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Small - and therefore pushed. Open in a devilish window Figure 2.
Only two datasets e. Select desired settings omline the drop-down list. Pre-populated parameters are available for different instrumentation setups, and these can be further modified for experimental needs. Submit and confirm job. Ensure that the LC alignment overlays any landmark peaks.
If any of the unaligned LC runs show large deviation clementian landmark peaks this may indicate a problem with the sample. For SIEVE, if internal standards are used, locate the corresponding peak group and normalize to the selected frame. Plot the data by coefficient of variation CV within a like sample population e. Any large CV values may indicate a problem with one or more sample. Sort the list based on desired traits e. Examine each peak for appropriate peak alignment across groups, peak integration, Gaussian peak shape, signal intensity, isotopes, and adduct assignment.
Export data as desired for further analysis or to generate targeted mass lists for confirmation and MSn experiments.
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Representative Results The results presented show selected data from a 6-hr onlihe of SH-SY5Y glioblastoma cells with the pesticide and mitochondrial complex I inhibitor rotenone. For brevity, only the organic phase positive mode data is presented. Although a large number of hits Figure 2, Figure 3 are identified by the two programs used for differential analysis these features include likely artifacts, poorly integrated peaks, and other features of questionable analytical value. This can be judged by screening the hits for appropriate signal intensity, low variation between samples of the same group, background levels, and good chromatographic peak shapes Figure 3.
To demonstrate the downstream confirmation of initial hits, we isolate a feature with a mass corresponding to our treatment compound rotenone within 3 ppm Figure 3, Figure 4.
onlinne We also identify a differentially abundant feature reduced in the rotenone treated group, tentatively identified as D-pantethine by accurate mass database searching Figure 3. Open in a separate window Figure 1. Phase Misss, liquid chromatography, and analysis by different ion modes in the mass spectrometer are outlined. Open in a separate window Table 1. General and compound optimized UPLC conditions. For organic phase 1, constant flow rate of 1. Open in a separate window Figure 2. Each dot represents a distinct "feature. Increasing size of the dot indicates increasing magnitude of fold change between the groups, and the intensity of color indicates a decreasing p-value with increasing saturation of color.
Open in a separate window Figure 3.
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