Mass spectrometry:

MS-imaging: (spatial metabolomics)

• ionizaton with specific treatment
• MS → visualize where a molecule is located.
• First you get distribution but not yet what molecule it is

iKnife

• MS during operation
• ionizes tissue → coupling to the MS
• very usefull for tumor surgerty

Metabolomic types

• metabolome: contains a wide range of molecules (structure, formula and concentrations)
• Isomere distinction is a challenge
• types:
  • MS
    • more sensitive, wider range can be studied
  • NMR
    • quick, less sample preparation
• Global vs targeted
  • Global: identify as many metabolites as possible
    • challenge: reliable identification (how m/z to actual compound?)
    • hypothesis generation
  • Targeted: quantify a given metabolit in (patient) samples
    • challenge: accurate quantification
    • hypothesis testing
• why couple LC to MS

  • LC is used to separate the compound (on whatever property) based on how well they stick to the collumn

  • in combination to MS, this means not all compounds reach the MS at the same time:

    • slower than without LC, but much more accurate measuring

  • Detection in MS

    • orbitrap: determining m/z ratio
    • quicly turning rod: ions orbit around and back and forth: this gives information about the m/z ratio

    

    • H+ can be added (+ ion) → + ESI, or H+ can be lost (- ion) - ESI. Different molecules prefere - or +
    • Determining the molecule: calculate the mass. Databases help with matching the mass to a molecule. → not necessarily a perfect match but veryvery close
      • might result in more possible matching molecules.
      • determine which is good based on hypothesis/literature, but you want to check it
        • ideally, link to a known compound with a know MS spectrum and retention time.
        • the small peak is not a small part of the molecule because this is not MS/MS
        • monoisotopic mass means: e.g. only 12C and not including the 13C

Identifying novel oncometabolites with untargeted MS

• linear profile WT signal intensity ???
• comparing the compound to known compound. Database shows molecule that differes like this between healthy and unhealthy cells.
  • molecules is fragmented. this database molecule (GSH) was found. But also fumarate was found → biologically it makes much more sense that the molecule is a GSH-fumarate combination
  • They made this suspected molecule and did NMR strucuture analyses → matches with the unknown compound and homemade compound.