Chapter 3 Pretreatment

Pretreatment will affect the results of metabolomics. For example, feces collected with 95% ethanol or FOBT would be more reproducible and stable.

Dmitri et.al(Sitnikov, Monnin, and Vuckovic 2016) thought the most orthogonal methods to methanol-based precipitation were ion-exchange solid-phase extraction and liquid-liquid extraction using methyl-tertbutyl ether.

3.1 Quenching

Quenching solvent is always used to stop stop enzymatic activity.

In this review(Lu et al. 2017), authors said:

A classical approach, which works well for many analytes, is boiling ethanol. Although the boiling solvent raises concerns about thermal degradation, it reliably denatures enzymes. In contrast, cold organic solvent may not fully denature enzymes or may do so too slowly such that some metabolic reactions continue, interconverting metabolites during the quenching process.

3.2 Extraction

According to this research(Bennett et al. 2009):

The total metabolome concentration is approximately 300 mM, whereas the protein concentration is approximately 7 mM., which implies that most cellular metabolites are in free form.

  • Tissue samples need to first be pulverized into fine powders

In this review(Lu et al. 2017), authors said:

In our experience, for both cell and tissue specimens, 40:40:20 acetonitrile:methanol:water with 0.1 M formic acid (and subsequent neutralization with ammonium bicarbonate) is generally an effective solvent system for both quenching and extraction, including for ATP and other high-energy phosphorylated compounds. We typically use approximately 1 mL of solvent mix to extract 25 mg of biological specimen. …Thus, although drying is acceptable for most metabolites, care must be taken with redox-active species.

(Luo and Li 2017) nano LC-MS could be used to analysis small numbers of cells

3.3 Instrumental analysis

To get more infomation in the samples, full scan is perferred on GC/LC-MS. Each scan would generat a mass spectrum to cover the setting mass range. If you narrow down your mass range and keep the same scan time, each mass would gain the collection time and you would get a higher sensitivity. However, if you expand your scan range, the sensitivity for each mass would decrease. You could also extend the collection time for each scan. However, it would affect the seperation process.

Full scan is performed synchronously with the seperation process. For a better seperation on chromotograph, each peak should have at least 10 point to get a nice peak shape. If you want to seperate two peaks with a retention time differences of 10s. Assuming the half peak width is 5s, you need to collect 10 mass spectrum within 10s. So the drwell time for each scan is 1s. If you use a high resolution column and the half peak width is 1s, you need to finish a scan within 0.2s. As we talked above, shorter drwell time would decrease the sensitivity. Thus there is a trade-off between seperation and sensitivity. If you use UPLC, the seperation could be finished within 20 min while you need to calculate if you mass spectrumetry could still show a good sensitivity.

References

Sitnikov, Dmitri G., Cian S. Monnin, and Dajana Vuckovic. 2016. “Systematic Assessment of Seven Solvent and Solid-Phase Extraction Methods for Metabolomics Analysis of Human Plasma by LC-MS.” Sci Rep 6 (December). doi:10.1038/srep38885.

Lu, Wenyun, Xiaoyang Su, Matthias S. Klein, Ian A. Lewis, Oliver Fiehn, and Joshua D. Rabinowitz. 2017. “Metabolite Measurement: Pitfalls to Avoid and Practices to Follow.” Annu. Rev. Biochem. 86 (1): 277–304. doi:10.1146/annurev-biochem-061516-044952.

Bennett, Bryson D., Elizabeth H. Kimball, Melissa Gao, Robin Osterhout, Stephen J. Van Dien, and Joshua D. Rabinowitz. 2009. “Absolute Metabolite Concentrations and Implied Enzyme Active Site Occupancy in Escherichia Coli.” Nat Chem Biol 5 (8): 593–99. doi:10.1038/nchembio.186.

Luo, Xian, and Liang Li. 2017. “Metabolomics of Small Numbers of Cells: Metabolomic Profiling of 100, 1000, and 10000 Human Breast Cancer Cells.” Anal. Chem. 89 (21): 11664–71. doi:10.1021/acs.analchem.7b03100.