Expert Insight: Essential technology for high-resolution untargeted metabolomics

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Dr. Chris Petucci, University of Pennsylvania

In this webinar, Dr. Chris Petucci provides an overview of the University of Pennsylvania Metabolomics Core and how it performs targeted and untargeted LC/MS metabolomics for internal and external research investigators and industry.

One of the platform’s key enabling technologies for in-depth understanding of biological pathways in disease and biomarker discovery is the Thermo Scientific™ Orbitrap ID-X™ Tribrid™ mass spectrometer. Using the Orbitrap ID-X mass spectrometer, examples of sample preparation, LC/MS method development, and untargeted metabolomics projects in medical research are presented, as Dr. Petucci covers: 

• Stable and reproducible sample preparation of cells, biological fluids, and tissues for metabolomics
• Essentials of chromatography theory and practice for high-resolution chromatography of metabolites
• Strengths of the Orbitrap ID-X LC/MS for untargeted metabolomics
• Basics of statistical data analysis of untargeted metabolomics data using Thermo Scientific™ Compound Discoverer™ software

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

Q: What is your output to users and clients for untargeted metabolomics? Do you only provide information on identified features or do you do it for unknowns as well?

CP: Our output is a formal report that lists both the metabolites that are identified and, through a database search using mzCloud and other databases, the unidentified metabolites as well. These are all provided with molecular formula predictions, retention times, peak areas, fold changes, Log2 fold changes, and adjusted p-values, with the usual corrections for a false discovery rate. We then create, using Compound Discoverer, relevant sampling of whisker plots, heat maps with hierarchical clustering, and volcano plots.

Q: Do you look to use the ID-X for quantitative information as well?

CP: No, we won't be using it for quantitation, only qualitative small molecule untargeted metabolomics and isotope tracers. To get the maximum sensitivity that we need, the Triple Quad is the best instrument to do trace detection. But for high-resolution, accurate mass work, structure elucidation work, and high-resolution isotope tracer work to resolve Carbon-13 and Nitrogen-15, the Orbitrap is the best instrument, especially with the ID-X tailored specifically for high coverage, deep annotation on targeted metabolomics.

Q: How critical is the higher resolution? What does it give you over a more common resolution?

CP: The critical feature of high resolution is that when you go into the high tens of thousands and over the 100,000 mark, your metabolite coverage increases. When you increase from the 40,000 to 60,000 resolution range up to 100,000 and over, the number of metabolite signatures that you can extract from the total ion chromatogram greatly increases. So, a high resolution of over 100,000 makes a difference in the number of metabolites that can be identified and resolved. 

Q: How do you combine the results tables from C18 and HILIC, in particular, for unknowns?

CP: Right now, we just give separate tables for HILIC and electrospray ionization mass spec. There's overlap with certain metabolites, but people can easily see that in the data tables. We keep things separate so that people see what the distinctions are between a PCA plot with data collected in electrospray ionization mode, with C18 chromatography, and data collected in HILIC mode with the ESI- and ESI+ ionization.

Q: How were your QC samples prepared?

CP: We take a 10-microliter aliquot of every study sample and combine it. Then we aliquot that and pull a bulk sample out into separate QC aliquots for extraction. Sometimes it's less than 10 microliters if you're sample limited. In the case of cell-based metabolomics, because sometimes people only give us around 100 microliters or so of frozen cell pellet, or even a little less, we might have to use 5 microliters of study sample per pool.

Q: Is manual inspection of peaks detected in Compound Discoverer important and necessary for reproducible quantitative and qualitative results?

CP: Yes. I tell people that we don't just do a data dump of numbers in an Excel spreadsheet and hope that people blindly believe what we report. In Compound Discoverer we actually go through and survey the data that is reported in the compounds list to see if the chromatography, alignment, and sensitivity above the baseline are sufficient and correct.

It's very, very important to do that because I've seen data from different people shown in Excel spreadsheets where, for example, they report a metabolite that is reported to elute at around two, three minutes. I told the professor: ‘Well, you're excited about that metabolite, but that annotation is wrong because that metabolite absolutely will not elute at two or three minutes. It's too un-polar. It will elute maybe in this particular gradient at five or six minutes.’ So, doing a review of the data as you go and visually inspecting it may be time-consuming, but it's necessary. You can't treat the statistical data processing and extraction programs as a black box and just expect that you're going to get everything with 100% accuracy.

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