Looking for ways to clean up your metabolomic and other omic data? Check out these tips.
Thanks for coming back for article 3 in a series dedicated to increasing awareness of factors that can affect metabolomic (and other omic) data quality. At this point I hope you’re glad that you didn’t have to spend 15 years conducting these experiments yourself!
Following the previous article on Sample Storage Time, today I’ll continue the 42 Factors Series with another cell-associated factor—detachment method. You may not have considered the biological impacts of this mundane process.
Factor – Adherent Cell Detachment Method
Trypsinization is the most popular method for detaching adherent cells from the plastic surfaces on which they are cultured. Add a little trypsin solution (which usually contains EDTA as well) to the flask or dish in which your cells are growing, wait a few minutes for the protease to cleave the extracellular adhesion proteins (and the EDTA to chelate metal atoms from adhesion proteins), then inactivate the trypsin by adding serum, which contains protease inhibitors. The cells are now ready for expanding, splitting, or conducting other procedures.
In the microarray days, our laboratory found that trypsin treatment caused significant up-regulation of multiple stress response pathways. It’s well-known that trypsin introduces artifacts in proteomics, too. It should come as no surprise, then, that trypsin causes artifacts in metabolomics.
We compared metabolite levels in cell samples prepared by trypsinization or scraping, and we observed a dramatic increase in metabolites associated with oxidative stress:
Since accurately measuring redox metabolites is a major goal of metabolomics, trypsin clearly should be avoided during preparation of cell samples. But the trypsin vs. scraping debate isn’t resolved by just avoiding trypsin during sample preparation—two additional issues should be considered.
One additional issue is that scraping cells appears to cause efflux of nucleotides (e.g., ATP) out of the cellular compartment, which we learned by examining extracellular fluid after scraping and centrifuging cell samples. We’re not sure why it happens, but the fix is easy—simply retain and measure the extracellular fluid. For example, add a suitable metabolite extraction solvent (this will become the extracellular fluid) to your adherent cells, scrape the cells, vortex-mix to facilitate metabolite extraction, centrifuge, then process the supernatant for analysis of metabolites (for instance, using mass spectrometry). That procedure will capture nucleotide metabolites that play a role in energy metabolism.
The final issue to consider is that trypsin is generally needed to prepare adherent cells for “seeding” to initiate a metabolomics experiment. If we can’t eliminate trypsin from the metabolomics workflow entirely, how can we minimize trypsin artifacts in our data? As a general rule, we wait two days after trypsinization before preparing cell samples for metabolomic analysis. For example, we trypsinize and seed cells into plastic dishes on Monday and wait until Wednesday to initiate a time course experiment in which time = 0 begins on Wednesday, allowing cells two days to recover from trypsin-induced stress. Note: we haven’t conducted detailed experiments to characterize the restoration of baseline metabolic conditions after trypsinization, so two days is only a recommendation.
Also note that Accutase® is a trypsin alternative that is marketed as being a milder alternative to trypsin. We haven’t yet assessed it, but many of our colleagues have suggested it, so we’ll try to do it in the future (unless someone else does it first).
Incidentally, if accurately measuring ATP is a priority, I generally recommend live cell assay kits (e.g.., Promega’s CellTiter-Glo). Promega also has great kits for difficult-to-measure metabolites including H2O2 (peroxide), NADH/NAD, NADPH/NADP, and GSH/GSSG (glutathione), among others.
Take-home messages: 1) allow sufficient time for cells to recover from trypsin stress; and 2) scrape cells directly into extraction solvent to improve accuracy and precision in metabolomics (and possibly other types of experiments).
Big thanks to Jennifer Dennison for pointing this factor out to us, and to present and former members of my team, especially Leslie Silva, who helped troubleshoot this issue.
Do you have any experience with trypsin artifacts? Any pressing metabolomics questions? Leave a comment.
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