Following this protocol allows one to characterize a peak of interest in an untargeted metabolomic experiment if it is a metabolite found in METLIN, or is an analog of a metabolite in METLIN. Metabolites that are not analogs of known metabolites are difficult to identify with this technique, although this protocol will provide information that would be valuable when used in combination with other analytical techniques. Some cases which have proved challenging when attempting to identify unknown metabolites are discussed below, they include examples of metabolites which have no exact match in METLIN and metabolites that co-elute with other metabolic peaks of similar m/z.
For our first example, the metabolic peak of interest has a m/z of 496.3409 and a RT of 24.5. The ion spectrum is extracted () from the TIC and upon inspection of the spectrum at m/z 496.3409, another peak is observed at m/z 518.3219, which is 21.981 amu larger. This is characteristic of the [M+Na]+ peak and supports that m/z 496.3409 is the [M+H]+ peak (Na+-H+ = 21.9820). Also noted in (), two isotope peaks for the m/z 496.3409 peak can be seen, m/z 497.3440 and m/z 498.3455. Since these peaks are approximately +1 and +2 from the [M+H]+ peak, it adds validation that this is a singly charged ion and that m/z 496.3409 is indeed the protonated monoisotopic mass of the molecule.
In order to determine the structure of the species at 480.3082 in , caution must be taken due to potential contamination from the species at m/z 479.7786 [M+2H]2+, m/z 480.2805, and m/z 482.2569[M+2H]2+. Indeed, when m/z 480.3082 is isolated and fragmented, the spectrum in is obtained which contains both m/z 480.2805 (isotope of m/z 479.7786) and m/z 482.2567 species. In this situation m/z 480.3082 cannot be identified since the MS/MS spectrum is suppressed and contaminated. If chromatography is used to separate these species like shown in , a pure MS/MS spectrum can be obtained for m/z 480.3084 (), which is characterized as LysoPE (18:1/0:0). Use of a narrow isolation window may also be useful to prevent contamination by other species if the mass difference of two species is sufficient.
The characterization of three metabolites, phenylalanine, arachidonic acid, and hypoxanthine are depicted in . The simple fragmentation of the experimental phenylalanine () and the more complex arachidonic acid () match the standard METLIN spectra in both intensity ratio and accurate mass of the fragments, supporting their identification. The experimental spectrum for hypoxanthine in negative mode () matches well with the METLIN spectrum, although there is significantly more contamination in the experimental sample than observed in positive mode (). Observing that hypoxanthine is dysregulated in both positive and negative mode also validates the characterization of this peak. In addition to the MS/MS pattern, the retention time is another important parameter to consider. For example, the precursor ion m/z 300.2889 is appropriate for both sphingosine C-18 and palmitoylethanolamide (PEA), which have the same formula of C18H37NO2 (). These molecules are indistinguishable by accurate mass alone. If these molecules were not resolved by chromatography, both species would be selected to fragment at the same time, generating a convoluted spectrum that would hinder identification of either species. When resolved, the individual species can be analyzed and structures assigned to each peak, as represented by peaks 1 and 3 in . The relative retention time can support a structural assignment. In , two additional peaks, 2 and 4, can be seen which are analogs of 1 and 3 but are an additional two carbon units long. In general, on C18 based columns, increasing chain number and increasing saturation increases the retention time for a group of molecules with the same functional group. Observing a later retention time for sphingosine C-20 over sphingosine C-18 and stearoyl ethanolamide over PEA is consistent with their characterization.
In a recent study 50
, an unknown endogenous compound was identified utilizing this process, which illustrates the power of LC-Q-ToF based characterization of metabolites. A species with a m/z
of 328.3213 was observed and was up-regulated in a rat model of neuropathic pain. At that time, searching m/z
328.3213 in METLIN returned two structural isomers, stearoyl ethanolamide and sphingosine C-20. The 20 V fragment experimental spectrum was compared against the stearoyl ethanolamide and sphingosine C-20 spectra in METLIN. Stearoyl ethanolamide was quickly ruled out since the experimental spectra lacked the m/z
62.060 ion characteristic of ethanolamides. Comparison to sphingosine C-20 revealed several fragments in common in the 250-310 m/z
range, although their ratios varied significantly. In the range of 40-120 m/z
, there were some low intensity ions that did not match well. Fragmentation at 40 V provided more intense signals in the 40-120 m/z
range, which did not match well to sphingosine C-20, ruling out this metabolite. Additionally, a peak with a m/z
and fragmentation pattern appropriate for sphingosine C-20 eluted a few minutes earlier than the unknown m/z
328.3213 species. Since matching to METLIN was exhausted, other databases were searched with the m/z
, which returned seven results within 0.01 Da. Searching again with the formula C20
, which is the same molecular formula for stearoyl ethanolamide and sphingosine C-20, and the most reasonable formula calculated from the accurate mass of 328.3213, stearoylethanolamide and N, N,-dimethylsphingosine (DMS) was found. DMS was purchased, and LC-Q-ToF analysis of the sample and standard provided matching retention times and MS/MS spectra as reported50
. This strongly supported the identification of the species as DMS and provided the first characterization and quantitation of DMS as a naturally occurring metabolite. Since this analysis was completed DMS has been added to METLIN. Investigators that have access to pure standards of compounds not currently characterized on METLIN can first metlin/at/scripps.edu