The EIPeptiDi tool: enhancing peptide discovery in ICAT-based LC MS/MS experiments-4

<p><b>Copyright information:</b></p><p>Taken from "The EIPeptiDi tool: enhancing peptide discovery in ICAT-based LC MS/MS experiments"</p><p>http://www.biomedcentral.com/1471-2105/8/255</p><p>BMC Bioinformatics 2007;8():255-255.</p><p>Published online 15 Jul 2007</p><p>PMCID:PMC1939855.</p><p></p

The EIPeptiDi tool: enhancing peptide discovery in ICAT-based LC MS/MS experiments-1

<p><b>Copyright information:</b></p><p>Taken from "The EIPeptiDi tool: enhancing peptide discovery in ICAT-based LC MS/MS experiments"</p><p>http://www.biomedcentral.com/1471-2105/8/255</p><p>BMC Bioinformatics 2007;8():255-255.</p><p>Published online 15 Jul 2007</p><p>PMCID:PMC1939855.</p><p></p> in the 7 samples data set A

The EIPeptiDi tool: enhancing peptide discovery in ICAT-based LC MS/MS experiments-3

<p><b>Copyright information:</b></p><p>Taken from "The EIPeptiDi tool: enhancing peptide discovery in ICAT-based LC MS/MS experiments"</p><p>http://www.biomedcentral.com/1471-2105/8/255</p><p>BMC Bioinformatics 2007;8():255-255.</p><p>Published online 15 Jul 2007</p><p>PMCID:PMC1939855.</p><p></p

PAGE analysis of inositol pyrophosphate during <i>D. Discoideum</i> development.

<p>Amoeba development program was induced as described in material and methods. The inositol phosphates extracted at the indicated time points were resolved on 35% PAGE and visualised with Toluidine. (A) The analysis of wild type (WT) <i>D. discoideum</i> developmental program reveal a 2.6fold increase in the IP₈/IP₆ ratio at the late stage of development, as quantified by densitometry quantified (Bottom), average +/− SD of four independent experiments. (B) To the contrary inositol pyrophosphates are not induced during IP₆-Kinase (ip6k null) developmental program. The figure shows the result of a representative experiment that was repeated four times for the WT and two times for ip6k1 null.</p

Treatment by <i>Phytase</i>, Ddp1 and acidic degradation define IP₆, IP₇, and IP₈, in <i>D. discoideum</i> cell extract.

<p>Wild type <i>D. discoideum</i> cell extract (-) was incubated with phytase (Phy) (A), recombinant diphosphoinositol polyphosphate phosphohydrolase (DDP1) (B) or treated with acid at high temperature (C). The inositol phosphate nature of the three major bands detectable by Toluidine stain is demonstrated by the Phytase treatment (A), an enzyme able to remove the phosphate group from any position of the inositol rings. The pyrophosphate nature of the two slower migrating bands is demonstrated by their disappearance after DDP1 treatment (B) and by the well known acidic sensitivity of the phosphoanhydride bond (C). The figure shows the result of a representative experiments repeated three to four times.</p

The EIPeptiDi tool: enhancing peptide discovery in ICAT-based LC MS/MS experiments-0

<p><b>Copyright information:</b></p><p>Taken from "The EIPeptiDi tool: enhancing peptide discovery in ICAT-based LC MS/MS experiments"</p><p>http://www.biomedcentral.com/1471-2105/8/255</p><p>BMC Bioinformatics 2007;8():255-255.</p><p>Published online 15 Jul 2007</p><p>PMCID:PMC1939855.</p><p></p>w represents a sample. Colored boxes indicate that a H/L ratio is available for the corresponding peptide

The EIPeptiDi tool: enhancing peptide discovery in ICAT-based LC MS/MS experiments-2

