Biomolecular Analysis

Small Molecule Mass Spectrometry

The Molecular Analysis facility exists primarily to support studies of plant and microbial metabolism across the John Innes Centre and the Norwich Research Park, where they collaborate with a range of groups to carry out metabolomic studies and small molecule analysis.

They also help those who prefer to carry out their own work, by providing access to instruments and training in their use and provide advice on metabolomic techniques, and training in basic data interpretation.

They are committed to giving the next generation of scientists the skills and knowledge they need for their future careers. Many scientists prefer to run their own samples, as such, they provide training in;

  • Basic chromatography
  • Mass spectrometry
  • Running our instruments

They can also help with the techniques necessary for interpretation of data.

Metabolomic technologies, and supporting expertise, enable us to identify the entire metabolite complement (metabolome) of a sample, whether from a cell or whole organism.

There are two different approaches to metabolite analysis: metabolite profiling and untargeted metabolomics. The most popular analytical approach is to utilise LC-MS or GC-MS.

For metabolite profiling, a very large number of known metabolites are targeted and measured. This is particularly appropriate, for example in medicine, where we know what chemicals are involved in human metabolism but want to know how they are affected by an illness or drug.

In untargeted metabolomics, the aim is to measure a wide range of metabolites without knowing their identity in advance, although they might have some idea about the type compound class (e.g. plant pigments, antibiotics from a microbe, etc.). However, they are typically working without any pre-conceptions. The primary goal is to investigate any metabolite changes in response to a specific treatment or metabolite differences between different types of plant or microbe.

The Molecular Analysis equipment and services at the John Innes Centre, include;

  • Synapt G2i Q-ToF equipped for nano-flow UPLC, conventional UPLC, Maldi, and DESI imaging
  • Bruker Maldi ToF
  • Shimadzu IT-ToF LC-MS equipped with UHPLC and PDA
  • Shimadzu 2020 single quadrupole MS detector, with UHPLC, also equipped with PDA, fluorescence and charged aerosol detectors and fraction collection
  • Waters Xevo TQS triple quadrupole LC-MS, with Acquity UPLC
  • Dionex HPLC with fixed-wavelength and charged aerosol detection
  • Dionex ion chromatography system with pulsed amperometry
  • Agilent 1290 HPLC with PDA and fraction collection
  • Two Agilent GC-MS systems, one equipped with a cutting-edge Gerstel sample preparation robot, capable of derivatisation, centrifugation, incubation, drying and automated SPME.EI

Molecular Analysis equipment

Thermo Dionex Ultimate 3000 HPLC

The Thermo Dionex Ultimate 3000 HPLC system allows conventional analytical chromatography of liquid samples.

Most sample components contain a chromaphore and so can be detected by UV-Vis absorbance. However, for non-volatile sample components that do not contain a chromaphore, an alternative mode of detection is provided: charged aerosol detection (CAD).

The system can be used to separate non-polar and polar samples with either reverse phase or normal phase chromatography.

Shimadzu LCMS-IT-TOF

The LCMS-IT-TOF combines IT (Ion Trap) and TOF (Time of Flight) MS. The IT technology enables MSn spectral capability and the TOF technology enables accurate mass to be measured.

The LCMS-IT-TOF system utilises a UHPLC system which provides fast, high resolution and highly reproducible chromatography. This complements the fast scanning capabilities of the IT-TOF-MS.

This system is best suited for qualitative, comparative sample analysis and/or structural elucidation e.g. metabolomics and/or metabolite identification.

Agilent GC-MS Single Quad (7890/5977) plus Gerstel MPS

GCMS is a powerful tool for separating, identifying and quantifying volatile and semi-volatile compounds in a variety of samples. The gas chromatograph functions to separate a mixture into its constituents, and the mass spectrometer performs the analysis which provides a “fingerprint” of the compound. This fingerprint can be searched for in libraries (e.g. NIST) to identify unknowns.

GC-MS is most suited to smaller molecules. Many are not normally volatile under GC conditions; they have to be derivatised before analysis.

This system is equipped for EI (Electron ionisation) or CI (Chemical Ionisation) and can work in either positive or negative mode. It has been used extensively for analysis of fatty acids, sugars, small polar molecules, and terpenes.

The system also includes a Gerstel MPS which provides a very flexible automated sample introduction system to provide capabilities such as: headspace analysis; sample agitation; sample centrigugation; sample heating; sample transfers; derivatisation; solid phase microextraction (SPME).

Agilent GC-MS Single Quad (6890/5973)

GCMS is a powerful tool for identifying and quantifying volatile and semi-volatile compounds in a variety of samples. The gas chromatograph functions to separate a mixture into its constituents, and the mass spectrometer performs the analysis which provides a “fingerprint” of the compound. This fingerprint can be searched for in libraries (e.g. NIST) to identify unknowns.

GC-MS is most suited to smaller molecules. Many are not normally volatile under GC conditions; they have to be derivatised before analysis.

This system is equipped for EI (Electron ionisation) and can work in either positive or negative mode. It has been used extensively for analysis of fatty acids, sugars, small polar molecules, and terpenes.

Shimadzu LC-MS Single Quad (2020)

The Shimadzu LC-MS 2020 is a bench-top single quadrupole LC-MS system. A wide variety of compound classes can be analysed using electrospray (API-ES) or also using atmospheric pressure chemical ionization (APCI). By using selected ion monitoring (SIM), sensitivity and selectivity can be greatly enhanced. The MS can run in positive mode, negative mode or positive/negative switching mode to collect both sorts of data simultaneously.

The associated Shimadzu UHPLC system can be configured with a variety of detectors i.e. Photodiode Array (PDA), Fluorescence, Charged Aerosol Detector (CAD) as well as the Mass Spectrometer (MS).

The system is capable of flow rates of at least 5ml/min so can be used with larger bore columns (<1cm diameter) for semi-preparative chromatography using the optional fraction collector. This can be triggered for sample collection using the appropriate detector signals including mass directed collection.

Waters Xevo TQ-S Tandem LC-MS

This instrument is best suited to quantitative measurement of known target chemicals, even at very low abundance. It offers enormous selectivity based on measuring an analyte that elutes at a particular time, has a particular precursor mass, and fragments to a known product mass (SRM or MRM).

The Xevo TQS has a very efficient system for transferring ions into the first quadrupole plus a sophisticated collision cell that can accumulate ions, fragment them and then release them synchronously into the final quadrupole as it is scanned. This results in significant improvements in sensitivity.

The instrument is also suitable for neutral loss and precursor ion studies and can be used for data-dependent MS-MS scans on unknown peaks. However, an ion trap MS might be more suited to the latter.

The Xevo TQS is equipped with a UPLC system and is capable of collecting data at UPLC rates, enabling very short run times.

The system can also be used for proteomics for which it has an alternative nano-flow chromatography system.