| Report Information | |
|---|---|
| Study Number | Weinstein_01152026_19475 |
| PI | John Weinstein |
| Institution | MD Anderson Cancer Center |
| Assay Type | IC-MS Targeted |
| Samples | cell |
| NGCHM Viewer Version | 2.26.0 |
| NGCHM R Version | 1.0.4 |
| MBatch Version | 1.7.6 |
| Template Version | v2.0.0 |
To determine the relative abundance of polar metabolites, extracts were prepared and analyzed by ion chromatography-mass spectrometry (IC-MS). Metabolites were extracted using ice-cold 0.1% ammonium hydroxide in methanol:water = 80:20. Extracts were centrifuged at 17,000 gfor 5 min at 4°C, and supernatants were transferred to clean tubes, followed by evaporation to dryness under nitrogen. Dried extracts were reconstituted in 200 µL deionized water, and 10 µL was injected for analysis. IC mobile phase A (MPA; weak) was water, and mobile phase B (MPB; strong) was water containing 100 mM KOH. A Thermo Dionex ICS-6000+ system included a Thermo IonPac AS11 column (4 µm particle size, 250 x 2 mm) with column compartment set at 35°C. The autosampler tray was chilled to 4°C. The mobile phase flow rate was 360 µL/min, and the gradient elution program was: 0-2 min, 1% MPB; 2-25 min, 1-40% MPB; 25-39 min, 40-100% MPB; 39-50 min, 100% MPB; 50-50.5, 100-1% MPB. The total run time was 55 min. To improve desolvation for better sensitivity, methanol was delivered by an external pump and combined with the eluent via a low dead volume mixing tee. Data were acquired using a Thermo Fisher Scientific Orbitrap IQ-X Tribrid Mass Spectrometer under ESI negative ionization mode at a resolution of 240,000 with a scan range of 70-800 m/z. All raw data files were imported into Skyline/Thermo Trace Finder 5.1 software for processing. The relative abundance (peak area) of each metabolite was normalized by total peak intensity.
The sample table contains metadata for the samples in the cohort. ID represents the sample name, and other column headers indicate additional metadata (e.g., time point, treatment group).
No Normalization: The peak area (raw relative abundance) of each metabolite is presented directly without normalization.
The data table provides normalized metabolite relative abundance. The tables are interactive and each column can be sorted in ascending or descending order. Users can search for a specific metabolite using the search bar and copy or download the data in CSV or Excel format.
Click the button below to show/hide the data distribution plots.
This plot presents Principal component analysis (PCA), with PC1 on the x-axis and PC2 on the y-axis, as is customary. Additionally, the median values are calculated and plotted for each group. The plot is interactive.
Dispersion Separability Criterion (DSC): DSC metric has been designed to quantify the dissimilarity between groups within the data, and can also be used to quantify batch effects. DSC is a ratio of dispersion across batches (Db) vs. dispersion within batches (Dw). A DSC value greater than one means that there is greater dispersion, or dissimilarity, across batches than within batches, whereas a DSC of zero means there is no dissimilarity across batches. Typically, values greater than 0.3 indicate the presence of moderate batch effects, whereas a value greater than 0.6 indicate severe batch effects.
DSC p-value: The p-value is derived empirically, using permutation tests. One thousand or more permutations are typically run on each data set, using different random permutations of the data each time. DSC values are computed for each permutation and at the end of all the runs, the proportion of values greater than the actual DSC value is computed to yield the p-value. The null hypothesis is that there are no batch effects and the data set is homogeneous in terms of batches. A p-value less than some significance threshold (usually 0.05) rejects the null hypothesis.
Plots created with MBatch from the Batch Effects Package
Please see https://pubmed.ncbi.nlm.nih.gov/38260566/.
