The Babraham Institute receives strategic funding from the BBSRC
The Babraham Institute receives strategic funding from the BBSRC
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A new service offered by the Babraham Institute is the analysis of lipids by mass spectroscopy, as described in a recent Nature Methods paper [1].
Download Babraham Lipidomics flier (pdf)
The method is particularly useful for the analysis of phosphatidylinositol (3,4,5)-trisphosphate (PI(3,4,5)P3) species, which are important signalling molecules within cells.
The structure of a typical PI(3,4,5)P3 is shown. The head group contains inositol which is phosphorylated at three positions. The head group is linked through another phosphate to a glycerol unit, which typically has one or two fatty acyl chains attached to it, shown as R1 and R2 in the figure.
The method developed at Babraham allows us to examine the composition of R1 and R2 as well as determining the absolute levels of these compounds in a typical biological sample.
We are also able to analyse other important molecules found in the lipid extracts, and routinely study PtdInsP2, PtdInsP, PtdIns, PtdA and PtdSer along with PtdInsP3. The chemistry facility at Babraham supports the mass spectroscopy work through the synthesis of standards, including materials with C17 fatty acyl-containing glycerols, and d6- and C13-labelled compounds.
The samples are analysed using an AB Sciex 4000 mass spectrometer with a Waters UPLC separation system. Each sample run usually takes about 20 minutes and we usually inject each sample twice so typically we run experiments of about 20 to 30 samples. We have found that for the protocol that we have developed, the best cell number to use is in the region of 100,000 to 300,000 cells, although we have shown that we can detect PI(3,4,5)P3 in 1,000 stimulated neutrophils.
The profile of d6 materials typically has the distribution of deuterium incorporation shown below. This allows us to alter the signal that we are monitoring quickly and easily if we find that there is a component in a particular biological matrix with a co-incidental parent and fragment mass.
[1] Clark J, Anderson KE, Juvin V, Smith TS, Karpe F, Wakelam MJO, Stephens LR, Hawkins PT (2011)
Quantification of PtdInsP3 molecular species in cells and tissues by mass spectrometry.
Nature Methods
http://dx.doi.org/10.1038/nmeth.1564
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