How much sample do I need for metabolomic profiling?

Minimum amount of material required:

• Cell culture: 1-2 million cells
• Microbial pellet: 5-25 mg
• Tissue: 5-25 mg
• Biofluids (plasma/ serum and urine): 50 μL

How can I be sure about submitting a correctly processed metabolite extract?

Discuss your sample extraction protocols with the MCF staff. Please follow the guide to sample preparation.

How many metabolites can I measure in my sample?

This depends on the kind of biological sample and metabolite extraction protocol followed. The list of detectable metabolites can be found in: EMBL-MCF Mass spectral library

Is it possible to do structural identification of the metabolites?

Yes, it is possible in a non-targeted workflow but would need additional time, data analysis tools and LC-MS runs. Please see the next FAQ and please discuss this with MCF staff in advance.

What can be the reasons for lesser identification of metabolites from my samples?

There can be several reasons, depending on the type of biological samples. If your sample is enriched with specific peaks compared to a control or blank sample, but no metabolite has been identified, then it’s chiefly the limitation of the database available. We can perform more elaborate studies using open source databases if required. Furthermore, the use of databases search or exact mass filtering (with HMDB, SwissLipids, LIPID MAPS, Mass frontier etc.) may help identify features for which fragmentation spectra have not been acquired or are not available yet in our EMBL-MCF Mass spectral library. The database search will give us an idea about what metabolite that could be. Then we may acquire and match spectra from the corresponding commercially available standards for confirmation.

Why were no metabolites detected in my sample?

The reason could be the dilution of samples, loss of metabolites during sample preparation (i.e. metabolite extraction procedure), or solubility issues during the reconstitution of the dried sample. Please make sure your sample amount meets the requirements and protocol verification was performed by MCF before proceeding with your precious samples. It is highly recommended to discuss your sample extraction protocols at the early phase with the facility staff.

How reliable is the provided information about identified metabolites?

We are performing all the identifications based on in-house generated metabolite spectral libraries via high accuracy mass (~1ppm), isotope pattern, fragmentation pattern (MS/MS), and retention time matching (level 1 confidence identification). Mass spectrometry has inherent limitations in identifying structural and chiral isomers unless they are well separated by chromatography or show selective MS/MS fragmentation. For such special applications, please discuss this with MCF staff in advance.

What kind of results will be provided for my samples?

You will receive a report in Excel format (as a Google spreadsheet) with a list of identified metabolites, mass over charge (m/z) values, chromatographic retention times (RT), and peak areas/intensities. In an untargeted workflow, you will be provided with a list of all detected features m/z, RT and peak areas/intensities) both with and without metabolite identifications. If you have an experimental design with specific sample groups then we can also provide you fold change along with p- and adjusted p-values. Depending on samples and replicates submitted the images of PCA or multivariate data analysis plots can also be provided along with the results. The raw data files, chromatograms and mass spectra can be shared and archived on request.

Would it be possible to perform absolute quantification?

Yes, but you need to add specific internal standards and prepare calibration curves for targeted analysis during your sample preparation. Such methods will need additional optimization and hence please contact the facility staff in advance to discuss it.