Mass Spectrometry facility
Mass Spectrometry facility
The aim of the Mass Spectrometry facility is to provide not just a service for routine metabolomics analysis, but to work in collaboration with academics to develop project lead methods to suit individual needs. We welcome input from researchers within, and external to, the University of Exeter. The facility, which is run by an Experimental Officer, specialises in small molecules (<1000 daltons).
The Biosciences MS facility operates three instruments. The two LC-MS instruments are an Agilent 6520 accurate mass QToF MS/MS and an Agilent 6410 triple quadrupole MS/MS. These instruments are interfaced to liquid chromatography columns via ESI or APCI sources. The LC systems comprise Agilent 1200 SL binary pumps capable of rapid high resolution chromatography, photodiode array, refractive index and fluorescence detectors. LC-MS is complemented by a GC-QToF MS/MS with EI and CI sources. An additional instrument outside the facility is equipped for hydrogen-deuterium exchange experiments. Chemical analysis by MS is complemented by a 400 MHz NMR spectrometer (Bruker) and IR spectroscopy.
Common applications of LC-MS (liquid chromatography-mass spectrometry)
Listed below are some common applications of LC-MS. The list is by no means exhaustive but may offer some guidance regarding your project. If you have an idea and you’re not sure how to go about it, please come and chat to Debbie Salmon.
- Targeted analysis (SIM/MRM) of specific compounds of interest. We currently have LC-MS methods set up for the analysis of amino acids, plant hormones, mammalian steroid hormones, monosaccharides, disaccharides, plant glucosinolates and polar organic acids.
- Concentrations can be calculated if we are provided with the pure compounds (standards), preferably stable isotope labelled standards that samples can be spiked with (internal standards).
- Metabolite profiling: identification of metabolites differentially expressed between samples and their characterisation by accurate mass and MS/MS.
- Comparative quantification of selected unknown metabolites by SIM/MRM.
The schemes described below are examples of various workflows which may be undertaken during a project. These can be used as a guide for your project, but schemes will be tailored to suit individual needs.
Exploratory analysis is performed using the quadrupole-time of flight (QTOF) MS where HPLC and MS parameters are optimized. Raw data is processed using MassHunter Qualitative and Quantitative software. Molecular Feature Extractor (MFE) deconvolves the data to create compound lists (*.mhd files). These can be exported to Mass Profiler or XCMS (The Scripps Research Institute) for alignment and statistical analysis to identify the mass of differentially expressed metabolites between samples.
Metabolite identification using accurate mass and retention time can be investigated using the METLIN Personal Database which can be configured to suit individual needs. Personal databases can be uploaded to the local server to aid searches.
The triple quadrupole (QQQ) MS is generally used for the task of performing multiple reaction monitoring experiments (MRMs) or targeted MS/MS. The HPLC and MS source parameters optimized on the QTOF can be directly transferred to the QQQ. Specified compounds shown to be of interest are isolated and fragmented. The product ions are detected and used for screening and/or comparative quantification.
Some form of sample clean-up is usually necessary (e.g. SPE, LLE, protein precipitation) – it is up to individuals to research the most appropriate methods and prepare their samples. However we are happy to give advice if needed. It is vital that all samples are free of particulate matter. Extractions should be centrifuged (15mins, 13K rpm) then filtered with a syringe ECONOSIL filter (small molecule samples).
If you are interested in undertaking LC-MS analysis, please contact Debbie Salmon, Experimental Officer for Mass Spectrometry.
Please contact Debbie Salmon to discuss your specific needs and we will arrange a quote. However, please note that run times can vary from 10 minutes to over an hour, so we work on a daily charge and cannot quote per sample until we have optimised a compound-specific method.