What is mass spectrometry?
Metabolomics can be run on almost any biological matrix using a variety of instruments, but here we will focus on mass spectrometry-based metabolomics. So what is mass spectrometry?
Essentially, a mass spectrometer is a very fancy scale. Top of the line instruments can measure differences in mass as small as a thousandth of the weight of a single atom (0.001 amu). With that kind of resolution, you can start to identify molecules simply by their mass.
In a normal metabolomics run, a sample is injected into the mass spectrometer over time. At each point in time, the mass spectrometer measures the mass of all molecules entering the mass spectrometer. It then creates a mass spectra with peaks that correspond to each molecule. Scientists can use the mass of the peak (m/z), the time it was collected (retention time, or RT), and the peak intensity to identify which molecule is being measured and its concentration.
At its best, mass spectrometry can give mass, intensity, and retention time information for tens of thousands of molecules in a single 10-minute run.
Example of mass spectrometry metabolomics results
Mass spec jargon:
A peak refers to a mass spec signal with a specific mass. Each molecule with a unique mass will thus have its own peak in a mass spectrum. Depending on your instrument and visualization software, a peak can look like anything from a smooth hill to a craggy mountain to a single vertical line.
The intensity (or height) of a peak is often measured relative to the Total Ion Current (TIC). While more abundant molecules will generally have a higher intensity, TIC is not tied to any physical value and can vary between instruments. Thus, to measure absolute concentrations using mass spectrometry, peak intensities must be compared to standards with known concentrations.
While it is possible to measure absolute concentrations (eg, ng/mol) using mass spectrometry, most experiments rely on relative concentrations. To do so, peak intensities are compared to find the relative abundance of a molecule in each sample. This method can tell you that Metabolite X is three times higher in Sample A than Sample B, but it can’t tell you that Metabolite X is 300 nM in Sample B.
Determining which peak corresponds to which molecule is called annotation.
m/z isn’t technically mass, but instead stands for mass-to-charge ratio. This is because most mass spectrometers measure the mass of positively or negatively charged ions, not neutral molecules. Thus, the m/z value of a molecule will often be its exact mass +/- 1.
You’ll often see metabolomics experiments described as GC-MS or LC-MS. MS stands for mass spectrometry, while LC and GC stand for liquid chromatography and gas chromatography, respectively. Chromatography refers to the way samples are separated before being injected into the mass spectrometer. LC and GC have their advantages and disadvantages depending on your sample type and the analytes you want to measure, but they are the two major forms of sample separation used today. What is LC-MS/MS? Read further to find out!
A metabolic feature refers to the signal corresponding to a molecule. In general, you can usually replace the word feature with metabolite, but feature is the preferred term when we don’t know exactly which metabolite a spectra represents.
