Introduction
During the last decade, the sequencing of genomes in different prokaryotic and eucaryotic species has revolutionised biology. The data that these efforts have yielded facilitate analyses that provide insights into the genetic basis of similarities and differences between diverse organisms. They also create new possibilities for investigating the fundamental biology of different organisms, as well as the genetic basis of various diseases. In the era of post-genomics, elucidation of gene function is a main target. Analysis of gene function by targeted knockouts and mutations and the measurement of gene products such as mRNA and protein species are currently the main methods used in functional genomics. However, these methods do not provide all the information needed to determine how changes in mRNA or proteins are linked to changes in biological function. Complex regulatory interactions occur at all levels in eukaryotic cells, and a change at one level in the network does not necessarily lead to a significant change in function or phenotype. Instead, single point mutations or alterations in gene expression may often lead to complex responses. Thus, metabolomic analysis is also needed if the final effects of upstream regulatory events on metabolism are to be determined accurately. So, techniques enabling metabolites to be identified and metabolic fluxes to be quantified are essential to complement the information provided by genetic experiments and the large-scale analysis of transcript and protein profiles in living organisms.
Access to the facility
The Metabolomics facility at Umeå Plant Science has received substantial support from the Wallenberg Consortium North (WCN; http://www.wcn.se/), and the facility is open for the universities associated to the WCN.
Organisation
Scientific responsible and contact person
Thomas Moritz,
UPSC, SLU, Umeå.
Tel:
Research engineer mass spectrometry
Krister Lundgren
UPSC, SLU, Umeå
Tel:
Research engineer chemometrics
Kjell Öberg
Research group for Chemometrics, UmU, Umeå
Tel:
Steering committee
Göran Sandberg, UPSC, Umeå (Chair); Stefan Marklund, UmU, Umeå, Johan Meijer, SLU, Uppsala; Thomas Moritz, UPSC, Umeå; Hans Ronne, SLU, Uppsala.
The steering committee will, if necessary, be responsible to allocate instrument time to the different projects.
Services at the Metabolomics facility
The main services at the facility are the following:
· Advice regarding design of metabolomics experiment
· Advice regarding extraction protocols
· Extraction of samples (depends on the type of samples)
· Mass spectrometry analysis
· Basic multivariate analysis on obtained results (PCA, PLS)
· Simple presentation of obtained results. Only the differences between samples will be presented
The services at the facility will not include:
· Extensive identification of unknown compounds.
· Extensive multivariate analysis on obtained results.
If above mentioned services are necessary, please contact the facility for information about collaborations within different research groups at UPSC or the Chemometrical group at UmU.
Instrumentation at the metabolomics facility
Mass spectrometers funded by WCN, and dedicated to metabolomics
LECO Pegasus III, Gas chromatography - mass spectrometry/time-of-flight analyser (GC/TOF)
Bruker Esquire plus, Liquid chromatography – mass spectrometer (LC/MS)
Mass spectrometers at the UPSC mass spectrometer facility
JEOL JMS-MStation GC/MS (magnetic-sector mass spectrometer)
JEOL JMS-SX/SX102 GC/MS (four-sector tandem mass spectrometer)
Micromass Quattro Ultima LC/MS (triple-stage quadrupole mass spectrometer)
Micromass Q-TOF Ultima LC/MS (quadrupole/time-of-flight mass spectrometer)
Applied Biosystem Voyager-DE STR (Maldi-TOF)
Above instruments might, when necessary, be used within the metabolomics facility.
Methodology
Sample extraction
Extraction involves solvent extraction with internal standards added to the extract prior extraction. The internal standards are isotope labelled compounds, representing classes of different compounds, e.g. amino acids, amines, fatty acids, mono- and disaccharides, sterols. The internal standards can be supplied from the facility, and are included in the cost of the analyses.
Different extraction protocols for different types of samples are under development. Protocols will be on this web-page as soon as possible.
Examples of extraction protocols:
Plant extract: This protocol is based on the possible to use a mixer mill, and doing all extraction in eppendorf tubes. (Ref: Gullberg et al. . Anal. Biochem. 331: 283-295)
1. Weight 10-50 mg plant samples in eppendorf tubes containing internal standards. Keep cold!! Ice bath or cold room.
2. Add 1 ml of chloroform:methanol:H2O (20:60:20) mixture including internal standards mixer beads. Shake 3 min in mixer (30 Hz).
3. Centrifuge in eppendorf centrifuge, 10 min, 14 000 rpm. Take out mixer beads before centrifugation.
4. Take out 200 µl of supernatant (volume depends on amount and type of plant material) and add to GC/MS vial (or LC/MS vial if samples will be analysed by LC/MS). Dry in speed-vac concentrator.
5. Derivatization and GC/MS analysis as described below
Plasma analysis: This protocol is based on the possible to use a mixer mill, and doing all extraction in eppendorf tubes.
1. Add 100 µl of plasma into an eppendorf tube.
2. Add 100 µl H2O including water soluble internal standards to the plasma. Shake vigorously.
3. Add 800 µl of MeOH including methanol soluble internal standards. Let the tubes stand on ice for 10 min.
4. Add mixer beads and shake in mixer for 1 min (30 Hz).
5. Remove the beads, and let the tubes stand in ice for 2 h.
6. Centrifuge in eppendorf centrifuge 5 min, 13 000 rpm.
7. Take out 200 µl of supernatant and add to GC/MS vial (or LC/MS vial if samples will be analysed by LC/MS). Dry in speed-vac concentrator.