Next-generation clustered heatmap (NG-CHM) software was used to process the data. NG-CHM plots are interactive and can be used for zooming, panning, searching, covariate bars, and link-outs that enable deep exploration of patterns and associations in heat maps. Note: if you do not have a wheel mouse, you can use two-finger swiping upward/downward on a touchpad to zoom.
The NG-CHM Heat Map Viewer contains two panels. The Summary Panel on the left provides a full view of your heat map. The Detail Panel on the right provides a zoomed-in view of a portion of the heat map. Click anywhere on the summary picture to see the detail of that portion of the map. The green box on the Summary Panel indicates the portion of the heat map currently displayed in the Detail Panel. You may zoom in or out with the mouse scroll wheel or zoom in/out buttons to see smaller or larger portions of the map in the Detail View. Double-clicking on a cell in the Detail Panel will also zoom in and center on that cell and shift-double click will zoom out. When the zoom level permits, row and column labels will be displayed in the Detail Panel. You may also use arrow keys to move up/down/left/right one row in the Detail Panel. The divider bar may be moved by clicking on it and dragging left or right to change the relative size of the Summary and Detail Panels. There are a variety of buttons on the header bar as well to manipulate the heat map view.
NG-CHMs created with the NG-CHM R package.
Please see https://pubmed.ncbi.nlm.nih.gov/32269754/.
Bidirectional Median Centering
A bidirectional median centered, or simply median centered, data matrix is where the column medians are subtracted from each column, then the row medians are subtracted from each row. Such normalization is performed to make patterns in the data matrix stand out in a heatmap. Without such normalization, one would typically observe some rows or columns that are saturated entirely by one color (red or blue) making patterns difficult to observe. However, a side-effect of visualizing median centered data is that because the data are altered, they may not directly correspond with patterns in other plots (like box plots) derived from non-median centered data. To circumvent that, we have included the option of visualizing non-median centered data in the NG-CHM as well by toggling to another layer using the “layer” button () in the upper right corner of the NG-CHM below.
ANOVA was performed for all pairs of samples. FDR-adjusted p-values across all metabolites were calculated and visualized as a dot plot. Metabolites with a p-value less than this displayed cutoff are shown in the dot plot. Complete ANOVA results of all metabolites can be obtained from the Table (Data matrix tab). Dot plots and data matrix tables are interactive. Column names indicate the compared pairwise comparison. The dot size represents the significance based on the adjusted p-value. There are a variety of buttons on the header bar to manipulate the dot plot view. One can zoom, pan, and download the plot.
Difference of abundance between normalized intensities of two time-points, upper and lower confidence interval along with respective p-values for each combination are listed in the table. ANOVA is separately performed for each time-point. Adj.Pvalues represent the p-value of each treatment group adjusted across other pairwise comparison at a single time-point. FDR corrected P-values across all compounds are also added to the table.
The volcano plot provides differentially modulated metabolites of pairwise comparisons by combining log2(Fold change) and -log10(p-value) on to a single graph. This plot allows the user to comprehensively identify significantly modulated metabolites based on either biological significance, statistical significance, or both. The volcano plot is also highly dynamic. You can use the dropdown buttons to choose the covariate, groups to compare, and additional comparison parameters in the ‘limit comparison’ dropdown. The p-value and fold change cutoffs can be changed via either the text boxes or sliders. The data can also be downloaded from the Data matrix tab.
The log2 fold change is the log2 of the ratio between the average values across samples of two conditions.’adjusted pvalue’ represent the p-value of each treatment group adjusted across other pairwise comparison.
To display the plot for a compound, click that compound’s name in the list below. This list is scrollable, and the search box can be used to refine the list of compounds shown. You can select multiple compounds using shift- or command-click to display multiple plots at once.
To display a pathway, click that pathway’s entry in the table below. Click on a column heading to sort entries by that column. Click on the column heading again to toggle the sort direction. If you enter a search term in the search box, only matching pathways will be included in the table. The table is paginated. You can move between pages using the controls at the lower right. You can also change the page size (number of pathways included per page).