is to stabilize metabolic bio-active peptides such as GLP-1, GIP, Glucagon and Ghrelin.
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Acknowledgments
We are grateful for the encouragement and support of our proteomics program from Robert Pierce. We are also thankful for continuous supports and helps from Frank Augello, Dr. Kesava Nagar-Anthal, Dr. Paul Holmes, Dr. David Warunek, Lena Khumush, and Lisa Gevirtz.
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Yi, J., Liu, Z., Gelfand, C.A., Craft, D. (2011). Investigation of Peptide Biomarker Stability in Plasma Samples Using Time-Course MS Analysis. In: Simpson, R., Greening, D. (eds) Serum/Plasma Proteomics. Methods in Molecular Biology, vol 728. Humana Press. https://doi.org/10.1007/978-1-61779-068-3_10
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DOI: https://doi.org/10.1007/978-1-61779-068-3_10
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