Reproducible ammonium ion adduct formation of PtdInsPs was evaluated, and the relative standard deviation for each type of PtdInsPs was shown in Table 2. Linearity of neutral loss scans of each PtdInsP was evaluated as shown in supplemental Fig. Ammonium ion adduct formation of PtdInsPs was stable and reproducible, and neutral loss scans provided a linear relationship between signal responses and amounts of PtdInsPs. Precursor ion scans also enabled reproducible quantitation of PtdInsPs; however, the ion abundances of precursor ion scans of sn- 1 MAG were lower than neutral loss scans of headgroups.
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Thus, we used neutral loss scans for relative quantitation of PtdInsPs from biological samples. Reproducibility in the quantification of ammonium ion adducts of PtdInsPs. Thus, neutral loss scans, corresponding to phosphoinositol headgroups, have been performed to profile PtdInsPs. In the present study, a neutral loss scan of was used to profile potential PtdInsP 3 isomers due to ammonium ion adduction.
Likewise, neutral loss scans of , , and were used to profile PtdInsP 2 , PtdInsP, and PtdIns species, respectively, from cells and mice livers. A neutral loss scan only provides the number of phosphate groups in the inositol phosphates of PtdInsPs, but fatty acyl compositions at the sn- 1 and sn- 2 positions could not be identified.
CID spectra for PtdInsPs should be obtained to confirm the complete structural characterization of the lipid species Potential PtdInsPs in biological samples should be selected from a neutral loss scan result for complete structural elucidation of the PtdInsPs, and this process is often complicated and tedious.
Then, MS 2 experiments for all potential PtdInsPs should be performed with frequent modification of the method depending on specific PtdInsPs observed in biological samples. Otherwise, the composition of fatty acyl chains in PtdInsPs could not be identified only with the neutral loss scan result. DAG-specific precursor ion scans could not elucidate the fatty acyl compositions at the sn- 1 and sn- 2 positions of PtdInsPs either In our present study, precursor ion scans of sn- 1 MAG were performed to identify sn- 1 fatty acid of PtdInsPs in positive ion mode.
Mass differences of Da among differentially phosphorylated PtdInsPs having the same fatty acyl compositions and comparison of LC retention times from neutral scan spectra enabled us identify the unknown PtdInsPs with sn- 1 fatty acid information. Biological samples were known to primarily contain , , and fatty acids at the sn- 1 position of phospholipids Thus, we included precursor ion scans for , , and fatty acids, as well as for internal standards, and the precursor ion scan could then be modified depending on the fatty acid compositions of any PtdInsPs.
The sn- 1 and sn- 2 fatty acids of PtdIns were assigned with specific fatty acid compositions using the result from precursor ion scans shown in Fig. PtdInsPs having fewer unsaturated fatty acids or longer chain fatty acids were likely to elute late as shown in Fig. Precursor ion scan of was performed to find PtdInsPs having stearic acid at the sn- 1 position shown in Fig. The peak eluted later However, the peak eluted earlier Precursor ion scans also enabled reproducible quantitation of PtdInsPs; however, the ion abundances of precursor ion scans of sn- 1 MAG were lower than neutral loss scans of headgroups in positive ion mode.
Mass spectra of precursor ion scans of to profile PtdInsPs containing fatty acids at sn- 1 position of glycerol backbone from insulin-treated cells, when 5 mM ammonium formate was included in the LC mobile phases. Insulin has been shown to stimulate Akt phosphorylation via activation of PI3K Western blot analysis shows Akt phosphorylation by insulin as shown in Fig. PtdInsP 3 was increased after insulin treatment, compared with the corresponding PtdInsP 3 from control cells. The ion abundances of precursor ion scans of sn- 1 MAG were lower than neutral loss scans of phosphoinositol headgroups.
Thus, we used neutral loss scans for the quantitation of PtdInsPs from biological samples.
A Western blot analysis of extracts from insulin-treated cells. Peak area was the normalized peak area of each PtdInsPs to the corresponding internal standard from neutral loss scan analysis. Changes of PtdInsPs were calculated by dividing the average peak areas of PtdInsPs in insulin-stimulated cells by those in control cells. Changes of PtdInsPs in insulin-stimulated mice livers are shown in Fig.
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Generally, PtdInsP expression in livers was increased after insulin treatment, but PtdIns expression was decreased. Interestingly, PtdInsP 2 levels in cells and livers were not increased when the PtdInsP 3 increment was obvious after insulin stimulation. Changes of PtdInsPs in insulin-stimulated mice livers. Changes of PtdInsPs were calculated by dividing the average peak areas of PtdInsPs in insulin-stimulated mice livers by those in control livers.
Our result shows that the combined strategy of the neutral loss scans of phosphoinositol headgroups and precursor ion scans of sn- 1 MAG is able to profile quantitatively PtdInsPs with structural information of the sn- 1 and sn- 2 fatty acids, and stable ammonium ion adduction is especially beneficial to observe more PtdInsPs. Quantitative profiling of PtdInsPs in cells and tissues was successfully performed with structural information of the sn- 1 and sn- 2 fatty acid compositions. Regioisomers of PtdInsPs, considering different phosphorylation sites on the inositol groups, could not be resolved with this method, and solving this problem remains part of our future work.
Separation of Labeled Inositol Phosphate Isomers by High-Pressure Liquid Chromatography (HPLC)
Previous Section Next Section. View this table: In this window In a new window. TABLE 1. Signal enhancement due to ammonium ion adduction. TABLE 2. Previous Section.
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Toker , A. Phosphoinositides and signal transduction. Life Sci. CrossRef Medline Google Scholar.
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Mayinger , P. Phosphoinositides and vesicular membrane traffic. Okada , M. Jang , and K. Akt phosphorylation and nuclear phosphoinositide association mediate mRNA export and cell proliferation activities by ALY. Rajala , R. Rajala , A. Morris , and R.
Phosphoinositides: minor lipids make a major impact on photoreceptor cell functions. Google Scholar. Vivanco , I. The phosphatidylinositol 3-kinase AKT pathway in human cancer. Kim , I. Kim , J. Kim , H. Kwak , and G. Di Paolo , G. De Camilli. Phosphoinositides in cell regulation and membrane dynamics. Krauss , M. Phosphoinositide-metabolizing enzymes at the interface between membrane traffic and cell signalling.
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