Robust metabolic profiling for routine quantitation and confident unknown identification

Metabolomics, the investigation of small molecules present in a biological system, finds application in diverse biomedical and industrial research. In large-scale applications, targeted profiling of metabolites is employed as it provides confident measurements for compounds of interest. This requires analytical rigor so that concentrations of select metabolites can be measured reproducibly. However, sometimes the observed phenotype cannot be explained by only monitoring a subset of metabolites. Instead, comprehensive metabolome coverage is needed in order to provide insights into underlying biochemical mechanisms.

Structural diversity of the metabolome necessitates data collection with multiple ionization modes for a complete picture. High-resolution, accurate-mass Thermo Scientific™ Orbitrap™ technology with polarity switching enables unbiased compound detection for the accurate analysis of target metabolites and the retro-mining of data to discover unknown biomarkers. In this on-demand webinar, Amanda Souza, Metabolomics Program Manager at Thermo Fisher Scientific, presents a targeted profiling workflow for the nutrient evaluation of bovine serum samples as supplements in cell culture.

Read on for highlights of the live Q&A session or register to watch the webinar at any time that suits you.

Q: Do you always need to have standards for metabolite profiling? What instrumentation can be used to identify and confirm metabolites if standards are not available in addition to Orbitrap?

AS: The first question here is about standards in metabolite profiling and I would highly recommend the use of standards, whether it is metabolite profiling for targeted or untargeted applications.

Standards allow us to understand if every injection went well, if we have prepared the samples well, even if this means I just must put one or two labeled standards in every injection at the same concentration. We highly recommend using standards.

In terms of the second question related to identification and confirmation of metabolites and the instrumentation, I would say to identify a metabolite, at least within the metabolomics community, we know we must always have the authentic chemical standard as a reference. Something to point out when dealing with unknowns is we can utilize a lot of information to be able to annotate that unknown compound. As I shared in the data, that's exactly what we did, where we used high-resolution accurate mass to give us accurate mass, but also assign isotope structure. Then, we applied fragmentation spectra to search against libraries. All of that information taken together gave us higher confidence in the annotation of the unknown.

Q: In the SBS experiments you showed, the Q-RES Standard was used as an internal standard and for quantitation. We have many other metabolites we want to profile. Can you suggest how we should go about this?

AS: For the Q-RES Standard, there are defined metabolites in the mix, but there are times when perhaps we want to hone in on other metabolites of interest, and certainly, we can incorporate those standards. We do not necessarily have to use those standards as internal standards, just utilize this in a niche standard mixture and define retention time and generate fragmentation for them. In our experiment, we used all the standards as internal standards such that they were spiked into every sample, but we did not have to do that. We could have used some of those compounds just to generate the retention time and the associated information. There is a lot of flexibility in how you can set this up, depending on what outcome you are trying to obtain.

Q: In the targeted SBS application example, can you describe your absolute quantitative approach further, and indicate if any alternate strategies were evaluated for comparison?

AS: We were able to determine the concentration values for the compound that were in the Q-RES mixture. We had a combination of labeled and unlabeled standards. The unlabeled standards were generated in a serial dilution of water, and that is what created the calibration curve. We labeled the corresponding standard as an internal standard. This is how we set up our quantitation, specifically absolute quantitation as the calibrants. We chose this as our only approach. We could have tried other approaches, for instance, creating the calibration curve with a matrix sample. For this experiment, we went ahead with just the water dilution.

Q: The data from this experiment was from an Orbitrap Exploris 120. How does this mass spectrometer differ from the other Exploris instruments?

AS: In this experiment, the data set was collected on the Orbitrap Exploris 120. Within the Exploris platform, I will mention there are three instruments, the Orbitrap Exploris 120, the Orbitrap Exploris 240, and the Orbitrap Exploris 480. The difference between these instruments is the resolution number. In this case, on the Orbitrap Exploris 120, the highest resolving power that is available to me is 120, whereas the other two systems that I have just mentioned have the capability to go to higher resolving power. That is one of the primary differences between these three systems.

Find out more about a targeted profiling workflow for the nutrient evaluation of bovine serum samples as supplements in cell culture here>>