<p><b>Copyright information:</b></p><p>Taken from "The EIPeptiDi tool: enhancing peptide discovery in ICAT-based LC MS/MS experiments"</p><p>http://www.biomedcentral.com/1471-2105/8/255</p><p>BMC Bioinformatics 2007;8():255-255.</p><p>Published online 15 Jul 2007</p><p>PMCID:PMC1939855.</p><p></p> in the 10 samples data set B

Analysis of <i>Dictyostelium discoideum</i> Inositol Pyrophosphate Metabolism by Gel Electrophoresis

<div><p>The social amoeba <i>Dictyostelium discoideum</i> was instrumental in the discovery and early characterization of inositol pyrophosphates, a class of molecules possessing highly-energetic pyrophosphate bonds. Inositol pyrophosphates regulate diverse biological processes and are attracting attention due to their ability to control energy metabolism and insulin signalling. However, inositol pyrophosphate research has been hampered by the lack of simple experimental procedures to study them. The recent development of polyacrylamide gel electrophoresis (PAGE) and simple staining to resolve and detect inositol pyrophosphate species has opened new investigative possibilities. This technology is now commonly applied to study <i>in vitro</i> enzymatic reactions. Here we employ PAGE technology to characterize the <i>D. discoideum</i> inositol pyrophosphate metabolism. Surprisingly, only three major bands are detectable after resolving acidic extract on PAGE. We have demonstrated that these three bands correspond to inositol hexakisphosphate (IP₆ or Phytic acid) and its derivative inositol pyrophosphates, IP₇ and IP₈. Biochemical analyses and genetic evidence were used to establish the genuine inositol phosphate nature of these bands. We also identified IP₉ in <i>D. discoideum</i> cells, a molecule so far detected only from <i>in vitro</i> biochemical reactions. Furthermore, we discovered that this amoeba possesses three different inositol pentakisphosphates (IP₅) isomers, which are largely metabolised to inositol pyrophosphates. Comparison of PAGE with traditional Sax-HPLC revealed an underestimation of the cellular abundance of inositol pyrophosphates by traditional methods. In fact our study revealed much higher levels of inositol pyrophosphates in <i>D. discoideum</i> in the vegetative state than previously detected. A three-fold increase in IP₈ was observed during development of <i>D. discoideum</i> a value lower that previously reported. Analysis of inositol pyrophosphate metabolism using ip6k null amoeba revealed the absence of developmentally-induced synthesis of inositol pyrophosphates, suggesting that the alternative class of enzyme responsible for pyrophosphate synthesis, PP-IP₅K, doesn’t’ play a major role in the IP₈ developmental increase.</p></div

No alteration of IP₇ and IP₈ metabolism after cAMP treatment.

<p>Vegetative growing <i>D. Discoideum</i> were incubated for the indicated time with 50 µM cAMP. The incubation was terminated with equal volume of 2M Percloric acid to extract the inositol phosphates. These were resolved on 35% PAGE and stained with Touilidine blue. Two independent experiments are shown with short (left) and long (right) cAMP incubation time. The figure shows the result of a representative experiment that was repeated three times.</p

Characterization of <i>D. discoideum</i> IP₅ species.

<p>Half of the acidic cell extract (from 5 ml culture) of WT <i>D. discoideum</i> was incubated on ice (-) while the second half was incubated at 90°C for 20 min (Acid). Both samples were thenneutralised and resolved on 35.5% PAGE together with the six possible IP₅ isomers. Inositol phosphates were visualised by Toluidine staining. Densitometry analysis of treated versus untreated sample was performed and IP₆ and IP₅s bands intensity compared. (A). Acidic treatment reveals the distinct presence of three IP₅ species, which are otherwise barely detectable, indicating that <i>D. discoideum</i> possesses a complex IP₅-derived inositol pyrophosphate metabolism. (B) Schematic representation of inositol pyrophosphate metabolism in <i>D. discoideum</i>. The gray arrow to (PP)₂-IP₃ indicates a likely potentiallye step. The dashed arrow from inositol to IP₃ indicates uncharacterized enzymatic steps. The figure shows the result of a representative experiment that was repeated three times.</p