8. Derivatization and GC/MS analysis as described below.
Derivatization for GC/MS analysis
With the GC/MS analysis we can detect organic acids, amino acids, fatty acids, amines, mono- and disaccharides, sterols and many more compounds that are volatile or can be converted to volatile derivatives. Samples undergoing GC/MS analysis must therefore be derivatized prior analysis. We use a two-step procedure: carbonyl moieties are first protected using methoximation (MO), and thereafter acidic protons are exchanged for trimethylsilyl groups (TMS).
Samples are dried before derivatisation. For methoximation, 30 µL of 15 mg/mL methoxyamine hydrochloride in dry pyridine is used at room temperature for 16 h. Afterwards, TMS derivatization is performed by the addition of 30 µL of N-methyl-N-trimethylsilyl-trifluoroacetamide (MSTFA1% TMCS) for 1 h at room temperature. Thereafter the sample can be diluted with heptane (e.g. 30 µl) to suitable dilution of sample prior GC/MS analysis. (Ref: Gullberg et al. . Anal. Biochem. 331: 283-295)
GC/MS analysis
One µL of the derivatized sample is injected splitless by an Agilent autosampler into an Agilent gas chromatograph equipped with a 15 m x 0.18 mm i.d. fused silica capillary column with a chemically bonded 0.18 µm DB 5-MS stationary phase (J&W Scientific). The injector temperature is 270°C, the purge flow-rate is 20 ml min-1 and the purge is turned on after 60 s. The gas flow rate through the column is 1 ml min-1, the column temperature is held at 70°C for 2 minutes, then increased by 40°C min-1 to 320°C, and held there for 1 min. The column effluent is introduced into the ion source of a Pegasus III time-of-flight mass spectrometer, GC/TOFMS (Leco Corp., St Joseph, MI, USA). The transfer line and the ion source temperatures are 250°C and 200°C, respectively. Ions are generated by a 70 eV electron beam at an ionization current of 2.0 mA, and 30 (15-30) spectra s-1 are recorded in the mass range 60 to 800 m/z. The acceleration voltage is turned on after a solvent delay of 170 s. The detector voltage is V.
GC/MS is a very robust technique, suitable for routine metabolomics analysis. Identification of compounds is based on comparison with mass spectra libraries (in-house database) as well as retention index.
LC/MS analysis
With LC/MS analysis, with on-line photodiode array detection, we can detect many compounds that are not possible to analyze by GC/MS, e.g. carotenoids, nucleotides, flavonoids, and other thermolabile or large molecules. Many compounds detected by GC/MS will also be detected by LC/MS.
Protocol will be added in the future.
Data analysis
Metabolomics projects generate large sets of data, and the accepted way of comparing large data sets is to use different multivariate tools, e.g. principal component analysis (PCA) or partial least squares projections to latent structures (PLS). PCA is an unsupervised method where no “a priori” knowledge of the class of samples is needed, and it is based on calculation of latent variables. The principle components are linear descriptions of the original descriptors, and are uncorrelated. The components also describe decreasing amount of data variance, i.e. P1>P2 and more. PCA will show the best representation of metabolic variation to be described in a limit number of dimensions. PLS is a supervised method so that the class of a sample from an independent data set can be predicted on the basis of a series of models that are derived from the original data. This will help us to maximize the separation between classes, but also enable us to validate data.
The mass spectrometry data will gain both qualitative and quantitative (relative) information. There are two possibilities to analyse the mass spectrometry data of the sample set:
-
Get quantitative data from every compound in each sample, and thereafter do the multivariate analysis.
- Time consuming
- Quantitative data for each compound detected
-
Do the multivariate analysis from the mass spectrometry raw data set, and thereafter get the quantitative data that are different between the samples.
- Quick
- Not quantitative value for every compound
The metabolomics facility will only use the second strategy, and thereby the data that will be presented are only the differences between samples within a sample set. (Ref: Jonsson et al., . Anal. Chem. 76: )
Submitting samples to the Metabolomics facility
Before submitting samples to the facility the principle investigators must contact the facility to discuss the metabolomics analysis. The facility will participate in the design of the experiments, since the design are very important for obtaining reliable information from the metabolomics analysis. The PI’s are encouraged to send a short description of the experiment in order to speed-up the procedure. When the facility approves to perform metabolomics analysis, the applicant will have information how to send samples, estimated time when the analysis can be done, advice regarding extraction protocols if the facility can not perform the extraction.
When submitting samples, a submittal form must be filled out. This can be obtained from Krister Lundgren (research engineer MS).
Price
The price is subsidised via grants from WCN and other sources. The cost for analysing samples will cover cost of columns, internal standards, reagents and data storage.
This price list is subject to change without notice.
| Administrative cost and starting cost for analysing each sample set: |
250 SEK
|
| Cost per sample 1-50 |
100 SEK
|
| Cost per sample 51- |
50 SEK
|
Links
Compound identification
• Retention index
RTI (PDF-format)
Retention index system (PDF-format)
• EI-TOFMS-library (will come)
Databases metabolic pathways
• KEGG (http://www.genome.jp/kegg/pathway.html)
• TAIR (http://www.arabidopsis.org/tools/aracyc/)
• EcoCyc (http://biocyc.org/ECOLI/class-subs-instances?object=Pathways)
Multivariate analysis and chemometrics
• http://www.acc.umu.se/~tnkjtg/chemometrics/
