Core Facility for Metabolomics

About mass spectrometry

Mass spectrometry is a group of analytical techniques that separate ions by their mass/charge ratio in an electrical or magnetic field.

Picture 1. Pipetting robot.
Picture 1. Pipetting robot.
Picture 2. HPLC columns.
Picture 2. HPLC columns.
Picture 3. Electrospray ionization (ESI) source.
Picture 3. Electrospray ionization (ESI) source.

Analytes must be ionizable, and chemical derivatization may be necessary to achieve this. Mass spectrometers have different properties with regard to mass resolution, mass range and sensitivity, and it may be necessary to use different types of instruments to analyze large (e.g. proteins) and small molecules. To ensure proper identification of unknown compounds instruments with high resolution should be used.

The core facility is equipped with two triple quadrupole mass spectrometers. Two quadrupoles serve as mass filters and are serially coupled with a collision cell in between. The instruments can select masses up to 2 000 Da. Mass resolution is 1 Da or slightly better. Quadrupole instruments are well suited for targeted quantification of small molecules. Methods have high selectivity compared to immunological methods, which makes it possible to analyze several compounds simultaneously in a small sample volume (often ≤ 100 µL). Applications of LC-MS/MS are therefore suitable for biobank studies and for the study of metabolic networks.

Small molecules are a heterogeneous group of compounds with variable physicochemical properties. Therefore methods must be developed and validated separately for every compound that is to be measured, which is often time consuming.

In our laboratory, nearly all sample processing is robotized (Picture 1). Comprehensive procedures may be necessary to remove proteins, which can occlude the chromatographic column, and interferents, for example phospholipids, which can suppress the ionization of compounds that are to be measured, so called ion suppression.

After processing, the sample is injected onto a column to chromatographically separate compounds of interest and interferents (Picture 2). The column effluent it is nebulized to form an aerosol when entering the ion source,  (Picture 3), and compounds to be measured are ionized in a strong electrical field at high temperature. This modality is called electrospray ionization. Charged particles are then pulled into the vacuumized part of the mass spectrometer and are selected in the first quadrupole according to their mass/charge ratio before being collided with gas molecules (argon) in the collision cell. Fragment ions that are characteristic for the compound are then selected in the last quadrupole and detected by a photomultiplier.